Patent Application
20230374500
This application claims priority based on Korean Patent Application No. 10-2020-0129937 filed on Oct. 8, 2020 and Korean Patent Application No. 10-2021-0050093 filed on Apr. 16, 2021, the disclosure of each of which is incorporated herein by reference in its entirety.
Disclosed herein is a technology for the field of using a CRISPR/Cas system, particularly a CRISPR/Cas12f1 system, for gene editing.
A CRISPR/Cas12f1 system is a CRISPR/Cas system classified as Class 2, Type V. A previous study (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)) has reported for the first time a CRISPR/Cas14a system which is a CRISPR/Cas system derived from Archaea. Since then, a subsequent study (Karvelis et al., Nucleic Acids Research, Vol. 48, No. 9 5017 (2020)) classified the CRISPR/Cas14 system as a CRISPR/Cas12f1 system. The CRISPR/Cas12f1 system belongs to a V-F1 system, which is a subtype of the CRISPR/Cas system classified as Class 2, Type V, and includes the CRISPR/Cas14a system having Cas14a as an effector protein. The CRISPR/Cas12f1 system is characterized in that a size of its effector protein is significantly smaller than a CRISPR/Cas9 system. However, as revealed in a previous study (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018), US 2020/0190494 A1), the CRISPR/Cas12f1 system, particularly the CRISPR/Cas14a system shows cleavage activity on single-stranded DNA and has no or extremely low cleavage activity on double-strand DNA, which limits its application to gene editing technology.
A recent prior literature (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) revealed the fact that a Cas12f1 protein forms a dimer in the CRISPR/Cas12f1 system, and a structure of its guide RNA. It was found by the prior literature that the guide RNA includes a so-called disordered region which does not directly interact with the Cas12f1 protein. In another prior literature (Xiao et al., Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease, bioRxiv (2020)), double-stranded DNA cleavage efficiency of the system was investigated in vitro with the disordered region removed.
However, the preceding studies did not show intracellular gene editing activity, nor were related to methods of increasing intracellular gene editing activity (for example, indel generation efficiency). Among the studies, there was an experiment in which the disordered region was removed, but it was reported in this experiment that the cleavage activity was even lowered. Moreover, the experiment performed in the above study was conducted in vitro and failed to reveal what modifications exhibit intracellular gene editing activity or increase efficiency.
The present disclosure intends to provide an engineered Cas12f1 guide RNA that can be used in a CRISPR/Cas12f1 system to increase gene editing efficiency.
The present disclosure intends to provide an engineered scaffold region and a U-rich tail which are included in the engineered Cas12f1 guide RNA and can increase gene editing efficiency.
The present disclosure intends to provide an engineered CRISPR/Cas12f1 complex with increased gene editing efficiency.
The present disclosure intends to provide an engineered CRISPR/Cas12f1 system with increased gene editing efficiency.
The present disclosure intends to provide a vector having nucleic acids that encode respective components of the engineered CRISPR/Cas12f1 system.
The present disclosure intends to provide a gene editing method using the engineered CRISPR/Cas12f1 system.
The present disclosure intends to provide a use of the engineered CRISPR/Cas12f1 system.
In an embodiment, there is provided herein an engineered guide RNA for a CRISPR/Cas12f1 system, comprising:
In an embodiment, there is provided herein an engineered guide RNA for a CRISPR/Cas12f1 system, comprising:
In an embodiment, there is provided in an engineered guide RNA for a CRISPR/Cas12f1 system, comprising:
In an embodiment, there is provided herein an engineered CRISPR/Cas12f1 complex, comprising:
In an embodiment, there is provided a vector that is capable of expressing respective components of a CRISPR/Cas12f1 system, comprising: a first sequence comprising a nucleic acid sequence encoding a Cas12f1 protein;
In an embodiment, there is provided herein a method of editing a target nucleic acid in a cell or a method of targeting a target nucleic acid in a cell, comprising:
In an embodiment, there is provided a use of an engineered Cas12f1 guide RNA for gene editing.
When a CRISPR/Cas12f1 system comprising an engineered Cas12f1 guide RNA having the engineered scaffold region and the U-rich tail provided herein, is used for gene editing, the CRISPR/Cas12f1 system exhibits higher gene editing efficiency than a naturally occurring CRISPR/Cas12f1 system.
As used herein, the term “about” refers to an amount, level, value, number, frequency, percent, dimension, size, amount, weight or length that varies by approximately 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% with respect to a reference amount, level, value, number, frequency, percent, dimension, size, amount, weight or length.
As used herein, the symbols A, T, C, G and U have the same meanings as commonly understood by those skilled in the art to which this invention belongs. It may be properly interpreted as a base, a nucleoside, or a nucleotide in DNA or RNA according to the context and description. For example, in a case where the symbols mean bases, they may be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), respectively; in a case where the symbols mean nucleosides, they may be interpreted as adenosine (A), thymidine (T), cytidine (C), guanosine (G) or uridine (U), respectively, and in a case where the symbols mean nucleotides, they may be interpreted to mean nucleotides including the respective nucleosides.
As used herein, the term “operably linked” means that, in gene expression, a particular component is linked to another component so that the particular component can perform its intended function. For example, in a case where a promoter sequence is operably linked to a coding sequence, it means that the promoter is linked thereto so as to affect transcription and/or expression of the coding sequence in a cell. In addition, the term includes all meanings recognized by those skilled in the art and may be appropriately interpreted according to the context.
As used herein, “target gene” or “target nucleic acid” basically means a gene or nucleic acid in a cell which becomes a target for gene editing. The target gene or target nucleic acid may be used interchangeably and may refer to the same target. Unless otherwise described, the target gene or target nucleic acid may refer to an endogenous gene or nucleic acid found in the cell, or an exogeneous gene or nucleic acid, and is not limited to particular embodiments as long as it can be a target for editing. The target gene or target nucleic acid may contain a target sequence or a region adjacent thereto. The target gene or target nucleic acid may be single-stranded DNA, double-stranded DNA, and/or RNA. In addition, the term includes all meanings recognized by those skilled in the art, and may be appropriately interpreted according to the context.
As used herein, “target sequence” or “recognition sequence” refers to a particular sequence recognized by a CRISPR/Cas complex to cleave a target gene or target nucleic acid. The target sequence may be appropriately selected depending on the purpose. Specifically, the “target sequence” may be a sequence included in the above-described target gene or target nucleic acid and refers to a sequence having complementarity with a spacer sequence included in the guide RNA or the engineered guide RNA provided herein. In general, the spacer sequence is determined in consideration of a sequence of a target gene or target nucleic acid and a protospacer adjacent motif (PAM) sequence recognized by an effector protein of a CRISPR/Cas system. In the present disclosure, the target sequence may refer only to a particular strand complementarily binding to a guide RNA of a CRISPR/Cas complex, or may refer to an entire target double strand including the particular strand. The term may be interpreted appropriately depending on the context. In addition, the term includes all meanings recognized by those skilled in the art and may be appropriately interpreted according to the context.
As used herein, unless otherwise specified, the “vector” refers collectively to any material capable of transporting a genetic material into a cell. For example, a vector may be a DNA molecule including a genetic material of interest, which is for example, a nucleic acid encoding an effector protein of a CRISPR/Cas system, and/or a nucleic acid encoding a guide RNA, but is not limited thereto. The term includes all meanings recognized by those skilled in the art and may be appropriately interpreted according to the context.
As used herein, the term “naturally occurring” refers to an object that is found in nature and is not artificially modified. The term is used to distinguish it from an “engineered object” obtained by artificial modification. The “naturally occurring” gene, nucleic acid, DNA, RNA, and the like are used as concepts that encompass all genes, nucleic acids, DNA, and RNA in wild type (original form) and mature form (active form). The term includes all meanings recognized by those skilled in the art and should be appropriately interpreted according to the context.
As used herein, the term “engineered” is used to distinguish it from a material, a molecule or the like whose configuration already exists in nature, and refers to a material, a molecule or the like which has undergone artificial modification. For example, the “engineered guide RNA” refers to a guide RNA obtained by applying artificial modification to the configuration of a naturally occurring guide RNA. In addition, the term includes all meanings recognized by those skilled in the art and may be appropriately interpreted according to the context.
The term “NLS” as used herein refers to a peptide of a certain length or a sequence thereof that is attached to a substance to be transported into the cell nucleus by nuclear transport and acts as a type of “tag.” Specifically, the NLS may be, but is not limited to, an NLS sequence derived from: the NLS of a SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 278); the NLS from a nucleoplasmin (for example, the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 279)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 280) or RQRRNELKRSP (SEQ ID NO: 281); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 282); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 283) of an IBB domain from importin alpha; the sequences VSRKRPRP (SEQ ID NO: 284) and PPKKARED (SEQ ID NO: 285) of myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 286) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 287) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 288) and PKQKKRK (SEQ ID NO: 289) of influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 290) of hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 291) of mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 292) of human poly (ADP-ribose) polymerase; or the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 293) of steroid hormone receptor (human) glucocorticoid. As used herein, the term “NLS” includes all meanings recognized by those skilled in the art and may be appropriately interpreted according to the context.
The term “NES” as used herein refers to a peptide of a certain length or a sequence thereof that is attached to a substance to be transported outside the nucleus by nuclear transport and acts as a type of “tag.” As used herein, the term “NES” includes all meanings recognized by those skilled in the art and may be appropriately interpreted according to the context.
As used herein, the term “tag” refers collectively to a functional domain added to facilitate transport, tracking, and/or separation and purification of a peptide or protein. Specifically, the tag includes, but is not limited to, tag proteins such as a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, an Myc tag, a VSV-G tag, and a thioredoxin (Trx) tag; autofluorescent proteins such as a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a cyan fluorescent protein (CFP), a blue fluorescent protein (BFP), HcRED, and DsRed; and reporter proteins such as a glutathione-S-transferase (GST), a horseradish peroxidase (HRP), a chloramphenicol acetyltransferase (CAT) beta-galactosidase, a beta-glucuronidase, and a luciferase. As used herein, the term “tag” includes all meanings recognized by those skilled in the art and may be appropriately interpreted according to the context.
A CRISPR/Cas12f system belongs to a V-F subtype among type V CRISPR/Cas systems, which is further divided into V-F1 to V-F3 variants. The CRISPR/Cas12f system includes a CRISPR/Cas14 system comprising Cas14a, Cas14b, or Cas14c variant among the effector proteins named Cas14 in a previous study (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)). Among them, the CRISPR/Cas14a system including a Cas14a effector protein is classified as a CRISPR/Cas12f1 system (Makarova et al., Nature Reviews, Microbiology volume 18, 67 (2020)). Recent previous studies (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021), Xiao et al., Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease, bioRxiv (2020)), etc. have revealed a structure of the CRISPR/Cas12f1 complex, which will be briefly described below.
It was found that within the CRISPR/Cas12f1 complex, two molecules of the Cas12f1 protein, which are in a form of a dimer, bind to a guide RNA to form a complex. The Cas12f1 protein contains an amino-terminal domain (NTD) and a carboxy-terminal domain (CTD) and has a structure in which the two domains are linked by a linker loop. The NTD consists of wedge (WED), recognition (REC), and zinc finger (ZF) domains, and the CTD consists of another ZF domain and an RuvC domain. The structure of the Cas12f1 dimeric protein may be largely divided into a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe consists of: a WED domain, a ZF domain, and an REC domain of one Cas12f1 protein constituting the dimer; and a WED domain, a ZF domain, and an REC domain of the other Cas12f1 protein constituting the dimer. The nuclease lobe consists of: an RuvC domain and a TNB domain of one Cas12f1 protein constituting the dimer; and an RuvC domain and a TNB domain of the other Cas12f1 protein constituting the dimer. All or part of each domain of the Cas12f1 protein respectively recognizes a specific portion of the scaffold region of the Cas12f1 guide RNA and forms a CRISPR/Cas12f1 complex.
In the present disclosure, the Cas12f1 guide RNA may be largely divided into a spacer region and a scaffold region by function. The scaffold region consists of five stems (named Stems 1 to 5) and one pseudoknot (PK).
Generally, it is well known to those skilled in the art that a naturally occurring Cas12f1 guide RNA is divided into a tracrRNA and a crRNA, in which the crRNA may be further divided into a crRNA repeat sequence portion and a spacer. Apart from the above criteria, in the present disclosure, parts of the Cas12f1 guide RNA, which interact with the Cas12f1 protein, are collectively referred to as a scaffold region. The scaffold region is a region including a tracrRNA and a portion of a crRNA, and functions to interact with a Cas12f1 protein. A detailed description thereof will be provided in the corresponding section below.
The Cas12f1 guide RNA includes two structures in which a part of a tracrRNA (tracrRNA anti-repeat) and a part of a crRNA repeat portion are complementarily bound to form a duplex, and this is named a crRNA repeat-tracrRNA anti-repeat (R:AR) portion. The Stem 5 (R:AR2), and PK (R:AR1) form this crRNA repeat-tracrRNA anti-repeat duplex structure. In the CRISPR/Cas12f1 complex, Stem 1 portion, a part of Stem 2, and Stem 5 (R:AR2) portion in the Cas12f1 guide RNA were found not to interact with the Cas12f1 dimer, and these are called a disordered region.
In order to use the CRISPR/Cas system for gene editing, a method is widely used in the art that introduces a vector, which has nucleotide sequences encoding respective components of the CRISPR/Cas system, into a cell so that the respective components of the CRISPR/Cas system are expressed in the cell. Hereinafter, the components of the vector, which allow the CRISPR/Cas system to be expressed in a cell, will be described.
Since the purpose of the vector is to express respective components of the CRISPR/Cas system in a cell, a sequence of the vector may comprise one or more of nucleic acids encoding the respective components of the CRISPR/Cas system. Specifically, the sequence of the vector comprises nucleic acids encoding a guide RNA and/or a Cas protein which are included in the CRISPR/Cas system to be expressed. Here, a nucleotide sequence of the vector may comprise nucleic acids encoding an engineered Cas12f1 guide RNA and a codon-optimized Cas protein or a nucleic acid encoding an engineered Cas protein according to the purpose, as well as nucleic acids encoding a wildtype guide RNA and a wildtype Cas protein. Here, the nucleic acid sequence encoding each component may be a DNA sequence.
In order to express the components in a cell, the vector needs to contain one or more regulatory/control elements. Specifically, the regulatory/control element may include, but is not limited to, a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor, a 2A sequence and/or a replication origin. The replication origin may be, but is not limited to, an f1 origin of replication, a SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, and/or a BBV origin of replication.
In order to cause the vector to express its target in a cell, a promoter sequence needs to be operatively linked to a sequence encoding each component so that a transcription factor can be activated in the cell. The promoter sequence may be designed differently depending on the corresponding transcription factor or expression environment and is not limited to any particular embodiments as long as it may properly express the components of the CRISPR/Cas system in a cell. The promoter sequence may be a promoter that promotes activation of an RNA polymerase (for example, RNA Pol I, Pol II, or Pol III). For example, the promoter may be, but is not limited to, one selected from: a SV40 early promoter, a mouse mammary tumor virus long terminal repeat (LTR) promoter, an adenovirus major late promoter (Ad MLP), a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as a CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1:31(17)), a human H1 promoter (H1), and a 7SK promoter.
A sequence, which induces termination of transcriptional activity of the transcription factor is referred to as a termination signal. The termination signal may vary depending on the type of promoter sequence. For example, when the promoter is a U6 or H1 promoter, the promoter recognizes a thymidine repeat sequence (for example, a TTTTTT (T6) sequence) as a termination signal.
The vector may comprise a nucleic acid sequence encoding an additional element as necessary in addition to components of a wildtype CRISPR/Cas system, and/or an engineered CRISPR/Cas system. For example, the additional element may be one of the tags described in the section “Tag,” but is not limited thereto. For example, the additional element may be a selection gene for selection, for example, an herbicide resistance gene such as glyphosate, glufosinate ammonium or phosphinothricin, or an antibiotic resistance gene such as ampicillin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, but is not limited thereto.
The expression vector may be designed in a form of a linear or circular vector.
The CRISPR/Cas12f1 system is an attractive system for gene editing technology due to its relatively small size. Since it was first reported (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)), several studies have been conducted on it (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018), US 2020/0190494 A1). However, the CRISPR/Cas12f1 system shows no or too low gene editing activity in cells (for example, eukaryotic cells), which is an obstacle to its utilization.
As a result of continuing studies, a recent prior literature (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) revealed the fact that a Cas12f1 protein forms a dimer in the CRISPR/Cas12f1 system, and a structure of its guide RNA. It was found by the prior literature that there is a so-called disordered region in the guide RNA that does not directly interact with the Cas12f1 protein. In another prior literature (Xiao et al., Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease, bioRxiv (2020)), double-stranded DNA cleavage efficiency of the system was investigated in vitro with the disordered region removed. However, the preceding studies did not show intracellular gene editing activity, nor were related to methods of increasing intracellular gene editing activity (for example, indel generation efficiency). Among the studies, there is an experiment in which the disordered region was removed, however, it was reported in this experiment that the cleavage activity was even lowered. Moreover, the experiment performed in the above study was performed in vitro and failed to reveal what modifications exhibit intracellular gene editing activity or increase efficiency.
Therefore, enhancing the intracellular gene editing activity of the CRISPR/Cas12f1 system remains an important task.
In the present disclosure, there is provided an engineered Cas12f1 guide RNA for increasing intracellular gene editing activity of a CRISPR/Cas12f1 system by overcoming limitations of the prior art. The engineered Cas12f1 guide RNA is a naturally occurring Cas12f1 guide RNA to which a new component is added and also in which a part of its structure is modified. The engineered Cas12f1 guide RNA is characterized by comprising a U-rich tail, which is a new component, at the 3′ end. In addition, the engineered Cas12f1 guide RNA is characterized in that at least a portion of the scaffold region, which serves to interact with a Cas12f1 protein, is modified.
In an embodiment, the engineered Cas12f1 guide RNA may comprise an engineered scaffold region, a spacer, and a U-rich tail. The engineered scaffold region is characterized by being different from a scaffold region of a naturally occurring guide RNA.
The engineered Cas12f1 guide RNA provided herein is characterized in that a U-rich tail is added to a naturally occurring guide RNA. The U-rich tail is located at the 3′ end portion of the engineered Cas12f1 guide RNA and is a portion rich in uridine.
In an embodiment, the engineered Cas12f1 guide RNA may comprise a U-rich tail, which is rich in uridine, at the 3′ end portion. In an embodiment, a sequence of the U-rich tail may be represented by (UaN)bUc. In this formula, N is selected from A, U, C, or G, and a, b, and c are each an integer, with a being between 1 and 5 inclusive, b being between 0 and 2 inclusive, and c being between 1 and 10 inclusive.
Characteristic 2 of Engineered Cas12f1 Guide RNA—One or More Parts of Scaffold Region being Modified
The engineered Cas12f1 guide RNA provided herein is characterized in that a part of its scaffold region is modified as compared with a naturally occurring guide RNA. The scaffold region comprises a tracrRNA and a part of a crRNA and has a function of interacting with a Cas12f1 protein. The scaffold region will be described in more detail below.
In an embodiment, the engineered Cas12f1 guide RNA may comprise an engineered scaffold region. The engineered scaffold region is obtained by modifying a scaffold region of a naturally occurring guide RNA. Therefore, the engineered scaffold region has a different sequence from the scaffold region of the naturally occurring guide RNA. In an embodiment, the engineered scaffold region may be obtained by removing a part of a scaffold region of a naturally occurring guide RNA. In an embodiment, the engineered scaffold region may be obtained by removing one or more nucleotides from a particular part included in a scaffold region of a naturally occurring guide RNA.
By using an engineered Cas12f1 guide RNA provided herein, the CRISPR/Cas12f1 system shows dramatically improved gene editing activity in a cell as compared with when a naturally occurring guide RNA is used. The present inventors revealed in detail through experiments what components need to be added to a naturally occurring guide RNA, or what modifications need to be applied to its scaffold region, to improve its gene editing efficiency. Use of the engineered Cas12f1 guide RNA makes it possible to edit a gene in a cell with high efficiency by overcoming limitations of the prior art. In addition, the engineered Cas12f1 guide RNA has a length equal to or shorter than a naturally occurring guide RNA, and thus has a high potential for application in the field of gene editing technology. The engineered Cas12f1 guide RNA of the present disclosure makes it possible to fully utilize the advantages of the CRISPR/Cas12f1 system (for example, the advantage of having a very small size) in gene editing technology.
The engineered Cas12f1 guide RNA provided herein may be used for gene editing and/or gene therapy together with a Cas12f1 protein. In addition, the engineered Cas12f1 guide RNA may be used for preparing a composition for gene editing.
In the present disclosure, there is provided a U-rich tail that can be introduced into a CRISPR/Cas12f1 system to improve gene editing efficiency thereof. A sequence of the U-rich tail is characterized by being linked to the 3′ end of a spacer of a crRNA in the engineered Cas12f1 guide RNA, and this sequence acts to increase cleavage efficiency, against a target nucleic acid, of a CRISPR/Cas12f1 system in which the engineered Cas12f1 guide RNA is used. The U-rich tail sequence basically comprises at least one uridine. The U-rich tail sequence may further comprise an additional nucleotide in addition to uridine according to an actual environment in which the engineered CRISPR/Cas12f1 system is used and expressed (for example, an environment in a eukaryotic cell or a prokaryotic cell).
The U-rich tail sequence may be a sequence of the U-rich tail sequence disclosed in the international application PCT/KR2020/014961. Hereinafter, when referring to the U-rich tail sequence in the present disclosure, it should be understood as including all of the contents and experimental results related to the U-rich tail sequence disclosed in the international application PCT/KR2020/014961, and the disclosure of which is incorporated herein by reference in its entirety.
In designing a U-rich tail sequence, the U-rich tail may comprise a sequence that abundantly contains one or more consecutive uridines. The present inventors have found through experiments that introduction of a U-rich tail sequence into a CRISPR/Cas12f1 system enables the CRISPR/Cas12f1 complex to show improved gene editing efficiency. Accordingly, the U-rich tail sequence provided herein comprises a sequence that contains one or more consecutive uridines.
In an embodiment, the U-rich tail sequence may comprise a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive uridines are contained.
The U-rich tail sequence provided herein may comprise a ‘modified uridine repeat sequence’ that contains at least one ribonucleoside (A, C, G) other than uridine for every repetition of 1 to 5 uridines. The modified uridine repeat sequence is particularly useful when designing a vector expressing an engineered crRNA.
In an embodiment, the U-rich tail sequence may comprise a sequence in which one or more UV, UUV, UUUV, UUUUV, and/or UUUUUV are repeated. Here, V is selected from adenosine (A), cytidine (C), and guanosine (G).
In an embodiment, the U-rich tail sequence may be represented by (UaN)bUc. Here, N is selected from adenosine (A), uridine (U), cytidine (C), and guanosine (G). Here, a, b, and c are each an integer, with a being between 1 and 5 inclusive, and b being 0 or more. In an embodiment, b may be between 0 and 2 inclusive. In an embodiment, c may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In an embodiment, c may be an integer within a range of two numbers selected from the immediately preceding sentence. For example, c may be between 1 and 6 inclusive.
In an embodiment, a sequence of the U-rich tail may be 5′-U-3′, 5′-UU-3′, 5′-UUU-3′, 5′-UUUU-3′, 5′-UUUUU-3′, 5′-UUUUUU-3′, 5′-UUURUUU-3′, 5′-UUURUUURUUU-3′ (SEQ ID NO: 646), 5′-UUUURU-3′, 5′-UUUURUU-3′, 5′-UUUURUUU-3′, 5′-UUUURUUUU-3′, 5′-UUUURUUUUU-3′ (SEQ ID NO: 647), or 5′-UUUURUUUUUU-3′ (SEQ ID NO: 648). In an embodiment, a sequence of the U-rich tail may be 5′-U-3′, 5′-UU-3′, 5′-UUU-3′, 5′-UUUU-3′, 5′-UUUUU-3′, 5′-UUUUUU-3′, 5′-UUUAUUU-3′, 5′-UUUAUUUAUUU-3′ (SEQ ID NO: 254), 5′-UUUUAU-3′, 5′-UUUUAUU-3′, 5′-UUUUAUUU-3′, 5′-UUUUAUUUU-3′, 5′-UUUUAUUUUU-3′ (SEQ ID NO: 255), 5′-UUUUAUUUUUU-3′ (SEQ ID NO: 256), 5′-UUUGUUU-3′, 5′-UUUGUUUGUUU-3′ (SEQ ID NO: 257), 5′-UUUUGU-3′, 5′-UUUUGUU-3′, 5′-UUUUGUUU-3′, 5′-UUUUGUUUU-3′, 5′-UUUUGUUUUU-3′ (SEQ ID NO: 258), or 5′-UUUUGUUUUUU-3′ (SEQ ID NO: 259).
In an embodiment, a sequence of the U-rich tail may be 5′-UUUUUU-3′, 5′-UUUUAUUUUUU-3′ (SEQ ID NO: 256), or 5′-UUUUGUUUUUU-3′ (SEQ ID NO: 259). In an embodiment, a sequence of the U-rich tail may be 5′-UUUUAUUUU-3′.
It is well known to those skilled in the art that the naturally occurring Cas12f1 guide RNA is divided into a tracrRNA and a crRNA, in which the crRNA may be further divided into a crRNA repeat sequence portion and a spacer.
Apart from the above criteria, in the present disclosure, parts of the Cas12f1 guide RNA, which interacts with a Cas12f1 protein, are collectively referred to as a scaffold region. The scaffold region comprises a tracrRNA and a part of a crRNA, and may not refer to a single molecule of RNA. Referring to
The “n-th region” or “naturally occurring n-th region” (n is an integer between 1 and 6 inclusive) as described below refers to each part of the naturally occurring Cas12f1 guide RNA. Specifically, the “n-th region” may refer to each region corresponding to a structure of a guide RNA transcribed in a prokaryotic cell, and/or a structure of a guide RNA that actually operates in a prokaryotic system (which is in an activated form, not a transcribed form, in a prokaryotic cell). The region in an engineered Cas12f1 guide RNA, which corresponds to the above classification criteria, is generally described as “modified n-th region” or “n-th region of an engineered scaffold region.”
However, an n-th region included in an engineered scaffold region may not be modified and thus may be identical to a naturally occurring n-th region; and only in that case, the term “n-th region” may be used interchangeably. What is referred to by the “n-th region” (for example, whether it is a region included in an engineered Cas12f1 guide RNA or a region included in a naturally occurring guide RNA) should be appropriately interpreted according to the context.
tracrRNA and crRNA
As used herein, the terms “tracrRNA” and “crRNA” include all meanings that can be recognized by those skilled in the field of CRISPR/Cas technology. The terms are generally used to refer to respective molecules of a naturally occurring dual guide RNA, and may also be used to refer to respective corresponding parts of a single guide RNA in which a tracrRNA and a crRNA are linked by a linker. Unless otherwise specified, in a case of being merely written as “tracrRNA” and “crRNA”, the terms refer to a tracrRNA and a crRNA constituting a CRISPR/Cas12f1 system, respectively.
In an embodiment, a sequence of the tracrRNA may be 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3′ (SEQ ID NO: 1) or 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUC UCCAAUUCUGCACAA-3′ (SEQ ID NO: 2). In an embodiment, the tracrRNA comprises a first region, a second region, a third region, and a fourth region. In an embodiment, the tracrRNA is one in which the first region, the second region, the third region, and the fourth region are sequentially linked to each other in a 5′ to 3′ direction.
In an embodiment, a sequence of the crRNA comprises a crRNA repeat sequence and a spacer sequence. The crRNA repeat sequence may be 5′-GAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 3) or 5′-GUUGCAGAACCCGAAUAGACGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 4). The crRNA repeat sequence comprises a fifth region and a sixth region. The spacer sequence may vary depending on a target sequence, and generally comprises 10 to 50 nucleotides. In an embodiment, the crRNA is one in which the fifth region, the sixth region, and the spacer are sequentially linked to each other in a 5′ to 3′ direction.
As used herein, the term “scaffold region” refers collectively to the rest of a naturally occurring guide RNA excluding the spacer. Specifically, the scaffold region comprises the tracrRNA, and a part of the crRNA. Specifically, the part of the crRNA may be a crRNA repeat sequence portion. The scaffold region is generally known as a portion capable of interacting with a Cas protein. In the present disclosure, the scaffold region is divided into first to sixth regions for description, and each region will be described in more detail below.
As used herein, the term “first region” refers to a region comprising the 5′ end of the tracrRNA. The first region may comprise nucleotides forming a stem structure in the CRISPR/Cas12f1 complex, and may comprise nucleotides adjacent thereto.
The first region comprises a Stem 1 portion (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)). The first region may comprise one or more nucleotides adjacent to the Stem 1 portion.
The first region comprises a disordered region that does not interact with a Cas12f1 protein in the CRISPR/Cas12f1 complex.
In an embodiment, the first region may refer to the 1st to 11th nucleotides from the 5′ end of the tracrRNA represented by SEQ ID NO: 1 or 2. In an embodiment, a sequence of the first region may be 5′-CUUCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 10).
As used herein, the term “second region” refers to a region located at the 3′ end of the first region in the tracrRNA. The second region may comprise nucleotides forming a stem structure in a CRISPR/Cas12f1 complex and may comprise nucleotides adjacent thereto. Here, the stem structure is different from the stem included in the first region.
The second region comprises a Stem 2 portion (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)). The second region may comprise one or more nucleotides adjacent to the Stem 2 portion. The second region may comprise one or more nucleotides that interact with an RuvC domain of one dimer-forming Cas12f1 protein and/or an RuvC domain of the other dimer-forming Cas12f1 protein in the CRISPR/Cas12f1 complex. The second region comprises a disordered region that does not interact with a Cas12f1 protein in the CRISPR/Cas12f1 complex.
In an embodiment, the second region may refer to the 22nd to 72nd nucleotides from the 5′ end of the tracrRNA represented by SEQ ID NO: 1 or 2. In an embodiment, a sequence of the second region may be 5′-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGU GG-3′ (SEQ ID NO: 11).
As used herein, the term “third region” refers to a region located at the 3′ end of the second region in a tracrRNA. The third region may comprise nucleotides forming a stem structure in the CRISPR/Cas12f1 complex and nucleotides forming complementary bonds with some nucleotides included in the crRNA and may comprise nucleotides adjacent thereto.
The third region comprises nucleotides which belong to the tracrRNA in a Stem 4 portion (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) and a Stem 3-PK (R:AR-1) portion (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)). The third region may comprise one or more nucleotides adjacent to the nucleotides which belong to the tracrRNA in the Stem 4 portion and/or the Stem 3-PK (R:AR-1) portion.
The third region comprises one or more nucleotides that interact with a WED domain and/or an RuvC domain of one dimer-forming Cas12f1 protein in the CRISPR/Cas12f1 complex. The nucleotides may be nucleotides which belong to the tracrRNA in the Stem 3-PK (R:AR-1) portion.
The third region comprises one or more nucleotides that interact with an RuvC domain of one dimer-forming Cas12f1 protein and/or an REC domain of the other dimer-forming Cas12f1 protein in the CRISPR/Cas12f1 complex. The nucleotides may be nucleotides included in the Stem 4 portion.
The third region may comprise one or more nucleotides complementarily binding to one or more nucleotides included in the sixth region of the crRNA.
In an embodiment, the third region may refer to the 73rd to 127th nucleotides from the 5′ end of the tracrRNA represented by SEQ ID NO: 1 or 2. In an embodiment, a sequence of the third region may be 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAAC CCUCGA-3′ (SEQ ID NO: 12).
As used herein, the term “fourth region” refers to a region located at the 3′ end of the third region in the tracrRNA. The fourth region may comprise nucleotides capable of forming complementary bonds with some nucleotides included in the crRNA in the CRISPR/Cas12f1 complex and may comprise nucleotides adjacent thereto.
The fourth region comprises nucleotides which belong to the tracrRNA in Stem 5 (R:AR-2) (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)). The fourth region may comprise one or more nucleotides adjacent to the nucleotides which belong to the tracrRNA in Stem 5 (R:AR-2).
The fourth region comprises one or more nucleotides that interact with a WED domain and/or a ZF domain of one dimer-forming Cas12f1 protein in the CRISPR/Cas12f1 complex. The nucleotides may be nucleotides which belong to the tracrRNA in Stem 5 (R:AR-2).
The fourth region may comprise one or more nucleotides complementarily binding to one or more nucleotides included in the fifth region of the crRNA. The fourth region comprises a disordered region that does not interact with a Cas12f1 protein in a CRISPR/Cas12f1 complex.
In an embodiment, the fourth region may refer to the 128th to 140th nucleotides from the 5′ end of the tracrRNA represented by SEQ ID NO: 1. In an embodiment, the fourth region may refer to the 128th to 162nd nucleotides from the 5′ end of the tracrRNA represented by SEQ ID NO: 2.
In an embodiment, a sequence of the fourth region may be 5′-AACAAAUUCAUUU-3′ (SEQ ID NO: 13) or 5′-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAA-3′ (SEQ ID NO: 14).
As used herein, the term “fifth region” refers to a region comprising the 5′ end of the crRNA. The fifth region may comprise nucleotides that form complementary bonds with one or more nucleotides of the fourth region in a CRISPR/Cas12f1 complex and may comprise any nucleotide adjacent thereto.
The fifth region comprises nucleotides which belong to the crRNA in Stem 5 (R:AR-2) (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)). The fifth region may comprise any one or more nucleotides adjacent to the nucleotides which belong to the crRNA in Stem 5 (R:AR-2).
The fifth region comprises one or more nucleotides that interact with a WED domain, an REC domain, and/or a ZF domain of one dimer-forming Cas12f1 protein in a CRISPR/Cas12f1 complex. Here, the nucleotides may be nucleotides which belong to the crRNA in Stem 5 (R:AR-2).
The fifth region may comprise one or more nucleotides complementarily binding to one or more nucleotides included in the fourth region. The fifth region comprises a disordered region that does not interact with a Cas12f1 protein in the CRISPR/Cas12f1 complex.
In an embodiment, the fifth region may refer to the 1st to 10th nucleotides from the 5′ end of the crRNA represented by SEQ ID NO: 3. In an embodiment, the fifth region may refer to the 1st to 30th nucleotides from the 5′ end of the crRNA represented by SEQ ID NO: 4. In an embodiment, a sequence of the fifth region may be 5′-GAAUGAAGGA-3′ (SEQ ID NO: 15) or 5′-GUUGCAGAACCCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 16).
As used herein, the term “sixth region” refers to a region located at the 3′ end of the fifth region in the crRNA. The sixth region may comprise nucleotides that form complementary bonds with one or more nucleotides of the third region in a CRISPR/Cas12f1 complex, and may comprise any nucleotide adjacent thereto.
The sixth region comprises nucleotides which belong to the crRNA in Stem 3-PK (R:AR-1) (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)). The sixth region may comprise any one or more nucleotides adjacent to the nucleotides which belong to the crRNA in Stem 3-PK (R:AR-1).
The sixth region comprises one or more nucleotides that interact with a WED domain, a ZF domain, and/or an RuvC domain of one dimer-forming Cas12f1 protein in the CRISPR/Cas12f1 complex. The nucleotides may be nucleotides which belong to the crRNA in Stem 3-PK (R:AR-1).
In an embodiment, the sixth region may refer to the 11th to 17th nucleotides from the 5′ end of the crRNA represented by SEQ ID NO: 3. In an embodiment, the sixth region may refer to the 31st to 37th nucleotides from the 5′ end of the crRNA represented by SEQ ID NO: 4. In an embodiment, a sequence of the sixth region may be 5′-AUGCAAC-3′.
As used herein, the term “spacer” refers to one or more nucleotides which hybridize with a target sequence in a CRISPR/Cas12f1 system. The spacer refers to 10 to 50 consecutive nucleotides near the 3′ end of the crRNA of the guide RNA in the CRISPR/Cas12f1 system. The spacer is designed to match a target sequence in the target nucleic acid to be edited using the CRISPR/Cas12f1 system. In other words, the spacer may have a different sequence depending on a target sequence of the target nucleic acid.
In the present disclosure, there is provided an engineered scaffold region that can be introduced into a CRISPR/Cas12f1 system to improve gene editing efficiency thereof. The engineered scaffold region synergizes with the above-described U-rich tail to improve gene editing efficiency of a CRISPR/Cas12f1 system in which an engineered Cas12f1 guide RNA is used. The engineered scaffold region is characterized in that it is obtained by applying one or more mutations in the scaffold region of a naturally occurring Cas12f1 guide RNA (hereinafter, naturally occurring scaffold region), and thus is different therefrom in terms of sequence and/or structure.
Here, functions of the engineered scaffold region are identical or similar to those of the naturally occurring scaffold region. Specifically, the engineered scaffold region has a function of interacting with a Cas12f1 protein dimer in the CRISPR/Cas12f1 complex.
In an embodiment, the engineered scaffold region comprises regions corresponding to respective portions of the naturally occurring scaffold region. Specifically, the engineered scaffold region includes a first region, a second region, a third region, a fourth region, a fifth region, and a sixth region, which respectively correspond to the first to sixth regions included in the naturally occurring scaffold region.
In an embodiment, the engineered scaffold region may not comprise regions corresponding to the first region and/or the second region included in the naturally occurring scaffold region.
The engineered Cas12f1 guide RNA provided herein may be a single guide RNA of one molecule. Accordingly, the engineered scaffold region provided herein may have a modification(s) in one or more of the respective regions, and additionally, the 3′ end of the fourth region of the tracrRNA and the 5′ end of the fifth region of the crRNA may be linked by a linker.
In an embodiment, the engineered scaffold region may be an engineered form of a naturally occurring scaffold region in which one or more regions are modified and the 3′ end of the fourth region and the 5′ end of the fifth region is linked by a linker. For example, the linker may be 5′-GAAA-3′.
The engineered scaffold region provided herein may be an engineered form of a naturally occurring scaffold region in which the first region is modified.
In an embodiment, the engineered scaffold region may comprise a ‘modified first region.’ Here, the modified first region is obtained by removing one or more nucleotides from the first region of the naturally occurring scaffold region. Here, the removed nucleotide(s) is a nucleotide(s) selected from a region forming a stem structure in a CRISPR/Cas12f1 complex.
In an embodiment, the engineered scaffold region in the engineered Cas12f1 guide RNA may be an engineered form of a naturally occurring scaffold region in which one or more nucleotides are removed from the first region and which may optionally have a modification(s) in other portions than the first region. In an embodiment, the removed nucleotide(s) may be a nucleotide(s) included in a portion, which forms a stem structure in the CRISPR/Cas12f1 complex, in the naturally occurring first region. In an embodiment, the removed nucleotide(s) may be a nucleotide(s), which belongs to Stem 1 (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)), in the naturally occurring first region. In an embodiment, the removed nucleotide(s) may be a nucleotide(s), which does not interact with a Cas12f1 protein in the CRISPR/Cas12f1 complex, in the naturally occurring first region.
In an embodiment, the modified first region comprises the sequence 5′-A-3′ at the 3′ end.
In an embodiment, the engineered scaffold region may be an engineered form of a naturally occurring scaffold region from which the first region is removed. In other words, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region.
The first region of the engineered scaffold region may be a modified form of a first region of the naturally occurring scaffold region from which one or more nucleotides are removed.
In an embodiment, the modified first region of the engineered scaffold region may be a modified form of a first region of the naturally occurring scaffold region from which 1 to 20 nucleotides at the 5 end are removed. In an embodiment, the modified first region may be a modified form of a first region of the naturally occurring scaffold region from which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides at the 5′ end are removed. In an embodiment, the modified first region may be a modified form of a first region of the naturally occurring scaffold region from which consecutive nucleotides at the 5′ end, the number of which is within a range of two numbers selected from the immediately preceding sentence, are removed. For example, the modified first region may be a modified form of a first region of the naturally occurring scaffold region from which 1 to 3 consecutive nucleotides at the 5′ end are removed.
In an embodiment, the modified first region comprises at least one nucleotide, which may be 5′-A-3′.
The engineered scaffold region provided herein may be a modified form of a naturally occurring scaffold region from which the first region is removed.
In an embodiment, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region.
Examples of Engineered Scaffold Sequence in which First Region is Modified
In an embodiment, a sequence of the modified first region may be selected from 5′-A-3′, 5′-AA-3′, 5′-GAA-3′, 5′-AGAA-3′, 5′-GAGAA-3′, 5′-GGAGAA-3′, 5′-UGGAGAA-3′, 5′-GUGGAGAA-3′, 5′-AGUGGAGAA-3′, 5′-AAGUGGAGAA-3′ (SEQ ID NO: 17), 5′-AAAGUGGAGAA-3′ (SEQ ID NO: 18), 5′-UAAAGUGGAGAA-3′ (SEQ ID NO: 19), 5′-AUAAAGUGGAGAA-3′ (SEQ ID NO: 20), 5′-GAUAAAGUGGAGAA-3′ (SEQ ID NO: 21), 5′-UGAUAAAGUGGAGAA-3′ (SEQ ID NO: 22), 5′-CUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 23), 5′-ACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 24), 5′-CACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 25), 5′-UCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 26), and 5′-UUCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 27).
In an embodiment, a sequence of the engineered scaffold region in which the first region is modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region is modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region is modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region is modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5′-GAAA-3′.
In an embodiment, a sequence of the engineered scaffold region in which a first region is modified may be a sequence selected from the group consisting of
Examples of Engineered Scaffold Sequence from which First Region is Removed
In an embodiment, a sequence of the engineered scaffold region from which the first region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region from which the first region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region from which the first region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region from which the first region is removed may be one in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5′-GAAA-3′.
In an embodiment, a sequence of the engineered scaffold region from which the first region is removed may be
The engineered scaffold region included in the engineered guide RNA provided herein may be an engineered form of a naturally occurring scaffold region in which the second region is modified.
In an embodiment, the engineered scaffold region may comprise a ‘modified second region.’ Here, the modified second region is a modified form of a second region of the naturally occurring scaffold region from which one or more nucleotides are removed. Here, the removed nucleotide(s) is a nucleotide(s) selected from a region forming a stem structure in a CRISPR/Cas12f1 complex.
In an embodiment, the engineered scaffold region included in the engineered Cas12f1 guide RNA may have an engineered form of a naturally occurring second region from which one or more nucleotides are removed, and which may optionally have a modification(s) in other portions than the second region. In an embodiment, removal of the nucleotides may occur in a portion forming a stem structure in the naturally occurring second region, in which the nucleotides may be removed in complementary pair. In an embodiment, the removed nucleotide may be a nucleotide included in a portion which forms a stem structure in the CRISPR/Cas12f1 complex in the naturally occurring second region.
In an embodiment, the removed nucleotide(s) may be a nucleotide(s), which belongs to Stem 2 (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)), in the naturally occurring second region. In an embodiment, the removed nucleotide(s) may be a nucleotide(s) which does not interact with a Cas12f1 protein in the CRISPR/Cas12f1 complex in the naturally occurring second region.
In an embodiment, the modified second region comprises a sequence of 5′-G-3′ at the 3′ end.
In an embodiment, the engineered scaffold region may be an engineered form of a naturally occurring scaffold region from which the second region is removed. In other words, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
The second region of the engineered scaffold region may be a modified form of a second region of the naturally occurring scaffold region from which one or more nucleotides are removed.
In an embodiment, the modified second region of the engineered scaffold region may be a modified form of a second region of the naturally occurring scaffold region from which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51 consecutive or nonconsecutive nucleotides are removed. In an embodiment, the modified second region may be a modified form of a second region of the naturally occurring scaffold region from which one or more nucleotides, of the 1st to 22nd nucleotides and/or the 27th to the 51st nucleotides from the 5′ end based on the sequence of SEQ ID NO: 11, are removed. In an embodiment, the modified second region may be a modified form of a second region of the naturally occurring scaffold region from which one or more nucleotides, of the 1st to 22nd nucleotides and/or the 27th to 51st nucleotides from the 5′ end based on the sequence of SEQ ID NO: 11, are removed, and in which the 23rd to 26th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 11 are substituted with other nucleotides. In an embodiment, the modified second region may be a modified form of a second region of the naturally occurring scaffold region from which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides, of the 1st to 22nd nucleotides from the 5 end based on the sequence of SEQ ID NO: 11, are removed. In an embodiment, the modified second region may be a modified form of a second region of the naturally occurring scaffold region from which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides, of the 27th to 51st nucleotides from the 5′ end based on the sequence of SEQ ID NO: 11, are removed.
In an embodiment, the modified second region comprises at least 5′-G-3′.
The modification of the second region may be removal of one or more pairs of nucleotides that are included in a portion forming a stem structure and complementarily bind to each other.
In an embodiment, the modified second region may be a modified form of a second region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs in the CRISPR/Cas12f1 complex, of the 1st to 22nd nucleotides and/or the 27th to 50th nucleotides from the 5 end based on the sequence of SEQ ID NO: 11, are removed.
In an embodiment, the modified second region may be a modified form of a second region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs and/or one or more nucleotides not forming a complementary pair in the CRISPR/Cas12f1 complex, of the 1st to 22nd nucleotides and/or the 27th to 50th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 11, are removed.
In an embodiment, the modified second region may be a modified form of a second region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs and/or one or more mismatched pairs of nucleotides in the CRISPR/Cas12f1 complex, of the 1st to 22nd nucleotides and/or the 27th to 50th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 11, are removed.
The engineered scaffold region provided herein may be an engineered form of a naturally occurring scaffold region from which the second region is removed.
In an embodiment, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
Examples of Engineered Scaffold Sequence in which Second Region is Modified
In an embodiment, a sequence of the modified second region may be a sequence selected from the group consisting of 5′-G-3′, 5′-UUAGG-3′, 5′-CUUAGGG-3′, 5′-CUUAGUGG-3′, 5′-CCUUAGGUGG-3′ (SEQ ID NO: 342), 5′-CCGUUAGGUGG-3′ (SEQ ID NO: 343), 5′-CCGCUUAGGGUGG-3′ (SEQ ID NO: 344), 5′-CCGCUUUAGAGGUGG-3′ (SEQ ID NO: 345), 5′-CCGCUUUUAGAAGGUGG-3′ (SEQ ID NO: 346), 5′-CCGCUUCUUAGGAAGGUGG-3′ (SEQ ID NO: 347), 5′-CCGCUUCAUUAGUGAAGGUGG-3′ (SEQ ID NO: 348), 5′-CCGCUUCACUUAGGUGAAGGUGG-3′ (SEQ ID NO: 349), 5′-CCGCUUCACUUAGAGUGAAGGUGG-3′ (SEQ ID NO: 350), 5′-CCGCUUCACCUUAGGAGUGAAGGUGG-3′ (SEQ ID NO: 351), 5′-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3′ (SEQ ID NO: 352), 5′-CCGCUUCACCAAUUAGUUGAGUGAAGGUGG-3′ (SEQ ID NO: 353), 5′-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3′ (SEQ ID NO: 354), 5′-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 355), 5′-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 356), 5′-CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 357), 5′-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 358), 5′-CCGCUUCACCAAAAGCUGUUAGUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 359), 5′-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 360), 5′-CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 361) and 5′-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 362).
In an embodiment, a sequence of the engineered scaffold region in which the second region is modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the second region is modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the second region is modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the second region is modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction: a sequence selected from the group consisting of SEQ ID NO: 10, 5′-G-3′, 5′-UUAGG-3′, 5′-CUUAGGG-3′, 5′-CUUAGUGG-3′, and SEQ ID NOS: 342 to 362, SEQ ID NO: 12, SEQ ID NO: 13, a linker, SEQ ID NO: 15, and 5′-AUGCAAC-3′.
Here, the linker may be 5′-GAAA-3′.
In an embodiment, a sequence of the engineered scaffold region in which the second region is modified may be a sequence selected from the group consisting of 5′-CUUCACUGAUAAAGUGGAGAAGGCUGCUUGCAUCAGCCUAAUGUCGAGA AGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAU GAAGGAAUGCAAC-3′ (SEQ ID NO: 408), 5′-CUUCACUGAUAAAGUGGAGAAUUAGGGCUGCUUGCAUCAGCCUAAUGUC GAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAA GAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 409), 5′-CUUCACUGAUAAAGUGGAGAACUUAGGGGCUGCUUGCAUCAGCCUAAUG UCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGA AAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 410), 5′-CUUCACUGAUAAAGUGGAGAACUUAGUGGGCUGCUUGCAUCAGCCUAAU GUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUG AAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 411), 5′-CUUCACUGAUAAAGUGGAGAACCUUAGGUGGGCUGCUUGCAUCAGCCU AAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAU UUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 412), 5′-CUUCACUGAUAAAGUGGAGAACCGUUAGGUGGGCUGCUUGCAUCAGCC UAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCA UUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 413), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUAGGGUGGGCUGCUUGCAUCAG CCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUU CAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 414), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUUAGAGGUGGGCUGCUUGCAUC AGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAA UUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 415), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUUUAGAAGGUGGGCUGCUUGCA UCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACA AAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 416), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCUUAGGAAGGUGGGCUGCUUG CAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAA CAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 417), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCAUUAGUGAAGGUGGGCUGCU UGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGA AACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 418), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACUUAGGUGAAGGUGGGCUG CUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUC GAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 419), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACUUAGAGUGAAGGUGGGCU GCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCU CGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 420), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCUUAGGAGUGAAGGUGGG CUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACC CUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 421), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAUUAGUGAGUGAAGGUGG GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAAC CCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 422), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAUUAGUUGAGUGAAGGU GGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGU AACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 423), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAUUAGCUUGAGUGAAG GUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAA GUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 424), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAUUAGACUUGAGUGA AGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGA AAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 425), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGUUAGAACUUGAGU GAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCG GAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 426), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUUAGGAACUUGA GUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUU CGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAA C-3′ (SEQ ID NO: 427), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUUUAGAGAACUU GAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUC UUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUG CAAC-3′ (SEQ ID NO: 428), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUUAGUAGAAC UUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUU UCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAA UGCAAC-3′ (SEQ ID NO: 429), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUUAGUUAGAA CUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCU UUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGA AUGCAAC-3′ (SEQ ID NO: 430), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUUUAGAUUAG AACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUG CUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAG GAAUGCAAC-3′ (SEQ ID NO: 431), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCUUAGGAUU AGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAG UGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGA AGGAAUGCAAC-3′ (SEQ ID NO: 432), and 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCUUAGGGA UUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGA AGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAU GAAGGAAUGCAAC-3′ (SEQ ID NO: 433).
Examples of Engineered Scaffold Sequence from which Second Region is Removed
In an embodiment, a sequence of the engineered scaffold region from which the second region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region from which the second region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region from which the second region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region from which the second region is removed may be a sequence in which SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, a linker, SEQ ID NO: 15, and 5′-AUGCAAC-3′ are linked to each other in a 5′ to 3′ direction.
Here, the linker may be 5′-GAAA-3′.
In an embodiment, a sequence of the engineered scaffold region in which the second region is removed may be 5′-CUUCACUGAUAAAGUGGAGAAGCUGCUUGCAUCAGCCUAAUGUCGAGAA GUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUG AAGGAAUGCAAC-3′ (SEQ ID NO: 380).
The engineered scaffold region included in the engineered guide RNA provided herein may be an engineered form of a naturally occurring scaffold region in which the third region is modified.
In an embodiment, the engineered scaffold region may comprise a modified third region. Here, the modified third region is obtained by removing one or more nucleotides from the third region of the naturally occurring scaffold region. Here, the removed nucleotide(s) is a nucleotide(s) selected from a region forming a stem structure in the CRISPR/Cas12f1 complex.
In an embodiment, the engineered scaffold region included in the engineered Cas12f1 guide RNA may comprise an engineered form of a naturally occurring scaffold region in which one or more nucleotides included in the third region are removed. In an embodiment, removal of the nucleotides may occur in a portion forming a stem structure in the naturally occurring third region, in which the nucleotides may be removed in complementary pair. In an embodiment, the removed nucleotide(s) may be a nucleotide(s) included in a portion, which forms a stem structure in a CRISPR/Cas12f1 complex, in the naturally occurring third region.
In an embodiment, the removed nucleotide(s) may be a nucleotide(s), which belongs to Stem 4 (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)), in the naturally occurring third region.
In an embodiment, the modified third region may be characterized by having
The third region of the engineered scaffold region may be a modified form of a third region of the naturally occurring scaffold region from which one or more nucleotides are removed.
In an embodiment, the modified third region of the engineered scaffold region may be a modified form of a third region of the naturally occurring scaffold region from which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive or nonconsecutive nucleotides are removed. In an embodiment, the modified third region may be a modified form of a third region of the naturally occurring scaffold region from which one or more nucleotides, of the 27th to 36th nucleotides and/or the 41st to 50th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 12, are removed. In an embodiment, the modified third region may be a modified form of a third region of the naturally occurring scaffold region from which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides of the 27th to 36th nucleotides from the 5 end based on the sequence of SEQ ID NO: 12, are removed. In an embodiment, the modified third region may be a third region of the naturally occurring scaffold region from which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides, of the 41st to 50th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 12, are removed.
The modification of the third region may be removal of one or more pairs of nucleotides that are included in a portion forming a stem structure and complementarily bind to each other.
In an embodiment, the modified third region may be a modified form of a third region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs in the CRISPR/Cas12f1 complex, of the 27th to 36th nucleotides and the 41st to 50th nucleotides from the 5′ end based on the sequence of SEQ ID NO: ID NO: 12, are removed.
In an embodiment, the modified third region may be a modified form of a third region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs and/or one or more nucleotides not forming a complementary pair in the CRISPR/Cas12f1 complex, of the 27th to 36th nucleotides and/or the 41st to 50th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 12, are removed.
In an embodiment, the modified third region may be a modified form of a third region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs and/or one or more mismatched pairs of nucleotides in the CRISPR/Cas12f1 complex, of the 27th to 36th nucleotides and the 41st to 50th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 12, are removed.
Examples of Engineered Scaffold Sequence in which Third Region is Modified
In an embodiment, a sequence of the modified third region may be a sequence selected from the group consisting of 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAAC CCUCGA-3′ (SEQ ID NO: 434), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUUCGAAAGUAACCC UCGA-3′ (SEQ ID NO: 435), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUUCGAAGUAACCCUC GA-3′ (SEQ ID NO: 436), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUCUUCGGAGUAACCCU CGA-3′ (SEQ ID NO: 437), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCGAGUAACCCUCG A-3′ (SEQ ID NO: 438), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUCGGUAACCCUCGA-3′ (SEQ ID NO: 439), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGUUCGUAACCCUCGA-3′ (SEQ ID NO: 440), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUUUCGAACCCUCGA-3′ (SEQ ID NO: 441), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUUCGACCCUCGA-3′ (SEQ ID NO: 442), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAGUUCGCCCUCGA-3′ (SEQ ID NO: 443), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAUUCGCCCUCGA-3′ (SEQ ID NO: 444), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAAUUCGCCUCGA-3′ (SEQ ID NO: 445), 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGAUUCGCCUCGA-3′ (SEQ ID NO: 446), and 5′-GCUGCUUGCAUCAGCCUAAUGUCGAGUUCGCUCGA-3′ (SEQ ID NO: 447).
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified may be one in which a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 434 to 447, a linker, SEQ ID NO: 15, and 5′-AUGCAAC-3′ are linked to each other in a 5′ to 3′ direction.
Here, the linker may be 5′-GAAA-3′.
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified may be a sequence selected from the group consisting of 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUUUCGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAU GAAGGAAUGCAAC-3′ (SEQ ID NO: 462), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUUCGAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGA AGGAAUGCAAC-3′ (SEQ ID NO: 463), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUCUUCGGAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGA AGGAAUGCAAC-3′ (SEQ ID NO: 464), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCGAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAG GAAUGCAAC-3′ (SEQ ID NO: 465), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUCGGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGA AUGCAAC-3′ (SEQ ID NO: 466), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGUUCGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAU GCAAC-3′ (SEQ ID NO: 467), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUUUCGAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGC AAC-3′ (SEQ ID NO: 468), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUUCGACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAA C-3′ (SEQ ID NO: 469), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUUCGCCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 470), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAUUCGCCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 471), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAUUCGCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 472), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAUUCGCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 473), and 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GUUCGCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 474).
The engineered scaffold region provided herein may be an engineered form of a naturally occurring scaffold region in which the fourth and fifth regions are modified. The fourth region and the fifth region correspond to a part of the tracrRNA and a part of the crRNA, respectively. These regions comprise parts that hybridize to each other to form a stem in the CRISPR/Cas12f1 complex, and thus the corresponding parts may be modified together to constitute an engineered scaffold region.
In an embodiment, the engineered scaffold region may comprise a modified fourth region and/or a modified fifth region.
The modified fourth region is characterized in that it is obtained by removing one or more nucleotides from the fourth region of the naturally occurring scaffold region. The modified fifth region is characterized in that it is obtained by removing one or more nucleotides from the fifth region of the naturally occurring scaffold region.
In an embodiment, an engineered scaffold region included in the engineered Cas12f1 guide RNA may be an engineered form of a naturally occurring scaffold region in which one or more nucleotides are removed from the fourth region and/or the fifth region.
In an embodiment, the modified fourth region has 5′-AACAAA-3′ at the 5 end. In an embodiment, the modified fifth region has 5′-GGA-3′ at the 3′ end.
The fourth region of the engineered scaffold region may be a modified form of a fourth region of the naturally occurring scaffold region from which one or more nucleotides are removed. The fifth region of the engineered scaffold region may be a modified form of a fifth region of the naturally occurring scaffold region from which one or more nucleotides are removed.
In an embodiment, the modified fourth region of the engineered scaffold region may be a modified form of a fourth region of the naturally occurring scaffold region from which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive or nonconsecutive nucleotides are removed. In an embodiment, the modified fourth region may be a modified form of a fourth region of the naturally occurring scaffold region from which one or more nucleotides, of the 7th to 13th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 13, are removed. In an embodiment, the modified fourth region may be a modified form of a fourth region of the naturally occurring scaffold region from which one or more nucleotides, of the 7th to 34th nucleotides from the 5 end based on the sequence of SEQ ID NO: 14, are removed.
In an embodiment, a sequence of the modified fourth region comprises at least 5′-AACAAA-3′.
In an embodiment, the modified fifth region of the engineered scaffold region may be a modified form of a fifth region of the naturally occurring scaffold region from which 1 to 7 nucleotides are removed. In an embodiment, the modified fifth region of the engineered scaffold region may be a modified form of a fifth region of the naturally occurring scaffold region from which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 consecutive or nonconsecutive nucleotides are removed. In an embodiment, the modified fifth region may be a modified form of a fifth region of the naturally occurring scaffold region from which one or more nucleotides, of the 1st to 7th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 15, are removed. In an embodiment, the modified fifth region may be a modified form of a fifth region of the naturally occurring scaffold region from which one or more nucleotides, of the 1st to 27th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 16, are removed.
In an embodiment, the modified fifth region comprises 5′-GGA-3′.
The fourth region and the fifth region are known to form a stem by complementarily binding to each other in the CRISPR/Cas12 complex. Since the above-described modifications of the fourth region and the fifth region are subject to one or more nucleotides constituting the stem, the modifications of the fourth region and the fifth region may be made to remove the nucleotides constituting the stem in complementary pair.
In an embodiment, the modified fourth and fifth regions may be engineered forms of a fourth region and a fifth region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs in the CRISPR/Cas12f1 complex, of the 7th to 13th nucleotides from the 5′ end based on SEQ ID NO: 13 and the 1st to 7th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 15, are removed.
In an embodiment, the modified fourth and fifth regions may be engineered forms of a fourth region and a fifth region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs and/or one or more nucleotides not forming a complementary pair in the CRISPR/Cas12f1 complex, of the 7th to 13th nucleotides from the 5′ end based on SEQ ID NO: 13 and the 1st to 7th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 15, are removed.
In an embodiment, the modified fourth and fifth regions may be engineered forms of a fourth region and a fifth region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs and/or one or more mismatched pairs of nucleotides in the CRISPR/Cas12f1 complex, of the 7th to 13th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 13 and the 1st to 7th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 15, are removed.
In an embodiment, the modified fourth and fifth regions may be engineered forms of a fourth region and a fifth region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs and/or one or more nucleotides not forming a complementary pair in the CRISPR/Cas12f1 complex, of the 7th to 34th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 14 and the 1st to 27th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 16, are removed.
In an embodiment, the modified fourth and fifth regions may be engineered forms of a fourth region and a fifth region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs and/or one or more nucleotides not forming a complementary pair in the CRISPR/Cas12f1 complex, of the 7th to 34th nucleotides from the 5′ end based on SEQ ID NO: 14 and the 1st to 27th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 16, are removed.
In an embodiment, the modified fourth and fifth regions may be engineered forms of a fourth region and a fifth region of the naturally occurring scaffold region from which one or more pairs of nucleotides forming complementary pairs and/or one or more mismatched pairs of nucleotides in the CRISPR/Cas12f1 complex, of the 7th to 37th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 14 and the 1st to 27th nucleotides from the 5′ end based on the sequence of SEQ ID NO: 16, are removed.
In an embodiment, a sequence of the modified fourth region may be selected from the group consisting of 5′-AACAAA-3′, 5′-AACAAAU-3′, 5′-AACAAAUU-3′, 5′-AACAAAUUC-3′, 5′-AACAAAUUCA-3′ (SEQ ID NO: 67), 5′-AACAAAUUCAU-3′ (SEQ ID NO: 68), and 5′-AACAAAUUCAUU-3′ (SEQ ID NO: 69).
In an embodiment, a sequence of the modified fourth region may be selected from the group consisting of 5′-AACAAA-3′, 5′-AACAAAU-3′, 5′-AACAAAUU-3′, 5′-AACAAAUUC-3′, 5′-AACAAAUUCA-3′ (SEQ ID NO: 67), 5′-AACAAAUUCAU-3′ (SEQ ID NO: 68), 5′-AACAAAUUCAUU-3′ (SEQ ID NO: 69), 5′-AACAAAUUCAUUU-3′ (SEQ ID NO: 70), 5′-AACAAAUUCAUUUU-3′ (SEQ ID NO: 71), 5′-AACAAAUUCAUUUUU-3′ (SEQ ID NO: 72), 5′-AACAAAUUCAUUUUUC-3′ (SEQ ID NO: 73), 5′-AACAAAUUCAUUUUUCC-3′ (SEQ ID NO: 74), 5′-AACAAAUUCAUUUUUCCU-3′ (SEQ ID NO: 75), 5′-AACAAAUUCAUUUUUCCUC-3′ (SEQ ID NO: 76), 5′-AACAAAUUCAUUUUUCCUCU-3′ (SEQ ID NO: 77), 5′-AACAAAUUCAUUUUUCCUCUC-3′ (SEQ ID NO: 78), 5′-AACAAAUUCAUUUUUCCUCUCC-3′ (SEQ ID NO: 79), 5′-AACAAAUUCAUUUUUCCUCUCCA-3′ (SEQ ID NO: 80), 5′-AACAAAUUCAUUUUUCCUCUCCAA-3′ (SEQ ID NO: 81), 5′-AACAAAUUCAUUUUUCCUCUCCAAU-3′ (SEQ ID NO: 82), 5′-AACAAAUUCAUUUUUCCUCUCCAAUU-3′ (SEQ ID NO: 83), 5′-AACAAAUUCAUUUUUCCUCUCCAAUUC-3′ (SEQ ID NO: 84), 5′-AACAAAUUCAUUUUUCCUCUCCAAUUCU-3′ (SEQ ID NO: 85), 5′-AACAAAUUCAUUUUUCCUCUCCAAUUCUG-3′ (SEQ ID NO: 86), 5′-AACAAAUUCAUUUUUCCUCUCCAAUUCUGC-3′ (SEQ ID NO: 87), 5′-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCA-3′ (SEQ ID NO: 88), 5′-AAACAAAUUCAUUUUUCCUCUCCAAUUCUGCAC-3′ (SEQ ID NO: 89), and 5′-AACAAAUUCAUUUUUCCUCUCCAAUUCUGCACA-3′ (SEQ ID NO: 90).
In an embodiment, a sequence of the modified fifth region may be selected from the group consisting of 5′-GGA-3′, 5′-AGGA-3′, 5′-AAGGA-3′, 5′-GAAGGA-3′, 5′-UGAAGGA-3′, 5′-AUGAAGGA-3′, and 5′-AAUGAAGGA-3′.
In an embodiment, a sequence of the modified fifth region may be selected from the group consisting of 5′-GGA-3′, 5′-AGGA-3′, 5′-AAGGA-3′, 5′-GAAGGA-3′, 5′-UGAAGGA-3′, 5′-AUGAAGGA-3′, 5′-AAUGAAGGA-3′, 5′-GAAUGAAGGA-3′ (SEQ ID NO: 91), 5′-CGAAUGAAGGA-3′ (SEQ ID NO: 92), 5′-ACGAAUGAAGGA-3′ (SEQ ID NO: 93), 5′-GACGAAUGAAGGA-3′ (SEQ ID NO: 94), 5′-AGACGAAUGAAGGA-3′ (SEQ ID NO: 95), 5′-UAGACGAAUGAAGGA-3′ (SEQ ID NO: 96), 5′-AUAGACGAAUGAAGGA-3′ (SEQ ID NO: 97), 5′-AAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 98), 5′-GAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 99), 5′-CGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 100), 5′-CCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 101), 5′-CCCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 102), 5′-ACCCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 103), 5′-AACCCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 104), 5′-GAACCCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 105), 5′-AGAACCCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 106), 5′-CAGAACCCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 107), 5′-GCAGAACCCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 108), 5′-UGCAGAACCCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 109), and 5′-UUGCAGAACCCGAAUAGACGAAUGAAGGA-3′ (SEQ ID NO: 110).
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified may be selected from the group consisting of 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAGAAAGGAAUGCAA C-3′ (SEQ ID NO: 200), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUGAAAAGGAAUGC AAC-3′ (SEQ ID NO: 201), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUGAAAAAGGAAU GCAAC-3′ (SEQ ID NO: 202), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCGAAAGAAGGA AUGCAAC-3′ (SEQ ID NO: 203), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAGAAAUGAAG GAAUGCAAC-3′ (SEQ ID NO: 204), 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUGAAAAUGA AGGAAUGCAAC-3′ (SEQ ID NO: 205), and 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUGAAAAAU GAAGGAAUGCAAC-3′ (SEQ ID NO: 206).
In the engineered scaffold provided herein, the sixth region is a region including nucleotides which belong to the crRNA in Stem 3-PK (R:AR-1). As described above, the sixth region comprises one or more nucleotides that interact with a WED domain, a ZF domain, and/or an RuvC domain of one dimer-forming Cas12f1 protein in the CRISPR/Cas12f1 complex. The sixth region of the engineered scaffold may be the same as the sixth region of the naturally occurring scaffold region, or may be a modified form of a sixth region of the naturally occurring scaffold region which is modified to the extent that a function of the sixth region is not impaired.
The engineered scaffold region included in the engineered Cas12f1 guide RNA provided herein may be a modified form of a naturally occurring scaffold region in which one or more of the above-mentioned modifications for respective regions are combined.
In an embodiment, the engineered scaffold region may comprise a modified first region and a modified second region.
In an embodiment, the engineered scaffold region may be one in which a modified second region is included and the first region is removed.
In an embodiment, the engineered scaffold region may be one in which a modified first region is included and the second region is removed.
In an embodiment, the engineered scaffold region may be one in which the first region and the second region are removed.
In an embodiment, the engineered scaffold region may comprise a modified first region and a modified third region.
In an embodiment, the engineered scaffold region may be one in which a modified third region is included and the first region is removed.
In an embodiment, the engineered scaffold region may comprise a modified first region and modified fourth and fifth regions.
In an embodiment, the engineered scaffold region may be one in which modified fourth and fifth regions are included and the first region is removed.
In an embodiment, the engineered scaffold region may comprise a modified second region and a modified third region.
In an embodiment, the engineered scaffold region may be one in which a modified third region is included and the second region is removed.
In an embodiment, the engineered scaffold region may comprise a modified second region and modified fourth and fifth regions.
In an embodiment, the engineered scaffold region may be one in which modified fourth and fifth regions are included and the second region is removed.
In an embodiment, the engineered scaffold region may comprise a modified third region and modified fourth and fifth regions.
In an embodiment, the engineered scaffold region may comprise a modified first region, a modified second region, and a modified third region.
In an embodiment, the engineered scaffold region may be one in which a modified second region and a modified third region are included and the first region is removed.
In an embodiment, the engineered scaffold region may be one in which a modified first region and a modified third region are included and the second region is removed.
In an embodiment, the engineered scaffold region may be one in which a modified third region is included and the first region and the second region are removed.
In an embodiment, the engineered scaffold region may comprise a modified first region, a modified second region, and modified fourth and fifth regions.
In an embodiment, the engineered scaffold region may be one in which a modified second region and modified fourth and fifth regions are included and the first region is removed.
In an embodiment, the engineered scaffold region may be one in which a modified first region and modified fourth and fifth regions are included and the second region is removed.
In an embodiment, the engineered scaffold region may be one in which modified fourth and fifth regions are included, and the first region and the second region are removed.
In an embodiment, the engineered scaffold region may comprise a modified first region, a modified third region, and modified fourth and fifth regions.
In an embodiment, the engineered scaffold region may be one in which a modified third region and modified fourth and fifth regions are included and the first region is removed.
In an embodiment, the engineered scaffold region may comprise a modified second region, a modified third region, and modified fourth and fifth regions.
In an embodiment, the engineered scaffold region may be one in which a modified third region and modified fourth and fifth regions are included and the second region is removed.
In an embodiment, the engineered scaffold region may comprise a modified first region, a modified second region, a modified third region, and modified fourth and fifth regions.
In an embodiment, the engineered scaffold region may be one in which a modified second region, a modified third region, and modified fourth and fifth regions are included, and the first region is removed.
In an embodiment, the engineered scaffold region may be one in which a modified first region, a modified third region, and modified fourth and fifth regions are included and the second region is removed.
In an embodiment, the engineered scaffold region may be one in which a modified third region and modified fourth and fifth regions are included and the first region and the second region are removed.
Here, the modified regions are as described above in the section for modification of each of the regions.
In an embodiment, the engineered scaffold region may comprise a modified first region and a modified second region. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.”
In an embodiment, a sequence of the engineered scaffold region in which the first region and the second region are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region and the second region are modified may comprise in a 5′ to 3′ direction:
In an embodiment, a sequence of the engineered scaffold region in which the first region and the second region are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5′-GAAA-3′.
In an embodiment, a sequence of the engineered scaffold region in which the first region and the second region are modified may be 5′-ACCGCUUCACCAUUAGUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAA UGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU GAAAGAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 207).
In an embodiment, the engineered scaffold region may be one in which a modified second region is included and the first region is removed. Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region. Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.”
In an embodiment, a sequence of the engineered scaffold region in which the second region is modified and the first region is removed may comprise: a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, a sequence of the engineered scaffold region in which the second region is modified and the first region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5′-GAAA-3′.
In an embodiment, the engineered scaffold region may be one in which a modified first region is included and the second region is removed. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the first region is modified and the second region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region is modified and the second region is removed may be one in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5′-GAAA-3′.
In an embodiment, the engineered scaffold region may be one in which the first region and the second region are removed. Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region. Here, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the first region and the second region are removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region and the second region are removed may be one in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5-GAAA-3′.
In an embodiment, the engineered scaffold region may comprise a modified first region and a modified third region. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.”
In an embodiment, a sequence of the engineered scaffold region in which the first region and the third region are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region and the third region are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5′-GAAA-3′.
In an embodiment, the engineered scaffold region may be one in which a modified third region is included and the first region is removed. Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified and the first region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified and the first region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5-GAAA-3′.
In an embodiment, the engineered scaffold region may comprise a modified first region and modified fourth and fifth regions. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.”
In an embodiment, a sequence of the engineered scaffold region in which the first region and the fourth and fifth regions are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region and the fourth and fifth regions are modified may comprise in a 5′ to 3′ direction:
In an embodiment, a sequence of the engineered scaffold region in which the first region and the fourth and fifth regions are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, a sequence of the engineered scaffold region in which the first region and the fourth and fifth regions are modified may be 5′-ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGG UGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAG UAACCCUCGAAACAAAGAAAGGAAUGCAAC-3′ (SEQ ID NO: 208).
In an embodiment, the engineered scaffold region may be one in which modified fourth and fifth regions are included and the first region is removed. Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.” Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified and the first region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified and the first region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may comprise a modified second region and a modified third region. Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.” Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.”
In an embodiment, a sequence of the engineered scaffold region in which the second region and the third region are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the second region and the third region are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5-GAAA-3.
In an embodiment, the engineered scaffold region may be one in which a modified third region is included and the second region is removed. Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified and the second region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified and the second region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5-GAAA-3′.
In an embodiment, the engineered scaffold region may comprise a modified second region and modified fourth and fifth regions. Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.”
In an embodiment, a sequence of the engineered scaffold region in which the second region and the fourth and fifth regions are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the second region and the fourth and fifth regions are modified may comprise in a 5′ to 3′ direction:
In an embodiment, a sequence of the engineered scaffold region in which the second region and the fourth and fifth regions are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, a sequence of the engineered scaffold region in which the second region and the fourth and fifth regions are modified may be 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACUUAGAGUGAAGGUGGGGC UGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCC UCGAAACAAAGAAAGGAAUGCAAC-3′ (SEQ ID NO: 209).
In an embodiment, the engineered scaffold region may be one in which modified fourth and fifth regions are included and the second region is removed. Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.” Here, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified and the second region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified and the second region is removed may be one in which the following sequences are linked to each other in a 5′ to 3′ direction,
In an embodiment, the engineered scaffold region may comprise a modified third region and modified fourth and fifth regions. Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.”
In an embodiment, a sequence of the engineered scaffold region in which the third region and the fourth and fifth regions are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the third region and the fourth and fifth regions are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may comprise a modified first region, a modified second region, and a modified third region. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.” Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.”
In an embodiment, a sequence of the engineered scaffold region in which the first region, the second region, and the third region are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region, the second region, and the third region are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5-GAAA-3.
In an embodiment, the engineered scaffold region may be one in which a modified second region and a modified third region are included and the first region is removed. Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.” Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the second region and the third region are modified and the first region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the second region and the third region are modified and the first region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5′-GAAA-3′.
In an embodiment, the engineered scaffold region may be one in which a modified first region and a modified third region are included and the second region is removed. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the first region and the third region are modified and the second region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region and the third region are modified and the second region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
Here, the linker may be 5-GAAA-3′.
In an embodiment, the engineered scaffold region may be one in which a modified third region is included and the first region and the second region are removed. Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region. Here, the engineered scaffold region may not comprise a region corresponding to the second region of the scaffold region occurring in nature.
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified and the first region and the second region are removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the third region is modified and the first region and the second region are removed may comprise:
Here, the linker may be 5′-GAAA-3′.
In an embodiment, the engineered scaffold region may comprise a modified first region, a modified second region, and modified fourth and fifth regions. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.”
In an embodiment, a sequence of the engineered scaffold region in which the first region, the second region, and the fourth and fifth regions are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region, the second region, and the fourth and fifth regions are modified may comprise in a 5′ to 3′ direction:
In an embodiment, a sequence of the engineered scaffold region in which the first region, the second region, and fourth and fifth regions are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, a sequence of the engineered scaffold region in which the first region, the second region, and the fourth and fifth regions are modified may be 5′-ACCGCUUCACUUAGAGUGAAGGUGGGGCUGCUUGCAUCAGCCUAAUGU CGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAGAAAGGAAUG CAAC-3′ (SEQ ID NO: 210).
In an embodiment, the engineered scaffold region may be one in which a modified second region and modified fourth and fifth regions are included and the first region is removed. Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.” Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the second region and the fourth and fifth regions are modified and the first region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the second region and the fourth and fifth regions are modified and the first region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may be one in which a modified first region and modified fourth and fifth regions are included, and the second region is removed. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.” Here, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the first region and the fourth and fifth regions are modified and the second region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region and the fourth and fifth regions are modified and the second region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may be one in which modified fourth and fifth regions are included and the first region and the second region are removed. Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.” Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region. Here, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified and the first region and the second region are removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the fourth and fifth regions are modified and the first region and the second region are removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may comprise a modified first region, a modified third region, and modified fourth and fifth regions. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.”
In an embodiment, the engineered scaffold sequence in which the first region, the third region, and the fourth and fifth regions are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region, the third region, and the fourth and fifth regions are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may be one in which a modified third region, and modified fourth and fifth regions are included, and the first region is removed. Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.” Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the third region, and the fourth and fifth regions are modified and the first region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the third region, and the fourth and fifth regions are modified and the first region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may comprise a modified second region, a modified third region, and modified fourth and fifth regions. Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.” Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.”
In an embodiment, the engineered scaffold sequence in which the second region, the third region, and the fourth and fifth regions are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the second region, the third region, and the fourth and fifth regions are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may be one in which a modified third region, and modified fourth and fifth regions are included and the second region is removed. Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.” Here, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the third region, and the fourth and fifth regions are modified and the second region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the third region, and the fourth and fifth regions are modified and the second region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may comprise a modified first region, a modified second region, a modified third region, and modified fourth and fifth regions. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.” Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.”
In an embodiment, the engineered scaffold sequence in which the first region, the second region, the third region, and the fourth and fifth regions are modified may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region, the second region, the third region, and the fourth and fifth regions are modified may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may be one in which a modified second region, a modified third region, and modified fourth and fifth regions are included and the first region is removed. Here, the modified second region includes any one of the modifications described in the section “Engineered scaffold region 2—Modification of second region.” Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.” Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the second region, the third region, and the fourth and fifth regions are modified and the first region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the second region, the third region, and the fourth and fifth regions are modified and the first region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may be one in which a modified first region, a modified third region, and modified fourth and fifth regions are included and the second region is removed. Here, the modified first region includes any one of the modifications described in the section “Engineered scaffold region 1—Modification of first region.” Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.” Here, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the first region, the third region, and the fourth and fifth regions are modified and the second region is removed may comprise:
In an embodiment, a sequence of the engineered scaffold region in which the first region, the third region, and the fourth and fifth regions are modified and the second region is removed may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered scaffold region may be one in which a modified third region and modified fourth and fifth regions are included and the first region and the second region are removed. Here, the modified third region includes any one of the modifications described in the section “Engineered scaffold region 3—Modification of third region.” Here, the modified fourth and fifth regions include any one of the modifications described in the section “Engineered scaffold region 4—Modification of fourth and fifth regions.” Here, the engineered scaffold region may not comprise a region corresponding to the first region of the naturally occurring scaffold region. Here, the engineered scaffold region may not comprise a region corresponding to the second region of the naturally occurring scaffold region.
In an embodiment, a sequence of the engineered scaffold region in which the third region, and the fourth and fifth regions are modified and the first region and the second region are removed may comprise:
In an embodiment, the engineered scaffold sequence, in which the third region, and the fourth and fifth regions are modified, and the first region and the second region are removed, may be a sequence in which the following sequences are linked to each other in a 5′ to 3′ direction:
As described above, since the sixth region may also be modified within a range in which its function is not impaired, the engineered scaffold region provided herein may be one in which the sixth region is additionally modified in addition to the modification(s) of the first region, the second region, the third region, the fourth region, and/or the fifth region, including the removal of the first region and/or the second region, as described above.
The engineered scaffold region provided herein comprises a sequence having identity with the sequences of the engineered scaffold region (hereinafter, referred to as the above-described engineered scaffold region) described in the sections of “Engineered scaffold region 1—Modification of first region”, “Engineered scaffold region 2—Modification of second region”, “Engineered scaffold region 3—Modification of third region”, “Engineered scaffold region 4—Modification of fourth and fifth regions”, and “Engineered scaffold region 6—Combination of respective modifications.”
In an embodiment, a sequence of the engineered scaffold region may be a sequence having sequence identity or sequence homology of 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, or 50% to any one of the sequences of the above-described engineered scaffold region. In an embodiment, the scaffold sequence may be a sequence that is identical to any one of the sequences of the above-described engineered scaffold region by a percentage falling within a range of two numbers selected from the immediately preceding sentence. For example, the scaffold sequence may be a sequence that is 90% to 100% identical to any one of the sequences of the above-described engineered scaffold region.
In the present disclosure, there is provided an engineered Cas12f1 guide RNA for increasing gene editing efficiency of the CRISPR/Cas12f1 system in a cell. The engineered Cas12f1 guide RNA comprises an engineered scaffold, a spacer, and a U-rich tail. Here, the engineered scaffold may be any one of those described in the above-described “engineered scaffold region.” Here, the U-rich tail may be any one of those described in the section “U-rich tail.”
The engineered Cas12f1 guide RNA may be a single guide RNA or a dual guide RNA. The dual guide RNA refers to a guide RNA which consists of two independent RNA molecules of a tracrRNA and a crRNA. The single guide RNA refers to a molecule formed by linking the 3′ end of a (engineered) tracrRNA and the 5′ end of a (engineered) crRNA through a linker. In other words, the single guide RNA means a molecule obtained by linking the 3′ end of a fourth region and the 5 end of a fifth region through a linker, wherein the fourth and fifth regions are included in the engineered scaffold of the dual guide RNA. Here, the respective regions of the engineered scaffold may include any one of the modifications, and specific sequences thereof, as described in the sections of “Engineered scaffold region.”
In an embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region, a spacer, and a U-rich tail are sequentially linked to each other in a 5′ to 3′ direction.
The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to a target sequence.
A sequence of the U-rich tail may comprise a uridine repeat sequence or a modified uridine repeat sequence. As an example, the U-rich tail sequence may comprise a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive uridines are contained. As another example, the U-rich tail sequence may comprise a sequence in which one or more UV, UUV, UUUV, UUUUV, and/or UUUUUV are repeated. Here, V is selected from adenosine (A), cytidine (C), and guanosine (G).
The engineered scaffold region is one in which a first region, a second region, a third region, a fourth region, a linker, a fifth region, and a sixth region, which correspond to those of the naturally occurring scaffold region, are sequentially linked to each other in a 5′ to 3′ direction, and one or more regions selected from the first region, the second region, the fourth region, and the fifth region are modified as compared with the naturally occurring scaffold region.
As an example, when a first region of the engineered scaffold region is modified, the modified first region may be a modified form of a first region of the naturally occurring scaffold region from which one or more nucleotides are removed. Here, the removed nucleotide(s) may be a nucleotide(s) belonging to Stem 1 (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) in the first region. Here, a sequence of the modified first region is characterized by comprising 5′-A-3′.
As another example, when a second region of the engineered scaffold region is modified, the modified second region may be a modified form of a second region of the naturally occurring scaffold region from which one or more nucleotides are removed. Here, the removal of the nucleotides may occur in a portion that forms a Stem 2 structure (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) in the second region, and such removal may be done in pairs of nucleotides that are complementary to each other. Here, a sequence of the modified second region is characterized by comprising at least 5′-CCGCUUCACCA-3′ (SEQ ID NO: 51) and 5′-UGAGUGAAGGUG-3′ (SEQ ID NO: 52). More specifically, a sequence of the modified second region may be one in which 5′-CCGCUUCACCA-3′ (SEQ ID NO: 51) and 5′-UGAGUGAAGGUG-3′ (SEQ ID NO: 52) are sequentially linked to each other in a 5′ to 3′ direction, wherein the sequences may be linked by an appropriate intermediate sequence. As an example, the intermediate sequence may be selected from the group consisting of 5′-UUAG-3′, 5′-AUUAGU-3′, 5′-AAUUAGCU-3′, 5′-AAAUUAGACU-3′ (SEQ ID NO: 58), 5′-AAAGUUAGAACU-3′ (SEQ ID NO: 59), 5′-AAAGCUUAGGAACU-3′ (SEQ ID NO: 60), 5′-AAAGCUUUAGAGAACU-3′ (SEQ ID NO: 61), 5′-AAAGCUGUUAGUUAGAACU-3′ (SEQ ID NO: 62), 5′-AAAGCUGUUAGUAGAACU-3′ (SEQ ID NO: 63), 5′-AAAGCUGUUUAGAUUAGAACU-3′ (SEQ ID NO: 64), 5′-AAAGCUGUCUUAGGAUUAGAACU-3′ (SEQ ID NO: 65), and 5′-AAAGCUGUCCUUAGGGAUUAGAACU-3′ (SEQ ID NO: 66).
As another example, when the fourth and fifth regions of the engineered scaffold region are modified, the modified fourth and fifth regions may be a modified form of a fourth region and/or a fifth region of the naturally occurring scaffold region from which one or more nucleotides are removed. Here, removal of the nucleotide may occur in a portion that forms a Stem 5 (R:AR-2) structure (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) in the fourth and fifth regions, and such removal may be done in pairs of nucleotides that are complementary to each other. Here, a sequence of the modified fourth region is characterized by comprising at least 5′-AACAAA-3′. Here, the modified fifth region is characterized by comprising at least 5′-GGA-3′.
In an embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region, a spacer, and a U-rich tail are sequentially linked to each other in a 5′ to 3′ direction.
The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to a target sequence.
A sequence of the U-rich tail may be represented by (UaN)bUc. Here, N is selected from adenosine (A), uridine (U), cytidine (C) and guanosine (G); and a, b, and c are each an integer, with a being between 1 and 5 inclusive, b being 0 or more, and c being between 1 to 10 inclusive. As an example, a sequence of the U-rich tail may be 5′-UUUUAUUUU-3′. As an example, a sequence of the U-rich tail may be 5′-UUUUGUUUU-3′.
A sequence of the engineered scaffold region is such that the following sequences are sequentially linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region, a spacer, and a U-rich tail are sequentially linked to each other in a 5′ to 3′ direction.
The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to a target sequence.
A sequence of the U-rich tail may be represented by (UaN)bUc. Here, N is selected from adenosine (A), uridine (U), cytidine (C), and guanosine (G). Here, a, b, and c are each an integer, with a being between 1 and 5 inclusive, b being 0 or more, and c being between 1 and 10 inclusive. As an example, a sequence of the U-rich tail may be 5′-UUUUAUUUU-3′. As an example, a sequence of the U-rich tail may be 5′-UUUUGUUUU-3′.
A sequence of the engineered scaffold region is such that the following sequences are sequentially linked to each other in a 5′ to 3′ direction:
As an example, the linker may be 5′-GAAA-3′.
As another example, the linker may be selected from the group consisting of 5′-GAAA-3′, 5′-UGAAAA-3′, 5′-UUGAAAAA-3′, 5′-UUCGAAAGAA-3′ (SEQ ID NO: 642), 5′-UUCAGAAAUGAA-3′ (SEQ ID NO: 643), 5′-UUCAUGAAAAUGAA-3′ (SEQ ID NO: 644), and 5′-UUCAUUGAAAAAUGAA-3′ (SEQ ID NO: 645).
As a specific example of the embodiment, a sequence of the engineered scaffold region may further comprise a ninth sequence selected from the group consisting of 5′-A-3′, 5′-GA-3′, 5′-AGA-3′, 5′-GAGA-3′, 5′-GGAGA-3′, 5′-UGGAGA-3′, 5′-GUGGAGA-3′, 5′-AGUGGAGA-3′, 5′-AAGUGGAGA-3′, 5′-AAAGUGGAGA-3′ (SEQ ID NO: 28), 5′-UAAAGUGGAGA-3′ (SEQ ID NO: 29), 5′-AUAAAGUGGAGA-3′ (SEQ ID NO: 30), 5′-GAUAAAGUGGAGA-3′ (SEQ ID NO: 31), 5′-UGAUAAAGUGGAGA-3′ (SEQ ID NO: 32), 5′-CUGAUAAAGUGGAGA-3′ (SEQ ID NO: 33), 5′-ACUGAUAAAGUGGAGA-3′ (SEQ ID NO: 34), 5′-CACUGAUAAAGUGGAGA-3′ (SEQ ID NO: 35), 5′-UCACUGAUAAAGUGGAGA-3′ (SEQ ID NO: 36), 5′-UUCACUGAUAAAGUGGAGA-3′ (SEQ ID NO: 37), and 5′-CUUCACUGAUAAAGUGGAGA-3′ (SEQ ID NO: 38). Here, the 3′ end of the ninth sequence may be linked to the 5′ end of the first sequence.
As another specific example of the embodiment, a sequence of the engineered scaffold region may further comprise a tenth sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-AAA-3′, 5′-AAAG-3′, 5′-AAAGC-3′, 5′-AAAGCU-3′, 5′-AAAGCUG-3′, 5′-AAAGCUGU-3′, 5′-AAAGCUGUC-3′, 5′-AAAGCUGUCC-3′ (SEQ ID NO: 53), and 5′-AAAGCUGUCCC-3′ (SEQ ID NO: 54). Here, the 3′ end of the second sequence and the 5 end of the third sequence may be linked by the tenth sequence.
As yet another specific example of the embodiment, a sequence of the engineered scaffold region may further comprise an eleventh sequence selected from the group consisting of 5′-U-3′, 5′-CU-3′, 5′-ACU-3′, 5′-AACU-3′, 5′-GAACU-3′, 5′-AGAACU-3′, 5′-UAGAACU-3′, 5′-UUAGAACU-3′, 5′-AUUAGAACU-3′, 5′-GAUUAGAACU-3′ (SEQ ID NO: 55), 5′-GGAUUAGAACU-3′ (SEQ ID NO: 56), and 5′-GGGAUUAGAACU-3′ (SEQ ID NO: 57). Here, the 3′ end of the third sequence and the 5′ end of the fourth sequence may be linked by the eleventh sequence.
As still yet another specific example of the embodiment, a sequence of the engineered scaffold region may further comprise a tenth sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-AAA-3′, 5′-AAAG-3′, 5′-AAAGC-3′, 5′-AAAGCU-3′, 5′-AAAGCUG-3′, 5′-AAAGCUGU-3′, 5′-AAAGCUGUC-3′, 5′-AAAGCUGUCC-3′ (SEQ ID NO: 53), and 5′-AAAGCUGUCCC-3′ (SEQ ID NO: 54), and an eleventh sequence selected from the group consisting of 5′-U-3′, 5′-CU-3′, 5′-ACU-3′, 5′-AACU-3′, 5′-GAACU-3′, 5′-AGAACU-3′, 5′-UAGAACU-3′, 5′-UUAGAACU-3′, 5′-AUUAGAACU-3′, 5′-GAUUAGAACU-3′ (SEQ ID NO: 55), 5′-GGAUUAGAACU-3′ (SEQ ID NO: 56), and 5′-GGGAUUAGAACU-3′ (SEQ ID NO: 57). Here, the 3′ end of the second sequence and the 5 end of the third sequence may be linked by the tenth sequence, and the 3′ end of the third sequence and the 5′ end of the fourth sequence may be linked by the eleventh sequence.
As an example, when the tenth sequence is 5′-A-3′, the eleventh sequence may be 5′-U-3′. As another example, when the tenth sequence is 5′-AA-3′, the eleventh sequence may be 5′-CU-3′. As yet another example, when the tenth sequence is 5′-AAA-3′, the eleventh sequence may be 5′-ACU-3′. As still yet another example, when the tenth sequence is 5′-AAAG-3′, the eleventh sequence may be 5′-AACU-3′. As still yet another example, when the tenth sequence is 5′-AAAGC-3′, the eleventh sequence may be 5′-GAACU-3′. As still yet another example, when the tenth sequence is 5′-AAAGCU-3′, the eleventh sequence may be 5′-AGAACU-3′. As still yet another example, when the tenth sequence is 5′-AAAGCUG-3′, the eleventh sequence may be 5′-UAGAACU-3′ or 5′-UUAGAACU-3′. As still yet another example, when the tenth sequence is 5′-AAAGCUGU-3′, the eleventh sequence may be 5′-AUUAGAACU-3′. As still yet another example, when the tenth sequence is 5′-AAAGCUGUC-3′, the eleventh sequence may be 5′-GAUUAGAACU-3′ (SEQ ID NO: 55). As still yet another example, when the tenth sequence is 5′-AAAGCUGUCC-3′ (SEQ ID NO: 53), the eleventh sequence may be 5′-GGAUUAGAACU-3′ (SEQ ID NO: 56). As still yet another example, when the tenth sequence is 5′-AAAGCUGUCCC-3′ (SEQ ID NO: 54), the eleventh sequence may be 5′-GGGAUUAGAACU-3′ (SEQ ID NO: 57).
As still yet another specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise a twelfth sequence selected from the group consisting of 5′-U-3′, 5′-UU-3′, 5′-UUC-3′, 5′-UUCA-3′, 5′-UUCAU-3′, 5′-UUCAUU-3′, and 5′-UUCAUUU-3′. Here, the 3′ end of the sixth sequence and the 5′ end of the linker may be linked by the twelfth sequence.
As still yet another specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise a thirteenth sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-GAA-3′, 5′-UGAA-3′, 5′-AUGAA-3′, 5′-AAUGAA-3′, and 5′-GAAUGAA-3′. Here, the 3′ end of the linker and the 5 end of the seventh sequence may be linked by the thirteenth sequence.
As still yet another specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise a twelfth sequence selected from the group consisting of 5′-U-3′, 5′-UU-3′, 5′-UUC-3′, 5′-UUCA-3′, 5′-UUCAU-3′, 5′-UUCAUU-3′, and 5′-UUCAUUU-3′, and a thirteenth sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-GAA-3′, 5′-UGAA-3′, 5′-AUGAA-3′, 5′-AAUGAA-3′, and 5′-GAAUGAA-3′. Here, the 3′ end of the sixth sequence and the 5 end of the linker may be linked by the twelfth sequence, and the 3′ end of the linker and the 5′ end of the seventh sequence may be linked by the thirteenth sequence.
As an example, when the twelfth sequence is 5′-U-3′, the thirteenth sequence may be 5′-A-3′. As another example, when the twelfth sequence is 5′-UU-3′, the thirteenth sequence may be 5′-AA-3′. As yet another example, when the twelfth sequence is 5′-UUC-3′, the thirteenth sequence may be 5′-GAA-3′. As still yet another example, when the twelfth sequence is 5′-UUCA-3′, the thirteenth sequence may be 5′-UGAA-3′. As still yet another example, when the twelfth sequence is 5′-UUCAU-3′, the thirteenth sequence may be 5′-AUGAA-3′. As still yet another example, when the twelfth sequence is 5′-UUCAUU-3′, the thirteenth sequence may be 5′-AAUGAA-3′. As still yet another example, when the twelfth sequence is 5′-UUCAUUU-3′, the thirteenth sequence may be 5′-GAAUGAA-3′.
In an embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region, a spacer, and a U-rich tail are linked to each other in a 5′ to 3′ direction.
The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to a target sequence.
A sequence of the U-rich tail may be a uridine repeat sequence, or a modified uridine repeat sequence. As an example, the U-rich tail sequence may comprise a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive uridines are contained. As another example, the U-rich tail sequence may comprise a sequence in which one or more UV, UUV, UUUV, UUUUV, and/or UUUUUV are repeated. Here, V is selected from adenosine (A), cytidine (C), and guanosine (G).
The engineered scaffold region is one in which a first region, a second region, a third region, a fourth region, a linker, a fifth region, and a sixth region, which correspond to those of the naturally occurring scaffold region, are sequentially linked to each other in a 5′ to 3′ direction, and one or more regions selected from the first region, the second region, the third region, the fourth region, and the fifth region are modified as compared with the naturally occurring scaffold region. Furthermore, the engineered scaffold region may be one from which a first region and/or a second region corresponding to those of the naturally occurring scaffold region are removed.
As an example, when a first region of the engineered scaffold region is modified, the modified first region may be a modified form of a first region of the naturally occurring scaffold region from which one or more nucleotides are removed. Here, the removed nucleotide may be a nucleotide belonging to Stem 1 (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) in the first region. Here, the modified first region is characterized by comprising 5′-A-3′.
As another example, when a second region of the engineered scaffold region is modified, the modified second region may be a modified form of a second region of the naturally occurring scaffold region from which one or more nucleotides are removed. Here, the removal of the nucleotide may occur in a portion that forms a Stem 2 structure (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) of the second region, and such removal may be done in pairs of nucleotides that are complementary to each other. Here, the modified second region is characterized by comprising at least 5′-G-3′.
As yet another example, when a third region of the engineered scaffold region is modified, the modified third region may be a modified form of a third region of the naturally occurring scaffold region from which one or more nucleotides are removed. Here, the removal of the nucleotide may occur in a portion that forms a Stem 4 structure (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) in the third region, and such removal may be done in pairs of nucleotides that are complementary to each other. Here, the sequence of the modified third region is characterized by comprising 5′-GCUGCUUGCAUCAGCCUAAUGUCGAG-3′ (SEQ ID NO: 475) and 5′-CUCGA-3′. More specifically, a sequence of the modified third region may be one in which 5′-GCUGCUUGCAUCAGCCUAAUGUCGAG-3′ (SEQ ID NO: 475) and 5′-CUCGA-3′ are sequentially linked to each other in a 5′ to 3′ direction, and the sequences may be linked by an appropriate intermediate sequence. In an example, the intermediate sequence may be selected from the group consisting of 5′-UUCG-3′, 5′-AUUCGC-3′, 5′-AAUUCGC-3′, 5′-AAUUCGCC-3′, 5′-AAGUUCGCC-3′, 5′-AAGUUCGACC-3′ (SEQ ID NO: 476), 5′-AAGUUUCGAACC-3′ (SEQ ID NO: 477), 5′-AAGUGUUCGUAACC-3′ (SEQ ID NO: 478), 5′-AAGUGCUUCGGUAACC-3′ (SEQ ID NO: 479), 5′-AAGUGCUUUCGAGUAACC-3′ (SEQ ID NO: 480), 5′-AAGUGCUCUUCGGAGUAACC-3′ (SEQ ID NO: 481), 5′-AAGUGCUUUUCGAAGUAACC-3′ (SEQ ID NO: 482), 5′-AAGUGCUUUUUCGAAAGUAACC-3′ (SEQ ID NO: 483), and 5′-AAGUGCUUUCUUCGGAAAGUAACC-3′ (SEQ ID NO: 484).
As still yet another example, when the fourth and fifth regions of the engineered scaffold region are modified, the modified fourth and fifth regions may be modified forms of a fourth region and/or a fifth region of the naturally occurring scaffold region from which one or more nucleotides are removed. Here, the removal of the nucleotide may occur in a portion that forms a Stem 5 (R:AR-2) structure (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) in the fourth and fifth regions, and such removal may be done in pairs of nucleotides that are complementary to each other. Here, a sequence of the modified fourth region is characterized by comprising at least 5′-AACAAA-3′. Here, a sequence of the modified fifth region is characterized by comprising at least 5′-GGA-3′.
In an embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region, a spacer, and a U-rich tail are sequentially linked to each other in a 5′ to 3′ direction.
The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to a target sequence.
A sequence of the U-rich tail may be represented by (UaN)bUc. Here, N is selected from adenosine (A), uridine (U), cytidine (C) and guanosine (G); and a, b, and c are each an integer, with a being between 1 and 5 inclusive, b being 0 or more, and c being between 0 and 10 inclusive. As an example, a sequence of the U-rich tail may be 5′-UUUUAUUUU-3′. As an example, a sequence of the U-rich tail may be 5′-UUUUGUUUU-3′.
A sequence of the engineered scaffold region is such that the following sequences are sequentially linked to each other in a 5′ to 3′ direction:
In an embodiment, the engineered Cas12f1 guide RNA may be one in which an engineered scaffold region, a spacer, and a U-rich tail are sequentially linked to each other in a 5′ to 3′ direction.
The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to a target sequence.
The sequence of the U-rich tail may be represented by (UaN)bUc. Here, N is selected from adenosine (A), uridine (U), cytidine (C), and guanosine (G). Here, a, b, and c are each an integer, with a being between 1 and 5 inclusive, b being 0 or more, and c being between 1 to 10 inclusive. As an example, a sequence of the U-rich tail may be 5′-UUUUAUUUU-3′. As an example, a sequence of the U-rich tail may be 5′-UUUUGUUUU-3′.
A sequence of the engineered scaffold region is such that the following sequences are sequentially linked to each other in a 5′ to 3′ direction:
As an example, the linker may be 5′-GAAA-3′.
As another example, the linker may be selected from the group consisting of 5′-GAAA-3′, 5′-UGAAAA-3′, 5′-UUGAAAAA-3′, 5′-UUCGAAAGAA-3′ (SEQ ID NO: 642), 5′-UUCAGAAAUGAA-3′ (SEQ ID NO: 643), 5′-UUCAUGAAAAUGAA-3′ (SEQ ID NO: 644), and 5′-UUCAUUGAAAAAUGAA-3′ (SEQ ID NO: 645).
As a specific example of the embodiment, a sequence of the engineered scaffold region may further comprise a seventh sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-GAA-3′, 5′-AGAA-3′, 5′-GAGAA-3′, 5′-GGAGAA-3′, 5′-UGGAGAA-3′, 5′-GUGGAGAA-3′, 5′-AGUGGAGAA-3′, 5′-AAGUGGAGAA-3′ (SEQ ID NO: 17), 5′-AAAGUGGAGAA-3′ (SEQ ID NO: 18), 5′-UAAAGUGGAGAA-3′ (SEQ ID NO: 19), 5′-AUAAAGUGGAGAA-3′ (SEQ ID NO: 20), 5′-GAUAAAGUGGAGAA-3′ (SEQ ID NO: 21), 5′-UGAUAAAGUGGAGAA-3′ (SEQ ID NO: 22), 5′-CUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 23), 5′-ACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 24), 5′-CACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 25), 5′-UCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 26), 5′-UUCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 27), and 5′-CUUCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 10). Here, the 3′ end of the seventh sequence may be linked to the 5′ end of the first sequence.
As a specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise an eighth sequence selected from the group consisting of 5′-G-3′, 5′-UUAGG-3′, 5′-CUUAGGG-3′, 5′-CUUAGUGG-3′, 5′-CCUUAGGUGG-3′ (SEQ ID NO: 342), 5′-CCGUUAGGUGG-3′ (SEQ ID NO: 343), 5′-CCGCUUAGGGUGG-3′ (SEQ ID NO: 344), 5′-CCGCUUUAGAGGUGG-3′ (SEQ ID NO: 345), 5′-CCGCUUUUAGAAGGUGG-3′ (SEQ ID NO: 346), 5′-CCGCUUCUUAGGAAGGUGG-3′ (SEQ ID NO: 347), 5′-CCGCUUCAUUAGUGAAGGUGG-3′ (SEQ ID NO: 348), 5′-CCGCUUCACUUAGGUGAAGGUGG-3′ (SEQ ID NO: 349), 5′-CCGCUUCACUUAGAGUGAAGGUGG-3′ (SEQ ID NO: 350), 5′-CCGCUUCACCUUAGGAGUGAAGGUGG-3′ (SEQ ID NO: 351), 5′-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3′ (SEQ ID NO: 352), 5′-CCGCUUCACCAAUUAGUUGAGUGAAGGUGG-3′ (SEQ ID NO: 353), 5′-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3′ (SEQ ID NO: 354), 5′-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 355), 5′-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 356), 5′-CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 357), 5′-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 358), 5′-CCGCUUCACCAAAAGCUGUUAGUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 359), 5′-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 360), 5′-CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 361), 5′-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 362) and 5′-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGU GG-3′ (SEQ ID NO: 11). Here, the 3′ end of the eighth sequence may be linked to the 5′ end of the first sequence.
As a specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise a seventh sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-GAA-3′, 5′-AGAA-3′, 5′-GAGAA-3′, 5′-GGAGAA-3′, 5′-UGGAGAA-3′, 5′-GUGGAGAA-3′, 5′-AGUGGAGAA-3′, 5′-AAGUGGAGAA-3′ (SEQ ID NO: 17), 5′-AAAGUGGAGAA-3′ (SEQ ID NO: 18), 5′-UAAAGUGGAGAA-3′ (SEQ ID NO: 19), 5′-AUAAAGUGGAGAA-3′ (SEQ ID NO: 20), 5′-GAUAAAGUGGAGAA-3′ (SEQ ID NO: 21), 5′-UGAUAAAGUGGAGAA-3′ (SEQ ID NO: 22), 5′-CUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 23), 5′-ACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 24), 5′-CACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 25), 5′-UCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 26), 5′-UUCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 27), and 5′-CUUCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 10), and an eighth sequence selected from the group consisting of 5′-G-3′, 5′-UUAGG-3′, 5′-CUUAGGG-3′, 5′-CUUAGUGG-3′, 5′-CCUUAGGUGG-3′ (SEQ ID NO: 342), 5′-CCGUUAGGUGG-3′ (SEQ ID NO: 343), 5′-CCGCUUAGGGUGG-3′ (SEQ ID NO: 344), 5′-CCGCUUUAGAGGUGG-3′ (SEQ ID NO: 345), 5′-CCGCUUUUAGAAGGUGG-3′ (SEQ ID NO: 346), 5′-CCGCUUCUUAGGAAGGUGG-3′ (SEQ ID NO: 347), 5′-CCGCUUCAUUAGUGAAGGUGG-3′ (SEQ ID NO: 348), 5′-CCGCUUCACUUAGGUGAAGGUGG-3′ (SEQ ID NO: 349), 5′-CCGCUUCACUUAGAGUGAAGGUGG-3′ (SEQ ID NO: 350), 5′-CCGCUUCACCUUAGGAGUGAAGGUGG-3′ (SEQ ID NO: 351), 5′-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3′ (SEQ ID NO: 352), 5′-CCGCUUCACCAAUUAGUUGAGUGAAGGUGG-3′ (SEQ ID NO: 353), 5′-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3′ (SEQ ID NO: 354), 5′-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 355), 5′-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 356), 5′-CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 357), 5′-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 358), 5′-CCGCUUCACCAAAAGCUGUUAGUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 359), 5′-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 360), 5′-CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 361), 5′-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 362), and 5′-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGU GG-3′ (SEQ ID NO: 11). Here, the 3′ end of the eighth sequence may be linked to the 5′ end of the first sequence, and the 3′ end of the seventh sequence may be linked to the 5′ end of the eighth sequence.
As a specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise a ninth sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-AAG-3′, 5′-AAGU-3′, 5′-AAGUG-3′, 5′-AAGUGC-3′, 5′-AAGUGCU-3′, 5′-AAGUGCUU-3′, 5′-AAGUGCUUU-3′, 5′-AAGUGCUUUC-3′ (SEQ ID NO: 485). Here, the 3′ end of the first sequence and the 5′ end of the second sequence may be linked by the ninth sequence.
As a specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise a tenth sequence selected from the group consisting of 5′-C-3′, 5′-CC-3′, 5′-ACC-3′, 5′-AACC-3′, 5′-UAACC-3′, 5′-GUAACC-3′, 5′-AGUAACC-3′, 5′-AAGUAACC-3′, 5′-AAAGUAACC-3′, and 5′-GAAAGUAACC-3′ (SEQ ID NO: 486). Here, the 3′ end of the second sequence and the 5′ end of the third sequence may be linked by the tenth sequence.
As a specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise a ninth sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-AAG-3′, 5′-AAGU-3′, 5′-AAGUG-3′, 5′-AAGUGC-3′, 5′-AAGUGCU-3′, 5′-AAGUGCUU-3′, 5′-AAGUGCUUU-3′, and 5′-AAGUGCUUUC-3′ (SEQ ID NO: 485) and may additionally comprise a tenth sequence selected from the group consisting of 5′-C-3′, 5′-CC-3′, 5′-ACC-3′, 5′-AACC-3′, 5′-UAACC-3′, 5′-GUAACC-3′, 5′-AGUAACC-3′, 5′-AAGUAACC-3′, 5′-AAAGUAACC-3′, and 5′-GAAAGUAACC-3′ (SEQ ID NO: 486). Here, the 3′ end of the first sequence and the 5 end of the second sequence may be linked by the ninth sequence, and the 3′ end of the second sequence and the 5′ end of the third sequence may be linked by the tenth sequence.
As an example, when the ninth sequence is 5′-A-3′, the tenth sequence may be 5′-C-3′. As another example, when the ninth sequence is 5′-AA-3′, the tenth sequence may be 5′-C-3′, or 5′-CC-3′. As yet another example, when the ninth sequence is 5′-AAG-3′, the tenth sequence may be 5′-CC-3′ or 5′-ACC-3′. As still yet another example, when the ninth sequence is 5′-AAGU-3′, the tenth sequence may be 5′-AACC-3′. As still yet another example, when the ninth sequence is 5′-AAGUG-3′, the tenth sequence may be 5′-UAACC-3′. As still yet another example, when the ninth sequence is 5′-AAGUGC-3′, the tenth sequence may be 5′-GUAACC-3′. As another example, when the ninth sequence is 5′-AAGUGCU-3′, the tenth sequence may be 5′-AGUAACC-3′. As still yet another example, when the ninth sequence is 5′-AAGUGCUC-3′, the tenth sequence may be 5′-GAGUAACC-3′. as still yet another example, when the ninth sequence is 5′-AAGUGCUU-3′, the tenth sequence may be 5′-AAGUAACC-3′. As still yet another example, when the ninth sequence is 5′-AAGUGCUUU-3′, the tenth sequence may be 5′-AAAGUAACC-3′. As still yet another example, when the ninth sequence is 5′-AAGUGCUUUC-3′ (SEQ ID NO: 485), the tenth sequence may be 5′-GAAAGUAACC-3′ (SEQ ID NO: 486).
As another specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise an eleventh sequence selected from the group consisting of 5′-U-3′, 5′-UU-3′, 5′-UUC-3′, 5′-UUCA-3′, 5′-UUCAU-3′, 5′-UUCAUU-3′, and 5′-UUCAUUU-3′. Here, the 3′ end of the fourth sequence and the 5′ end of the linker may be linked by the eleventh sequence.
As yet another specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise a twelfth sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-GAA-3′, 5′-UGAA-3′, 5′-AUGAA-3′, 5′-AAUGAA-3′, and 5′-GAAUGAA-3′. Here, the 3′ end of the linker and the 5′ end of the fifth sequence may be linked by the twelfth sequence.
As still yet another specific example of the embodiment, a sequence of the engineered scaffold region may additionally comprise an eleventh sequence selected from the group consisting of 5′-U-3′, 5′-UU-3′, 5′-UUC-3′, 5′-UUCA-3′, 5′-UUCAU-3′, 5′-UUCAUU-3′, and 5′-UUCAUUU-3′ and may additionally comprise a twelfth sequence selected from 5′-A-3′, 5′-AA-3′, 5′-GAA-3′, 5′-UGAA-3′, 5′-AUGAA-3′, 5′-AAUGAA-3′, and 5′-GAAUGAA-3′. Here, the 3′ end of the fourth sequence and the 5′ end of the linker may be linked by the eleventh sequence, and the 3′ end of the linker and the 5′ end of the fifth sequence may be linked by the twelfth sequence.
As an example, when the eleventh sequence is 5′-U-3′, the twelfth sequence may be 5′-A-3′. As another example, when the eleventh sequence is 5′-UU-3′, the twelfth sequence may be 5′-AA-3′. As another example, when the eleventh sequence is 5′-UUC-3′, the twelfth sequence may be 5′-GAA-3′. As yet another example, when the eleventh sequence is 5′-UUCA-3′, the twelfth sequence may be 5′-UGAA-3′. As still yet another example, when the eleventh sequence is 5′-UUCAU-3′, the twelfth sequence may be 5′-AUGAA-3′. As still yet another example, when the eleventh sequence is 5′-UUCAUU-3′, the twelfth sequence may be 5′-AAUGAA-3′. As still yet another example, when the eleventh sequence is 5′-UUCAUUU-3′, the twelfth sequence may be 5′-GAAUGAA-3′.
In an embodiment, the engineered single guide RNA may have a sequence selected from the group consisting of SEQ ID NOS: 211 to 253, SEQ ID NOS: 296 to 308, SEQ ID NOS: 311 to 323, SEQ ID NOS: 326 to 338, SEQ ID NOS: 488 to 541, and SEQ ID NOS: 545 to 551.
In an embodiment, the engineered Cas12f1 guide RNA may comprise an engineered scaffold region, a spacer, and a U-rich tail.
The spacer has a length of 10 to 50 nucleotides and has a sequence complementary to a target sequence.
A sequence of the U-rich tail may be represented by (UaN)bUc. Here, N is selected from adenosine (A), uridine (U), cytidine (C), and guanosine (G). Here, a, b, and c are each an integer, with a being between 1 and 5 inclusive, and b being 0 or more.
As an example, a sequence of the U-rich tail may be 5′-UUUUAUUUU-3′.
A sequence of the engineered scaffold region comprises in a 5′ to 3′ direction:
Here, a sequence of the engineered tracrRNA may be different from 5′-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGG GAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGA GAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3′ (SEQ ID NO: 1) and/or the engineered crRNA repeat sequence portion may be different from 5′-GAAUGAAGGAAUGCAAC-3′ (SEQ ID NO: 3).
As an example, a sequence of the engineered tracrRNA may be the same as the sequence of SEQ ID NO: 1, and the engineered crRNA repeat sequence portion may be different from the sequence of SEQ ID NO: 3. As another example, a sequence of the engineered tracrRNA may be different from the sequence of SEQ ID NO: 1, and the engineered crRNA repeat sequence portion may be the same as the sequence of SEQ ID NO: 3. As yet another example, a sequence of the engineered tracrRNA may be different from the sequence of SEQ ID NO: 1, and the engineered crRNA repeat sequence portion may be different from the sequence of SEQ ID NO: 3.
As an example, a sequence of the engineered tracrRNA may not comprise the first sequence and/or the second sequence.
Specifically, a sequence of the engineered tracrRNA may be selected from the group consisting of:
As an example, when the engineered tracrRNA comprises 5′-AACAAA-3′, the engineered crRNA may comprise 5′-GGA-3′. As another example, when the engineered tracrRNA comprises 5′-AACAAAU-3′, the engineered crRNA may comprise 5′-AGGA-3′. As yet another example, when the engineered tracrRNA comprises 5′-AACAAAUU-3′, the engineered crRNA may comprise 5′-AAGGA-3′. As still yet another example, when the engineered tracrRNA comprises 5′-AACAAAUUC-3′, the engineered crRNA may comprise 5′-GAAGGA-3′. As still yet another example, when the engineered tracrRNA comprises 5′-AACAAAUUCA-3′, the engineered crRNA may comprise 5′-UGAAGGA-3′. As still yet another example, when the engineered tracrRNA comprises 5′-AACAAAUUCAU-3′, the engineered crRNA may comprise 5′-AUGAAGGA-3′. As still yet another example, when the engineered tracrRNA comprises 5′-AACAAAUUCAUU-3′, the engineered crRNA may comprise 5′-AAUGAAGGA-3′. As still yet another example, when the engineered tracrRNA comprises 5′-AACAAAUUCAUUU-3′, the engineered crRNA may comprise 5′-GAAUGAAGGA-3′.
In the present disclosure, there is provided an engineered CRISPR/Cas12f1 complex. The engineered CRISPR/Cas12f1 complex comprises a Cas12f1 protein and an engineered Cas12f1 guide RNA. Here, the engineered Cas12f1 guide RNA is as described in the section “Engineered Cas12f1 guide RNA.”
In an embodiment, there is provided herein an engineered CRISPR/Cas12f1 complex capable of editing a target sequence-containing nucleic acid, the complex comprising a Cas12f1 protein and an engineered Cas12f1 guide RNA. Here, the engineered Cas12f1 guide RNA may be any one of those described in the section “Engineered Cas12f1 guide RNA.”
The engineered CRISPR/Cas12f1 complex provided herein comprises a Cas12f1 protein. Basically, the Cas12f1 protein may be a wildtype Cas12f1 protein occurring in nature. The sequence encoding the Cas12f1 protein may be a human codon-optimized Cas12f1 sequence for the wildtype Cas12f1 protein. In addition, the Cas12f1 protein may have the same function as the wildtype Cas12f1 protein occurring in nature. However, unless specifically limited, the term “Cas12f1 protein” as used herein may refer not only to a wildtype or codon-optimized Cas12f1 protein, but also encompass a modified Cas12f1 protein and a Cas12f1 fusion protein. In addition, the “Cas12f1 protein” may refer collectively not only to those having the same function as the wildtype Cas12f1 protein occurring in nature, but to those in which all or a part of the function is modified, those in which all or a part of the function is lost, and/or those to which an additional function is added. A meaning of the “Cas12f1 protein” may be appropriately interpreted according to the context and is interpreted in the broadest sense except for special cases. Hereinafter, a structure or function of the Cas12f1 protein will be described in detail.
The engineered CRISPR/Cas12f1 complex provided herein may comprise a Cas12f1 protein.
In an embodiment, the Cas12f1 protein may be a wildtype Cas12f1 protein. In an embodiment, the Cas12f1 protein may be derived from the Cas14 family (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)). In an embodiment, the Cas12f1 protein may be a Cas14a protein derived from an uncultured archaeon (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)). In an embodiment, the Cas12f1 protein may be a Cas14a1 protein.
The engineered CRISPR/Cas12f1 complex provided herein may comprise a modified Cas12f1 protein. The modified Cas12f1 means a sequence of a wildtype or codon-optimized Cas12f1 protein, at least some of which are modified. Modification of the Cas12f1 protein may be made in its individual amino acid unit or its functional domain unit.
In an embodiment, modification of the protein may be made by individual substitution, deletion, and/or addition of one or more amino acids, peptides, polypeptides, proteins, and/or domains in the sequence of the wildtype or codon-optimized Cas12f1 protein. In an embodiment, the Cas12f1 protein may be a wildtype Cas12f1 protein whose RuvC domain includes substitution, deletion, and/or addition of one or more amino acids, peptides, and/or polypeptides.
The engineered CRISPR/Cas12f1 complex provided herein may comprise a Cas12f1 fusion protein. Here, the Cas12f1 fusion protein refers to a protein in which an additional amino acid, peptide, polypeptide, protein, and/or domain is fused to a wildtype or modified Cas12f1 protein.
In an embodiment, the Cas12f1 protein may be one in which a base editor and/or a reverse transcriptase is fused to a wildtype Cas12f1 protein. In an embodiment, the base editor may be an adenosine deaminase, and/or a cytidine deaminase. In an embodiment, the reverse transcriptase may be Moloney murine leukemia virus (M-MLV) reverse transcriptase, and/or a variant thereof. Here, the Cas12f1 protein fused with the reverse transcriptase may function as a prime editor.
In an embodiment, the Cas12f1 protein may be one in which various enzymes, which can be involved in a gene expression process in a cell, are fused to a wildtype Cas12f1 protein. Here, the Cas12f1 protein fused with the enzyme may cause various quantitative and qualitative changes in gene expression in a cell. In an embodiment, the enzyme may be VP64, DNMT, TET, KRAB, DHAC, LSD, and/or p300.
The Cas12f1 protein included in the engineered CRISPR/Cas12f1 complex provided herein may have the same function as a wildtype Cas12f1 protein. The Cas12f1 protein included in the engineered CRISPR/Cas12f1 complex provided herein may have an altered function as compared with a wildtype Cas12f1 protein. Specifically, the alteration may be modification of all or a part of the functions, loss of all or a part of the functions, and/or addition of an additional function. In an embodiment, the Cas12f1 protein is not particularly limited as long as such alteration can be applied to a Cas protein of a CRISPR/Cas system by those skilled in the art. Here, the alteration may be made using a known technique in the art.
In an embodiment, the Cas12f1 protein may be a Cas12f1 protein that is altered to cleave only one strand of the double strand of a target nucleic acid. Moreover, the Cas12f1 protein may be a Cas12f1 protein altered so that it is able to cleave only one strand of the double strand of a target nucleic acid, and to perform base editing or prime editing on the uncleaved strand. In an embodiment, the Cas12f1 protein may be a Cas12f1 protein altered so that it is unable to cleave neither strand of the double strand of a target nucleic acid. Furthermore, the Cas12f1 protein may be a Cas12f1 protein altered so that it is unable to cleave neither strand of the double strand of a target nucleic acid, and is able to perform base editing, prime editing, or a function of regulating gene expression on the target nucleic acid.
In an embodiment, the Cas12f1 protein may comprise a nuclear localization sequence (NLS) or a nuclear export sequence (NES). Specifically, the NLS may be any one of those exemplified in the section for NLS among “Definitions of terms,” but is not limited thereto. In an embodiment, the Cas12f1 protein may comprise a tag. Specifically, the tag may be any one of those exemplified in the section for tag in “Definition of terms,” but is not limited thereto.
Two conditions are required for a CRISPR/Cas12f1 complex to cleave a target gene or target nucleic acid.
First, it is required that there be a nucleotide sequence of a certain length, which can be recognized by a Cas12f1 protein, in the target gene or target nucleic acid. Here, the nucleotide sequence of a certain length recognized by the Cas12f1 protein is referred to as a protospacer adjacent motif (PAM) sequence. The PAM sequence is a distinctive sequence determined according to the Cas12f1 protein. Second, it is required that there be a sequence, which is capable of complementarily binding to a spacer sequence included in the guide RNA, around the PAM sequence of a certain length.
When these two conditions are satisfied, that is, 1) the Cas12f1 protein recognizes the PAM sequence of a certain length, and 2) the spacer sequence portion complementarily binds to a sequence around the PAM sequence, the Cas12f1 protein/guide RNA complex (CRISPR/Cas12f1 complex) cleaves a target gene or target nucleic acid. Therefore, when determining a target sequence of the CRISPR/Cas12f1 complex, there is a constraint that the target sequence has to be determined within sequences adjacent to the PAM sequence.
In an embodiment, a PAM sequence of the Cas12f1 protein may be a T-rich sequence.
In an embodiment, a PAM sequence of the Cas12f1 protein may be THTN in a 5′ to 3′ direction. Here, N may be one of deoxythymidine (T), deoxyadenosine (A), deoxycytidine (C), or deoxyguanosine (G), and H may be one of deoxythymidine (T), deoxyadenosine (A), and deoxycytidine (C). In an embodiment, a PAM sequence of the Cas12f1 protein may be TTTN in a 5′ to 3′ direction. Here, N is one of deoxythymidine (T), deoxyadenosine (A), deoxycytidine (C), or deoxyguanosine (G). In an embodiment, a PAM sequence of the Cas12f1 protein may be TTTA, TTTT, TTTC, or TTTG in a 5′ to 3′ direction. In an embodiment, a PAM sequence of the Cas12f1 protein may be TATA, TATT, TATC, or TATG in a 5′ to 3′ direction. In an embodiment, a PAM sequence of the Cas12f1 protein may be TCTA, TCTT, TCTC, or TCTG in a 5′ to 3′ direction. In an embodiment, a PAM sequence of the Cas12f1 protein may be TTTA or TTTG in a 5′ to 3′ direction. In an embodiment, a PAM sequence of the Cas12f1 protein may be different from a PAM sequence of the wildtype Cas12f1 protein.
In an embodiment, the Cas12f1 protein may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 260 to 267.
In an embodiment, a DNA sequence encoding the Cas12f1 protein may be a human codon-optimized sequence.
In an embodiment, a DNA sequence encoding the Cas12f1 protein may be a DNA sequence selected from the group consisting of SEQ ID NOS: 268 to 277.
The engineered Cas12f1 guide RNA constituting the CRISPR/Cas12f1 complex provided herein has the same characteristics and structure as described in the section “Engineered Cas12f1 guide RNA.”
In an embodiment, the Cas12f1 protein may have an amino acid sequence of SEQ ID NO: 260, and the engineered Cas12f1 guide RNA may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 211 to 253, SEQ ID NOS: 296 to 308, SEQ ID NOS: 311 to 323, SEQ ID NOS: 326 to 338, SEQ ID NOS: 488 to 541, and SEQ ID NOS: 545 to 551. Here, the Cas12f1 protein and the engineered Cas12f1 guide RNA may be combined to form a CRISPR/Cas12f1 complex.
In an embodiment, the Cas12f1 protein has an amino acid sequence selected from the group consisting of SEQ ID NOS: 264 to 267, and the engineered Cas12f1 guide RNA may have a sequence selected from the group consisting of SEQ ID NOS: 211 to 253, SEQ ID NOS: 296 to 308, SEQ ID NOS: 311 to 323, SEQ ID NOS: 326 to 338, SEQ ID NOS: 488 to 541, and SEQ ID NOS: 545 to 551. Here, the CRISPR/Cas12f1 complex formed by combination of the Cas12f1 protein with the engineered Cas12f1 guide RNA may have a base editing function.
In the present specification, there is provided a vector for expressing components of a CRISPR/Cas12f1 system. The vector is constructed to express a Cas12f1 protein, and/or an engineered Cas12f1 guide RNA. A sequence of the vector may comprise a nucleic acid sequence encoding one of the components of the CRISPR/Cas12f1 system or may comprise a nucleic acid sequence encoding two or more of the components thereof. A sequence of the vector comprises a nucleic acid sequence encoding the Cas12f1 protein and/or a nucleic acid sequence encoding the engineered Cas12f1 guide RNA. A sequence of the vector comprises one or more promoter sequences. The promoter is operatively linked with a nucleic acid sequence encoding the Cas12f1 protein and/or a nucleic acid sequence encoding the engineered Cas12f1 guide RNA, so that transcription of the nucleic acid sequence(s) in a cell can be promoted. The Cas12f1 protein has the same characteristics and structure as the Cas12f1 protein, the modified Cas12f1 protein, and/or the Cas12f1 fusion protein as described in the section “Engineered CRISPR/Cas12f1 complex.” The engineered Cas12f1 guide RNA has the same characteristics and composition as the engineered Cas12f1 guide RNA as described in the section “Engineered Cas12f1 guide RNA.”
A sequence of the vector may comprise a nucleic acid sequence encoding the Cas12f1 protein and/or a nucleic acid sequence encoding the engineered Cas12f1 guide RNA. In an embodiment, a sequence of the vector may comprise a first sequence comprising a nucleic acid sequence encoding the Cas12f1 protein and a second sequence comprising a nucleic acid sequence encoding the engineered Cas12f1 guide RNA. The sequence of the vector comprises a promoter sequence for expressing a nucleic acid sequence encoding the Cas12f1 protein in a cell, and a promoter sequence for expressing a nucleic acid sequence encoding the engineered Cas12f1 guide RNA in a cell, wherein each of the promoters is operably linked to each target to be expressed. In an embodiment, a sequence of the vector may comprise a first promoter sequence operably linked to the first sequence, and a second promoter sequence operably linked to the second sequence.
A sequence of the vector may comprise a nucleic acid sequence encoding the Cas12f1 protein and/or a nucleic acid sequence encoding two or more engineered Cas12f1 guide RNAs that are different from each other. In an embodiment, a sequence of the vector may comprise a first sequence comprising a nucleic acid sequence encoding the Cas12f1 protein, a second sequence comprising a nucleic acid sequence encoding a first engineered Cas12f1 guide RNA, and a third sequence comprising a nucleic acid sequence encoding a second engineered Cas12f1 guide RNA. Furthermore, the sequence of the vector may comprise a first promoter sequence operably linked to the first sequence, a second promoter sequence operably linked to the second sequence, and a third promoter sequence operably linked to the third sequence.
In the vector, the nucleic acid sequence encoding each component may be a DNA sequence.
The vector may be constructed to express a Cas12f1 protein. Here, the Cas12f1 protein may have the same structure and characteristics as each of those described in the section “Engineered CRISPR/Cas12f1 complex.”
In an embodiment, the vector may be constructed to express a wildtype Cas12f1 protein. Here, the wildtype Cas12f1 protein may be Cas14a1. In an embodiment, the vector may be constructed to express a Cas12f1 protein altered so that it cleaves only one strand of the double strand of a target nucleic acid. Furthermore, the modified Cas12f1 protein may be a Cas12f1 protein altered so that it is able to cleave only one strand of the double strand of a target nucleic acid and is able to perform base editing or prime editing on the uncleaved strand. In an embodiment, the Cas12f1 protein may be a Cas12f1 protein altered so that it is unable to cleave neither strand of the double strand of a target nucleic acid. Furthermore, the Cas12f1 protein may be a Cas12f1 protein altered so that it is unable to cleave neither strand of the double strand of a target nucleic acid and is able to perform base editing, prime editing, or a function of regulating gene expression on a target nucleic acid.
The vector may be constructed to express an engineered Cas12f1 guide RNA. The engineered Cas12f1 guide RNA may have the same characteristics and composition as the engineered Cas12f1 guide RNA as described in the section “Engineered Cas12f1 guide RNA.” The vector may be constructed to express two or more engineered Cas12f1 guide RNAs that are different from each other.
The vector may be constructed to express an additional component such as an NLS and a tag protein in addition to the above-described targets to be expressed. In an embodiment, the additional component may be expressed independently of the Cas12f1 protein, the modified Cas12f1 protein, and/or the engineered Cas12f1 guide RNA. In another embodiment, the additional component may be expressed in conjunction with the Cas12f1 protein, the modified Cas12f1 protein, and/or the engineered Cas12f1 guide RNA. Here, the additional component may be a component that is generally expressed when it is intended to express a CRISPR/Cas system. In this regard, reference may be made to the prior art. The additional component may be one or more of the components as described in the section “Background art—Design of vector expressing CRISPR/Cas system.”
A sequence of the vector may comprise a nucleic acid sequence encoding the Cas12f1 protein. Here, the Cas12f1 protein may have the same structure and characteristics as each of those described in the section “Engineered CRISPR/Cas12f1 complex.”
In an embodiment, a sequence of the vector may comprise a sequence encoding a wildtype Cas12f1 protein. Here, the wildtype Cas12f1 protein may be Cas14a1. In an embodiment, a sequence of the vector may comprise a human codon-optimized nucleic acid sequence encoding a Cas12f1 protein. Here, the human codon-optimized nucleic acid sequence encoding a Cas12f1 protein may be a human codon-optimized nucleic acid sequence encoding a Cas14a1 protein. In an embodiment, a sequence of the vector may comprise a sequence encoding a modified Cas12f1 protein or a Cas12f1 fusion protein. In an embodiment, a sequence of the vector may comprise a sequence encoding a Cas12f1 fusion protein altered so that it is able to cleave only one strand of the double strand of a target nucleic acid, and is able to perform base editing or prime editing on the uncleaved strand. In an embodiment, a sequence of the vector may comprise a sequence encoding a Cas12f1 fusion protein altered so that it is unable to cleave neither strand of the double strand of a target nucleic acid, and is able to perform base editing, prime editing, or a function of regulating gene expression on the uncleaved strand.
In an embodiment, a sequence of the vector may comprise a sequence encoding an engineered Cas12f1 guide RNA. For example, a sequence of the vector may comprise a sequence selected from the group consisting of SEQ ID NOS: 211 to 253, SEQ ID NOS: 296 to 308, SEQ ID NOS: 311 to 323, SEQ ID NOS: 326 to 338, SEQ ID NOS: 488 to 541, and SEQ ID NOS: 545 to 551.
In an embodiment, a sequence of the vector may comprise a sequence encoding two or more engineered Cas12f1 guide RNAs that are different from each other. For example, a sequence of the vector may comprise a sequence encoding a first engineered Cas12f1 guide RNA and a sequence encoding a second engineered Cas12f1 guide RNA, each of which is selected from the group consisting of SEQ ID NOS: 211 to 253, SEQ ID NOS: 296 to 308, SEQ ID NOS: 311 to 323, SEQ ID NOS: 326 to 338, SEQ ID NOS: 488 to 541, and SEQ ID NOS: 545 to 551.
A sequence of the vector may comprise a promoter sequence operably linked to a sequence encoding each component. Specifically, the promoter sequence may be one of the promoters disclosed in the promoter part of the section “Background art—Design of vector expressing CRISPR/Cas system,” but is not limited thereto.
In an embodiment, a sequence of the vector may comprise a sequence encoding a Cas12f1 protein and a promoter sequence. Here, the promoter sequence is operably linked to the sequence encoding a Cas12f1 protein. In an embodiment, a sequence of the vector may comprise a sequence encoding an engineered Cas12f1 guide RNA and a promoter sequence. Here, the promoter sequence may be operably linked to the sequence encoding an engineered Cas12f1 guide RNA. In an embodiment, the vector sequence may comprise a sequence encoding a Cas12f1 protein, a sequence encoding an engineered Cas12f1 guide RNA, and a promoter sequence. Here, the promoter sequence is operatively linked to the sequence encoding a Cas12f1 protein and the sequence encoding an engineered Cas12f1 guide RNA sequence, wherein a transcription factor activated by the promoter sequence causes expression of the Cas12f1 protein and the engineered Cas12f1 guide RNA.
In an embodiment, a sequence of the vector may comprise a first sequence encoding a Cas12f1 protein, a first promoter sequence, a second sequence encoding an engineered Cas12f1 guide RNA, and a second promoter sequence. Here, the first promoter sequence is operably linked to the first sequence, the second promoter sequence is operatively linked to the second sequence, transcription of the first sequence is induced by the first promoter sequence, wherein transcription of the second sequence is induced by the second promoter sequence. Here, the first promoter and the second promoter may be the same type of promoters. Here, the first promoter and the second promoter may be different kinds of promoters.
In an embodiment, a sequence of the vector may comprise a first sequence encoding a Cas12f1 protein, a first promoter sequence, a second sequence encoding a first engineered Cas12f1 guide RNA, a second promoter sequence, a third sequence encoding a second engineered Cas12f1 guide RNA, and a third promoter sequence. Here, the first promoter sequence is operatively linked to the first sequence, the second promoter sequence is operably linked to the second sequence, and the third promoter sequence is operably linked to the third sequence, wherein transcription of the first sequence is induced by the first promoter sequence, transcription of the second sequence is induced by the second promoter sequence, and transcription of the third sequence is induced by the third promoter sequence. Here, the second promoter and the third promoter may be the same type of promoters. Specifically, the second promoter sequence and the third promoter sequence may be a U6 promoter sequence, but are not limited thereto. Here, the second promoter and the third promoter may be different types of promoters. Specifically, the second promoter may be a U6 promoter sequence, and the third promoter may be an H1 promoter sequence, but these promoters are not limited thereto.
The vector may comprise a termination signal operably linked to the promoter sequence. Here, the termination signal may be one of the termination signals disclosed in the termination signal portion of the section “Background art—Design of vector expressing CRISPR/Cas system,” but is not limited thereto. The termination signal may vary depending on the type of promoter sequence.
In an embodiment, when a sequence of the vector comprises a U6 promoter sequence, a thymidine repeat sequence operably linked to the U6 promoter sequence may serve as a termination signal. In an embodiment, the thymidine repeat sequence may be a sequence in which five or more thymidines are continuously linked. In an embodiment, when a sequence of the vector comprises an H1 promoter sequence, a thymidine repeat sequence operably linked to the H1 promoter sequence may serve as a termination signal. In an embodiment, the thymidine repeat sequence may be a sequence in which five or more thymidines are continuously linked.
A sequence of the vector may comprise a component necessary according to the purpose in addition to the above components.
In an embodiment, a sequence of the vector may comprise a sequence of a regulatory/control element, and/or a sequence of an additional component. In an embodiment, the additional component may be added for the purpose of distinguishing transfected cells from non-transfected cells. Here, each of the sequence of the regulatory/control element and the additional component may be one of those disclosed in the section “Background art—Design of vector expressing CRISPR/Cas system,” but is not limited thereto.
The vector may be a viral vector.
In an embodiment, the viral vector may be at least one selected from the group consisting of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus. In an embodiment, the viral vector may be an adeno-associated virus.
The vector may be a non-viral vector. In an embodiment, the non-viral vector may be one or more selected from the group consisting of a plasmid, a phage, naked DNA, a DNA complex, and mRNA. In an embodiment, the plasmid may be selected from the group consisting of pcDNA series, pS456, pG1806, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19. In an embodiment, the phage may be selected from the group consisting of λgt4λB, λ-Charon, λΔz1, and M13. In an embodiment, the vector may be a PCR amplicon.
The vector may have a circular or linear form. When the vector is a linear vector, RNA transcription is terminated at the 3′ end thereof even if a sequence of the linear vector does not separately comprise a termination signal. In contrast, when the vector is a circular vector, RNA transcription is not terminated unless the circular vector sequence separately comprises a termination signal. Therefore, when the vector is used in a form of a circular vector, a termination signal corresponding to a transcription factor related to each promoter sequence has to be included in order for the vector to express an intended target.
In an embodiment, the vector may be a linear vector. In an embodiment, the vector may be a linear amplicon. In an embodiment, the vector may be a linear amplicon comprising a sequence selected from the group consisting of SEQ ID NOS: 211 to 253, SEQ ID NOS: 296 to 308, SEQ ID NOS: 311 to 323, SEQ ID NOS: 326 to 338, SEQ ID NOS: 488 to 541, and SEQ ID NOS: 545 to 551. In an embodiment, the vector may be a circular vector. In an embodiment, the vector may be a circular amplicon including a sequence selected from the group consisting of SEQ ID NOS: 211 to 253, SEQ ID NOS: 296 to 308, SEQ ID NOS: 311 to 323, SEQ ID NOS: 326 to 338, SEQ ID NOS: 488 to 541, and SEQ ID NOS: 545 to 551.
In an embodiment, a sequence of the vector may comprise a sequence selected from the group consisting of SEQ ID NOS: 211 to 253, SEQ ID NOS: 296 to 308, SEQ ID NOS: 311 to 323, SEQ ID NOS: 326 to 338, SEQ ID NOS: 488 to 541, and SEQ ID NOS: 545 to 551.
In the present disclosure, there is provided a composition for gene editing comprising respective components of a CRISPR/Cas12f1 system is disclosed. In an embodiment, there is provided herein a composition for gene editing comprising: a Cas12f1 protein or a nucleic acid encoding the same, and an engineered Cas12f1 guide RNA or a nucleic acid encoding the same. Here, the Cas12f1 protein may be one of those described in the section “Engineered CRISPR/Cas12f1 complex.” Here, the engineered Cas12f1 guide RNA may be one of those described in the section “Engineered Cas12f1 guide RNA.”
The composition for gene editing may further comprise an appropriate material necessary for gene editing in addition to the respective components of the CRISPR/Cas12f1 system.
In the present disclosure, there is provided a component that comprises or consists of a nucleic acid such as an engineered crRNA or a nucleic acid encoding the same, an engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and/or a vector for expressing components of a CRISPR/Cas12f1 system. Here, the “nucleic acid” in the component may be naturally occurring DNA or RNA, or a modified nucleic acid in which a part of or all of a constituent nucleic acid is chemically modified. In an embodiment, the constituent nucleic acid may be naturally occurring DNA and/or RNA. In an embodiment, the constituent nucleic acid may be one in which one or more nucleotides are chemically modified. Here, the chemical modification includes any of modifications of a nucleic acid known to those skilled in the art. Specifically, the chemical modification may include any of modifications of a nucleic acid as described in WO 2019/089820 A1, but is not limited thereto.
In the present disclosure, there is provided a method of editing a target gene or target nucleic acid in a target cell by using an engineered crRNA. The target gene or target nucleic acid contains a target sequence. The target nucleic acid may be single-stranded DNA, double-stranded DNA, and/or RNA. The gene editing method comprises delivering an engineered Cas12f1 guide RNA and a Cas12f1 protein, or nucleic acids, each of which encodes each of them, into a target cell including a target gene or target nucleic acid. As a result, an engineered CRISPR/Cas12f1 complex is introduced into the target cell, or formation of an engineered CRISPR/Cas12f1 complex is induced, so that the target gene is edited by the engineered CRISPR/Cas12f1 complex. The engineered Cas12f1 guide RNA has the same characteristics and structure as described in the section “Engineered Cas12f1 guide RNA.” The Cas12f1 protein has the same characteristics and structure as the Cas12f1 protein and/or the modified Cas12f1 protein as described in the section “Engineered CRISPR/Cas12f1 complex.”
In an embodiment, the gene editing method may comprise delivering a Cas12f1 protein or a nucleic acid encoding the same, and an engineered Cas12f1 guide RNA or a nucleic acid encoding the same into a target cell.
Here, the engineered Cas12f1 guide RNA may comprise an engineered scaffold region, a spacer, and a U-rich tail.
Here, the engineered scaffold region has the same characteristics and structure as each of those described in any one of the above-described “Engineered scaffold region” sections. As an example, the engineered scaffold region may be represented by a sequence selected from the group consisting of SEQ ID NOS: 168 to 187. As another example, the engineered scaffold region may be represented by a sequence selected from the group consisting of SEQ ID NOS: 188 to 199. As yet another example, the engineered scaffold region may be represented by a sequence selected from the group consisting of SEQ ID NOS: 200 to 206. As still yet another example, the engineered scaffold region may be represented by a sequence selected from the group consisting of SEQ ID NOS: 207 to 210.
Here, the spacer sequence may complementarily bind to a target gene or target nucleic acid included in the target cell.
Here, a sequence of the U-rich tail is represented by (UaN)bUc wherein N is selected from adenosine (A), uridine (U), cytidine (C) and guanosine (G); and a, b, c are each an integer, with a being between 1 and 5 inclusive, b being 0 or more, and c being between 1 to 10 inclusive. As an example, a sequence of the U-rich tail may be 5′-UUUUAUUUU-3′. As an example, the sequence of the U-rich tail may be 5′-UUUUGUUUU-3′.
In an embodiment, the target cell may be a prokaryotic cell. In an embodiment, the target cell may be a eukaryotic cell. Specifically, the eukaryotic cell may be, but is not limited to, a plant cell, an animal cell, and/or a human cell.
A target gene or target nucleic acid, and a target sequence to be edited by a CRISPR/Cas12f1 complex may be determined in consideration of the purpose of gene editing, environment of a target cell, a PAM sequence recognized by a Cas12f1 protein, and/or other variables. Here, a method of determining the target sequence is not particularly limited as long as it is capable of determining a target sequence of an appropriate length, and a technique known in the art may be used therefor.
Once the target sequence is determined, a spacer sequence corresponding thereto is designed. The spacer sequence is designed as a sequence capable of complementarily binding to the target sequence. In an embodiment, the spacer sequence may be designed as a sequence capable of complementarily binding to the target gene. In an embodiment, the spacer sequence may be designed to be capable of complementarily binding to the target nucleic acid. In an embodiment, the spacer sequence may be designed as a sequence complementary to a target sequence included in a target strand sequence of the target nucleic acid. In an embodiment, the spacer sequence is designed as an RNA sequence corresponding to a DNA sequence of a protospacer included in a non-target strand sequence of the target nucleic acid. Specifically, the spacer sequence is designed to have the same nucleotide sequence as the protospacer sequence, except that every thymidine included in the nucleotide sequence is substituted by a uridine.
In an embodiment, the spacer has a length of 10 to 50 nucleotides. For example, the spacer has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Preferably the spacer has a length of 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.
In an embodiment, the spacer sequence may be complementary to the target sequence by 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In an embodiment, the spacer sequence may be a sequence complementary to the target sequence within a numerical range selected from the immediately preceding sentence. As an example, the spacer sequence may be a sequence that is 60% to 90% complementary to the target sequence. As another example, the spacer sequence may be a sequence that is 90% to 100% complementary to the target sequence.
In an embodiment, the spacer sequence may be a sequence that is complementary to the target sequence and has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches therewith. In an embodiment, the spacer sequence may have mismatches within a numerical range selected from the immediately preceding sentence. As an example, the spacer sequence may have 0, 1, 2, 3, 4, or 5 mismatches with the target sequence. As another example, the spacer sequence may have 6 to 10 mismatches with the target sequence.
The gene editing method provided herein utilizes the fact that the engineered CRISPR/Cas12f1 complex has activity of cleaving a gene or nucleic acid in a target-specific manner. The engineered CRISPR/Cas12f1 complex has the same characteristics and composition as the engineered CRISPR/Cas12f1 complex as described in the section “Engineered CRISPR/Cas12f1 complex.”
Delivery of Respective Components of CRISPR/Cas12f1 Complex into Cell
The gene editing method provided herein comprises bringing an engineered CRISPR/Cas12f1 complex in contact with a target gene or target nucleic acid in a target cell. Accordingly, in order to induce the engineered CRISPR/Cas12f1 complex to come in contact with the target gene or target nucleic acid, the gene editing method comprises delivering respective components of the engineered CRISPR/Cas12f1 complex into a target cell.
In an embodiment, the gene editing method may comprise delivering into a target cell an engineered Cas12f1 guide RNA or a nucleic acid encoding the same and a Cas12f1 protein or a nucleic acid encoding the same. In an embodiment, the gene editing method may comprise delivering an engineered Cas12f1 guide RNA and a Cas12f1 protein into a target cell. In an embodiment, the gene editing method may comprise delivering a nucleic acid encoding an engineered Cas12f1 guide RNA and a Cas12f1 protein into a target cell. In an embodiment, the gene editing method may comprise delivering an engineered Cas12f1 guide RNA and a nucleic acid encoding a Cas12f1 protein into a target cell. In an embodiment, the gene editing method may comprise delivering a nucleic acid encoding an engineered Cas12f1 guide RNA and a nucleic acid encoding a Cas12f1 protein into a target cell.
The engineered Cas12f1 guide RNA or the nucleic acid encoding the same, and the Cas12f1 protein or the nucleic acid encoding the same may be delivered into the target cell in various forms of delivery using various delivery methods.
As the form of delivery, a ribonucleoprotein particle (RNP), in which an engineered Cas12f1 guide RNA and a Cas12f1 protein are bound to each other, may be used. In an embodiment, the gene editing method may comprise introducing, into a target cell, a CRISPR/Cas12f1 complex in which the engineered Cas12f1 guide RNA and the Cas12f1 protein are bound to each other.
As another form of delivery, a non-viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein may be used. In an embodiment, the gene editing method may comprise introducing, into a target cell, a non-viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein. Specifically, the non-viral vector may be a plasmid, naked DNA, a DNA complex, or mRNA, but is not limited thereto. In another embodiment, the gene editing method comprises introducing into a target cell, a first non-viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA, and a second non-viral vector comprising a nucleic acid sequence encoding a Cas12f1 protein. Specifically, each of the first non-viral vector and the second non-viral vector may be one selected from the group consisting of a plasmid, naked DNA, a DNA complex, and mRNA, but is not limited thereto.
As another form of delivery, a viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein may be used. In an embodiment, the gene editing method may comprise introducing, into a target cell, a viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein. Specifically, the viral vector may be one selected from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus, but is not limited thereto. In an embodiment, the viral vector may be an adeno-associated virus.
In another embodiment, the gene editing method may comprise introducing into a target cell, a first viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA, and a second viral vector comprising a nucleic acid sequence encoding a Cas12f1 protein. Specifically, each of the first viral vector and the second viral vector may be one selected from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus, but is not limited thereto.
The delivery method is not particularly limited as long as it is capable of delivering, into a cell, an engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a Cas12f1 protein or a nucleic acid encoding the same in an appropriate form of delivery. In an embodiment, the delivery method may be electroporation, gene gun, sonoporation, magnetofection, and/or transient cell compression or squeezing.
The delivery method may be delivering at least one component, which is included in the CRISPR/Cas12f1 system, using nanoparticles. Here, the delivery method may be a method known in the art which can be appropriately selected by those skilled in the art. For example, the nanoparticle delivery method may be a method disclosed in WO 2019/089820 A1, but is not limited thereto.
In an embodiment, the delivery method may be delivering, using nanoparticles, a Cas12f1 protein or a nucleic acid encoding the same and/or an engineered Cas12f1 guide RNA or a nucleic acid encoding the same. In an embodiment, the delivery method may be delivering, using nanoparticles, a Cas12f1 protein or a nucleic acid encoding the same, a first engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and/or a second engineered Cas12f1 guide RNA or a nucleic acid encoding the same. Here, the delivery method may be a cationic liposome method, a lithium acetate-dimethyl sulfoxide (DMSO) method, lipid-mediated transfection, calcium phosphate precipitation, lipofection, polyethyleneimine (PEI)-mediated transfection, diethylaminoethyl (DEAE)-dextran-mediated transfection, and/or nanoparticle-mediated nucleic acid delivery (see Panyam et., al Adv Drug Deliv Rev. 2012 Sep. 13. pii: S0169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023), but is not limited thereto. Here, the component of the CRISPR/Cas12f1 system may be in a form of an RNP, a non-viral vector, and/or a viral vector. For example, each of the components of the CRISPR/Cas12f1 system may be in a form of mRNA encoding the same, but is not limited thereto.
Form and Method of Delivery—Combination being Possible
The gene editing method comprises delivering, into a cell, an engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a Cas12f1 protein or a nucleic acid encoding the same, wherein delivery forms and/or delivery methods of respective components may be the same as or different from each other. In an embodiment, the gene editing method may comprise delivering an engineered Cas12f1 guide RNA or a nucleic acid encoding the same in a first form of delivery and delivering a Cas12f1 protein or a nucleic acid encoding the same in a second form of delivery. Here, each of the first form of delivery and the second form of delivery may be any one of the above-described forms of delivery. In an embodiment, the gene editing method may comprise delivering an engineered Cas12f1 guide RNA or a nucleic acid encoding the same in a first form of delivery, and delivering a Cas12f1 protein or a nucleic acid encoding the same in a second form of delivery. Here, each of the first form of delivery and the second form of delivery may be any one of the above-described forms of delivery.
The gene editing method comprises delivering, into a cell, an engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a Cas12f1 protein or a nucleic acid encoding the same, wherein the components may be delivered into the cell simultaneously or sequentially with a time interval.
In an embodiment, the gene editing method may comprise delivering, into a target cell, an engineered Cas12f1 guide RNA or a nucleic acid encoding the same and a Cas12f1 protein or a nucleic acid encoding the same simultaneously. In an embodiment, the gene editing method may comprise delivering an engineered Cas12f1 guide RNA or a nucleic acid encoding the same into a cell, and then delivering a Cas12f1 protein or a nucleic acid encoding the same into the cell. In an embodiment, the gene editing method may comprise delivering a Cas12f1 protein or a nucleic acid encoding the same into a cell, and then delivering an engineered Cas12f1 guide RNA into the cell. In an embodiment, the gene editing method may comprise delivering a nucleic acid encoding a Cas12f1 protein into a cell, and then delivering an engineered Cas12f1 guide RNA into the cell.
The gene editing method provided herein may comprise delivering, into a target cell, a Cas12f1 protein or a nucleic acid encoding the same, and two or more engineered Cas12f1 guide RNAs or nucleic acids encoding the same. By using the above method, two or more CRISPR/Cas12f1 complexes, which target different sequences, may be introduced into a target cell or formed in a target cell. As a result, the method enables editing of two or more different target genes or target nucleic acids included in the cell. In an embodiment, the gene editing method comprises delivering, into a target cell including a target gene or target nucleic acid, a Cas12f1 protein or a nucleic acid encoding the same, a first engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a second engineered Cas12f1 guide RNA or a nucleic acid encoding the same. Here, each of the components may be delivered into the cell using one or more of the above-described forms of delivery and methods of delivery. Here, two or more of the components may be delivered simultaneously or sequentially into the cell.
Bringing CRISPR/Cas12f1 Complex in Contact with Target Nucleic Acid
In the gene editing method provided herein, editing of a target gene or target nucleic acid in a target cell is performed as an engineered CRISPR/Cas12f1 complex comes in contact with the target gene or target nucleic acid. Accordingly, the gene editing method may comprise bringing the engineered CRISPR/Cas12f1 complex in contact with the target gene or target nucleic acid in the target cell or inducing the engineered CRISPR/Cas12f1 complex to come in contact therewith.
In an embodiment, the gene editing method may comprise bringing an engineered CRISPR/Cas12f1 complex in contact with a target nucleic acid in a target cell. In an embodiment, the gene editing method may comprise inducing an engineered CRISPR/Cas12f1 complex to come in contact with a target nucleic acid in a target cell. Here, the induction method is not particularly limited as long as it allows the engineered CRISPR/Cas12f1 complex to come in contact with the target nucleic acid in the cell. In an embodiment, the induction may be achieved by delivering, into a cell, an engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a Cas12f1 protein or a nucleic acid encoding the same.
As a result of performing the gene editing method provided herein, indel may occur in a target gene or target nucleic acid. Here, the indel may occur inside and/or outside of a target sequence portion and/or a protospacer sequence portion. The indel refers to a mutation caused by deletion of some nucleotides in the nucleotide sequence of the nucleic acid, insertion of any nucleotide thereinto, and/or both of the deletion and the insertion, before the gene editing. In general, when an indel occurs in a target gene or target nucleic acid sequence, the gene or nucleic acid is inactivated. In an embodiment, as a result of performing the gene editing method, deletion and/or addition of one or more nucleotides may occur in the target gene or target nucleic acid.
As a result of performing the gene editing method provided herein, base editing may occur in a target gene or target nucleic acid. Base editing means intentionally altering one or more specific nucleotides in a nucleic acid, unlike the indel caused by deletion or addition of any nucleotide in a target gene or target nucleic acid. In other words, the base editing causes an intended point mutation at a specific location in the target gene or target nucleic acid. In an embodiment, as a result of performing the gene editing method, substitution of one or more nucleotides by other nucleotides may occur in the target gene or target nucleic acid.
As a result of performing the gene editing method provided herein, a knock-in may occur in a target gene or target nucleic acid. The knock-in means inserting an additional nucleic acid sequence into a target gene or target nucleic acid sequence. To cause the knock-in, a donor including the additional nucleic acid sequence is further required in addition to the CRISPR/Cas12f1 complex. When the CRISPR/Cas12f1 complex cleaves a target gene or target nucleic acid in a cell, repair of the cleaved target gene or target nucleic acid occurs. Here, the donor participates in the repair process so that the additional nucleic acid sequence can be inserted into the target gene or target nucleic acid. In an embodiment, the gene editing method may further comprise introducing a donor into a target cell. For example, the donor comprises an exogeneous DNA sequence to be inserted into an intracellular genome, and induces the exogeneous DNA sequence to be inserted into the target gene or target nucleic acid. Here, when delivering the donor into the target cell, the above-described forms of delivery and/or delivery methods may be used.
As a result of performing the gene editing method provided herein, all or a part of a target gene or target nucleic acid sequence may be deleted. The deletion means removing a part of the nucleotide sequence over a certain length or longer in the target gene or target nucleic acid. The deletion refers to an effect capable of completely removing a specific region of a gene, for example, a first exon region, as compared with an effect of the above-described indel.
In an embodiment, the gene editing method comprises introducing, into a target cell including a target gene or target nucleic acid, a Cas12f1 protein or a nucleic acid encoding the same, a first engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a second engineered Cas12f1 guide RNA or a nucleic acid encoding the same. Thus, as a result of the gene editing, removal of a specific sequence portion in the target gene or target nucleic acid occurs.
In an embodiment, the gene editing method may comprise delivering, into a eukaryotic cell, a CRISPR/Cas12f1 complex in a form of a ribonucleoprotein particle in which an engineered Cas12f1 guide RNA and a Cas12f1 protein are bound to each other. Here, the delivery may be achieved by electroporation or lipofection.
In an embodiment, the gene editing method may comprise delivering, into a eukaryotic cell, a nucleic acid encoding an engineered Cas12f1 guide RNA and a nucleic acid encoding a Cas12f1 protein. Here, the delivery may be achieved by electroporation or lipofection.
In an embodiment, the gene editing method may comprise delivering, into a eukaryotic cell, an adeno-associated viral (AAV) vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein.
In an embodiment, the gene editing method may comprise delivering, into a eukaryotic cell, an adeno-associated viral (AAV) vector comprising a nucleic acid sequence encoding a first engineered Cas12f1 guide RNA, a nucleic acid sequence encoding a second engineered Cas12f1 guide RNA, and a nucleic acid sequence encoding a Cas12f1 protein.
An engineered guide RNA for a CRISPR/Cas12f1 system, comprising:
The engineered guide RNA of Example 1, wherein the sequence of the U-rich tail is represented by 5′-UUUURUUUU-3′.
The engineered guide RNA of Example 1, wherein the sequence of the scaffold region is such that the following sequences are sequentially linked to each other in a 5′ to 3′ direction:
The engineered guide RNA of Example 1, wherein the sequence of the scaffold region is such that the following sequences are sequentially linked to each other in a 5′ to 3′ direction:
DNA encoding the engineered guide RNA of any one of Examples 1 to 4.
An engineered guide RNA for a CRISPR/Cas12f1 system, comprising:
The engineered guide RNA of Example 6, wherein the linker is 5′-GAAA-3′.
The engineered guide RNA of Example 6, wherein the linker is selected from the group consisting of 5′-GAAA-3′, 5′-UGAAAA-3′, 5′-UUGAAAAA-3′, 5′-UUCGAAAGAA-3′ (SEQ ID NO: 642), 5′-UUCAGAAAUGAA-3′ (SEQ ID NO: 643), 5′-UUCAUGAAAAUGAA-3′ (SEQ ID NO: 644), and 5′-UUCAUUGAAAAAUGAA-3′ (SEQ ID NO: 645).
The engineered guide RNA of Example 6, wherein the sequence of the engineered scaffold region further comprises a seventh sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-GAA-3′, 5′-AGAA-3′, 5′-GAGAA-3′, 5′-GGAGAA-3′, 5′-UGGAGAA-3′, 5′-GUGGAGAA-3′, 5′-AGUGGAGAA-3′, 5′-AAGUGGAGAA-3′ (SEQ ID NO: 17), 5′-AAAGUGGAGAA-3′ (SEQ ID NO: 18), 5′-UAAAGUGGAGAA-3′ (SEQ ID NO: 19), 5′-AUAAAGUGGAGAA-3′ (SEQ ID NO: 20), 5′-GAUAAAGUGGAGAA-3′ (SEQ ID NO: 21), 5′-UGAUAAAGUGGAGAA-3′ (SEQ ID NO: 22), 5′-CUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 23), 5′-ACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 24), 5′-CACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 25), 5′-UCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 26), 5′-UUCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 27), and 5′-CUUCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 10), and the 3′ end of the seventh sequence is linked to the 5′ end of the first sequence.
The engineered guide RNA of Example 6, wherein the sequence of the U-rich tail is represented by 5′-UUUURUUUU-3′.
The engineered guide RNA of Example 6, wherein a sequence of the engineered scaffold region further comprises an eighth sequence selected from the group consisting of 5′-G-3′, 5′-UUAGG-3′, 5′-CUUAGGG-3′, 5′-CUUAGUGG-3′, 5′-CCUUAGGUGG-3′ (SEQ ID NO: 342), 5′-CCGUUAGGUGG-3′ (SEQ ID NO: 343), 5′-CCGCUUAGGGUGG-3′ (SEQ ID NO: 344), 5′-CCGCUUUAGAGGUGG-3′ (SEQ ID NO: 345), 5′-CCGCUUUUAGAAGGUGG-3′ (SEQ ID NO: 346), 5′-CCGCUUCUUAGGAAGGUGG-3′ (SEQ ID NO: 347), 5′-CCGCUUCAUUAGUGAAGGUGG-3′ (SEQ ID NO: 348), 5′-CCGCUUCACUUAGGUGAAGGUGG-3′ (SEQ ID NO: 349), 5′-CCGCUUCACUUAGAGUGAAGGUGG-3′ (SEQ ID NO: 350), 5′-CCGCUUCACCUUAGGAGUGAAGGUGG-3′ (SEQ ID NO: 351), 5′-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3′ (SEQ ID NO: 352), 5′-CCGCUUCACCAAUUAGUUGAGUGAAGGUGG-3′ (SEQ ID NO: 353), 5′-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3′ (SEQ ID NO: 354), 5′-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 355), 5′-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 356), 5′-CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 357), 5′-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 358), 5′-CCGCUUCACCAAAAGCUGUUAGUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 359), 5′-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 360), 5′-CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 361), 5′-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 362) and 5′-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGU GG-3′ (SEQ ID NO: 11), and the 3′ end of the eighth sequence is linked to the 5′ end of the first sequence.
The engineered guide RNA of Example 6, wherein the sequence of the engineered scaffold region further comprises a seventh sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-GAA-3′, 5′-AGAA-3′, 5′-GAGAA-3′, 5′-GGAGAA-3′, 5′-UGGAGAA-3′, 5′-GUGGAGAA-3′, 5′-AGUGGAGAA-3′, 5′-AAGUGGAGAA-3′ (SEQ ID NO: 17), 5′-AAAGUGGAGAA-3′ (SEQ ID NO: 18), 5′-UAAAGUGGAGAA-3′ (SEQ ID NO: 19), 5′-AUAAAGUGGAGAA-3′ (SEQ ID NO: 20), 5′-GAUAAAGUGGAGAA-3′ (SEQ ID NO: 21), 5′-UGAUAAAGUGGAGAA-3′ (SEQ ID NO: 22), 5′-CUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 23), 5′-ACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 24), 5′-CACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 25), 5′-UCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 26), 5′-UUCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 27), and 5′-CUUCACUGAUAAAGUGGAGAA-3′ (SEQ ID NO: 10), and an eighth sequence selected from the group consisting of 5′-G-3′, 5′-UUAGG-3′, 5′-CUUAGGG-3′, 5′-CUUAGUGG-3′, 5′-CCUUAGGUGG-3′ (SEQ ID NO: 342), 5′-CCGUUAGGUGG-3′ (SEQ ID NO: 343), 5′-CCGCUUAGGGUGG-3′ (SEQ ID NO: 344), 5′-CCGCUUUAGAGGUGG-3′ (SEQ ID NO: 345), 5′-CCGCUUUUAGAAGGUGG-3′ (SEQ ID NO: 346), 5′-CCGCUUCUUAGGAAGGUGG-3′ (SEQ ID NO: 347), 5′-CCGCUUCAUUAGUGAAGGUGG-3′ (SEQ ID NO: 348), 5′-CCGCUUCACUUAGGUGAAGGUGG-3′ (SEQ ID NO: 349), 5′-CCGCUUCACUUAGAGUGAAGGUGG-3′ (SEQ ID NO: 350), 5′-CCGCUUCACCUUAGGAGUGAAGGUGG-3′ (SEQ ID NO: 351), 5′-CCGCUUCACCAUUAGUGAGUGAAGGUGG-3′ (SEQ ID NO: 352), 5′-CCGCUUCACCAAUUAGUUGAGUGAAGGUGG-3′ (SEQ ID NO: 353), 5′-CCGCUUCACCAAAUUAGCUUGAGUGAAGGUGG-3′ (SEQ ID NO: 354), 5′-CCGCUUCACCAAAAUUAGACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 355), 5′-CCGCUUCACCAAAAGUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 356), 5′-CCGCUUCACCAAAAGCUUAGGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 357), 5′-CCGCUUCACCAAAAGCUUUAGAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 358), 5′-CCGCUUCACCAAAAGCUGUUAGUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 359), 5′-CCGCUUCACCAAAAGCUGUUAGUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 360), 5′-CCGCUUCACCAAAAGCUGUUUAGAUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 361), 5′-CCGCUUCACCAAAAGCUGUCUUAGGAUUAGAACUUGAGUGAAGGUGG-3′ (SEQ ID NO: 362), and 5′-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGU GG-3′ (SEQ ID NO: 11), the 3′ end of the eighth sequence is linked to the 5 end of the first sequence, and the 3′ end of the seventh sequence is linked to the 5′ end of the eighth sequence.
The engineered guide RNA of Example 6, wherein the sequence of the engineered scaffold region further comprises a ninth sequence selected from the group consisting of 5′-A-3′, 5′-AA-3′, 5′-AAG-3′, 5′-AAGU-3′, 5′-AAGUG-3′, 5′-AAGUGC-3′, 5′-AAGUGCU-3′, 5′-AAGUGCUU-3′, 5′-AAGUGCUUU-3′, 5′-AAGUGCUUUC-3′ (SEQ ID NO: 485), and a tenth sequence selected from the group consisting of 5′-C-3′, 5′-CC-3′, 5′-ACC-3′, 5′-AACC-3′, 5′-UAACC-3′, 5′-GUAACC-3′, 5′-AGUAACC-3′, 5′-AAGUAACC-3′, 5′-AAAGUAACC-3′, and 5′-GAAAGUAACC-3′ (SEQ ID NO: 486), the 3′ end of the first sequence and the 5′ end of the second sequence are linked by the ninth sequence, and the 3′ end of the second sequence and the 5′ end of the third sequence are linked by the tenth sequence.
The engineered guide RNA of Example 13, wherein
The engineered guide RNA of Example 6, wherein the sequence of the engineered scaffold region further comprises an eleventh sequence selected from the group consisting of 5′-U-3′, 5′-UU-3′, 5′-UUC-3′, 5′-UUCA-3′, 5′-UUCAU-3′, 5′-UUCAUU-3′, and 5′-UUCAUUU-3′, and a twelfth sequence selected from 5′-A-3′, 5′-AA-3′, 5′-GAA-3′, 5′-UGAA-3′, 5′-AUGAA-3′, 5′-AAUGAA-3′, and 5′-GAAUGAA-3′, and the 3′ end of the fourth sequence and the 5′ end of the linker are linked by the eleventh sequence, and the 3′ end of the linker and the 5′ end of the fifth sequence are linked by the twelfth sequence.
The engineered guide RNA of Example 15, wherein
The engineered guide RNA of Example 6, wherein the sequence of the engineered scaffold region further comprises a seventh sequence represented by 5′-A-3′, an eighth sequence represented by 5′-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGU GG-3′ (SEQ ID NO: 11), a ninth sequence represented by 5′-AAGUGCUUUC-3′ (SEQ ID NO: 485), and a tenth sequence represented by 5′-GAAAGUAACC-3′ (SEQ ID NO: 486), the 3′ end of the eighth sequence is linked to the 5′ end of the first sequence, the 3′ end of the seventh sequence is linked to the 5′ end of the eighth sequence, the 3′ end of the first sequence and the 5′ end of the second sequence are linked by the ninth sequence, and the 3′ end of the second sequence and the 5′ end of the third sequence are linked by the tenth sequence.
The engineered guide RNA of Example 6, wherein the sequence of the engineered scaffold region further comprises a seventh sequence represented by 5′-A-3′, an eighth sequence represented by 5′-CCGCUUCACUUAGAGUGAAGGUGG-3′ (SEQ ID NO: 350), a ninth sequence represented by 5′-AAGUGCUUUC-3′ (SEQ ID NO: 485), and a tenth sequence represented by 5′-GAAAGUAACC-3′ (SEQ ID NO: 486), the 3′ end of the eighth sequence is linked to the 5′ end of the first sequence, the 3′ end of the seventh sequence is linked to the 5′ end of the eighth sequence, the 3′ end of the first sequence and the 5′ end of the second sequence are linked by the ninth sequence, and the 3′ end of the second sequence and the 5′ end of the third sequence are linked by the tenth sequence.
DNA encoding the engineered guide RNA of any one of Examples 6 to 18.
An engineered guide RNA for a CRISPR/Cas12f1 system, comprising:
DNA encoding the engineered guide RNA of Example 20.
An engineered CRISPR/Cas12f1 complex capable of editing a target sequence-containing nucleic acid, comprising:
The engineered CRISPR/Cas12f1 complex of Example 22, wherein the sequence of the U-rich tail is represented by 5′-UUUURUUUU-3′.
The engineered CRISPR/Cas12f1 complex of Example 22, wherein the sequence of the scaffold region included in the engineered guide RNA is such that the following sequences are sequentially linked to each other in a 5′ to 3′ direction:
The engineered CRISPR/Cas12f1 complex of Example 22, wherein the sequence of the scaffold region included in the engineered guide RNA is such that the following sequences are sequentially linked to each other in a 5′ to 3′ direction:
An engineered CRISPR/Cas12f1 complex capable of editing a target sequence-containing nucleic acid, comprising:
A vector capable of expressing respective components of a CRISPR/Cas12f1 system, comprising:
The vector of Example 27, wherein the sequence of the scaffold region included in the engineered guide RNA is such that the following sequences are sequentially linked to each other in a 5′ to 3′ direction:
The vector of Example 27, wherein the sequence of the scaffold region included in the engineered guide RNA is such that the following sequences are sequentially linked to each other in a 5′ to 3′ direction:
The vector of Example 27, wherein the second promoter sequence is a U6 promoter sequence.
The vector of Example 27, wherein the vector is at least one selected from the group consisting of a plasmid, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus.
A vector capable of expressing respective components of a CRISPR/Cas12f1 system, comprising:
A method of editing a target sequence-containing nucleic acid in a cell, comprising:
The method of Example 33, wherein the delivery is achieved by introducing, into the cell, the Cas12f1 protein and the engineered guide RNA as a CRISPR/Cas12f1 complex.
The method of Example 33, wherein the delivery is achieved by introducing, into the cell, a vector comprising a nucleic acid encoding the Cas12f1 protein and a nucleic acid encoding the engineered guide RNA.
The vector of Example 35, wherein the vector is at least one selected from the group consisting of a plasmid, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus.
The method of Example 33, wherein the cell is a eukaryotic cell.
A method of editing a target sequence-containing nucleic acid in a cell, comprising:
Hereinafter, the present disclosure will be described in more detail through experimental examples and examples. These examples are only for illustrating the present disclosure, and it would be obvious to those skilled in the art that a scope of the disclosure is not to be construed as being limited by these examples.
A Cas12f1 gene was codon-optimized for expression in human cells, and the optimized sequence was synthesized for vector construction. Finally, to the Cas12f1 protein-encoding sequence were added a chicken p-actin promoter, a nuclear localization signal sequence at the 5′-end and the 3′-end, and a sequence encoding an enhanced green fluorescent protein (eGFP) linked by a self-cleaving T2A peptide. An amino acid sequence of the Cas12f1 protein and a DNA sequence encoding the same are shown in Table 01.
A template DNA encoding a (engineered) Cas12f1 guide RNA was synthesized and cloned into a pTwist Amp plasmid vector (Twist Bioscience). When necessary, the vector was used as a template for amplifying a sequence encoding the guide RNA using a U6-complementary forward primer and a protospacer-complementary reverse primer. Using a Gibson assembly, an oligonucleotide encoding the engineered Cas12f1 guide RNA was cloned into the vector comprising the codon-optimized Cas12f1 gene, so that a vector for an engineered CRISPR/Cas12f1 system was constructed.
Linking a U-rich tail to the 3′ end of the engineered Cas12f1 guide RNA was performed using Pfu PCR Master Mix5 (Biofact) in the presence of a sequence-modified primer and the Cas12f1 guide RNA plasmid vector. The PCR amplicon was purified using a HiGene™ Gel&PCR Purification System (Biofact). Modification of the second region, and the fourth and fifth regions of the engineered scaffold region of the engineered Cas12f1 guide RNA was performed by cloning synthetic oligonucleotides, each of which delivers a modified sequence (Macrogen) into a linearized guide RNA-encoding vector, using Apo I and BamH I restriction enzymes. Modification of the first region of the engineered scaffold region of the engineered Cas12f1 guide RNA was performed by PCR amplification of a canonical or engineered template plasmid vector using a forward primer targeting the 5′ end of the tracrRNA and a reverse primer targeting the U6 promoter region. The PCR amplification was performed using a Q5 Hot Start high-fidelity DNA polymerase (NEB), and ligation of the PCR products was performed using a KLD Enzyme Mix (NEB). The ligated PCR product was transformed into DH5a E. coli cells. Mutagenesis was identified by a Sanger sequencing analysis. The modified plasmid vector was purified using a NucleoBond® Xtra Midi EF kit (MN). 1 microgram of the purified plasmid was used as a template for mRNA synthesis using T7 RNA polymerase (NEB) and NTPs (Jena Bioscience). The engineered Cas12f1 guide RNA prepared above was purified using a Monarch® RNA cleanup kit (NEB), aliquoted into cryogenic vials and stored in liquid nitrogen.
HEK293 T cells (LentX-293T, Takara) were cultured under a condition of 5% of CO2 in Dulbecco's modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Corning) and penicillin/streptomycin. Cell transfection was performed by electroporation or lipofection. For the electroporation, each 2 μg to 5 μg of the plasmid vector encoding the Cas12f1 protein and DNA encoding the guide RNA (or the engineered guide RNA) produced in Experimental Example 1.2 were transfected into 4×105 HEK-293 T cells using a Neon transfection system (Invitrogen). The electroporation was performed under conditions of 1300V, 10 mA, and 3 pulses. For the lipofection, 6 μL to 15 μL of FuGene reagent (Promega) was mixed for 15 minutes with 2 μg to 5 μg of the plasmid vector encoding a Cas12f1 protein and 1.5 μg to 5 μg of the PCR amplicon. The mixture (300 μL) was added to 1.5 ml DMEM medium plated with 1×106 cells 1 day before transfection. The cells were cultured in the presence of the mixture for 1 day to 10 days. After culturing, the cells were collected, and genomic DNA of the cells was manually isolated using a PureHelix™ genomic DNA preparation kit (NanoHelix) or a Maxwell RSC Cultured cells DNA Kit (Promega).
PCR was performed using target-specific primers in the presence of KAPA HiFi HotStart DNA polymerase (Roche) on a region including a protospacer in the genomic DNA isolated from HEK-293 T cells. The amplification was performed following the manufacturer's instructions. The PCR amplicon, which is a resulting product of the amplification and contains Illumina TruSeq HT dual indexes, was subjected to 150-bp paired end sequencing using Illumina iSeq 100. Indel frequencies were calculated using MAUND. The MAUND is provided at https://github.com/ibs-cge/maund.
A guide RNA (or engineered guide RNA) or a genomic DNA was each extracted from HEK293 T cells using an RNeasy Miniprep kit (Qiagen), a Maxwell RSC miRNA Tissue Kit (Promega), or a DNeasy Blood & Tissue Kit (Qiagen). To quantify the guide RNA, ligation of an RNA-specific primer was performed and cDNA was synthesized using a crRNA-specific primer. The cDNA was used as a template for quantitative real-time PCR. The real-time PCR was analyzed using a KAFA SYBR FAST qPCR Master Mix (2×) Kit (KAPAbiosystems).
For each experimental example, the experiment was performed three times, and an average of the respective values was used for analysis.
In order to measure indel efficiency of an engineered CRISPR/Cas12f1 system using an engineered Cas12f1 guide RNA, each of the examples was prepared by Experimental Examples 1.1 to 1.2. The target sequences used for the experiments are shown in Table 02 below.
Sequences of the engineered Cas12f1 guide RNAs used in the respective examples are shown in Table 03 to Table 08.
Here, for each target sequence,
Here, n is 1, 2, or 3 according to the target sequence, wherein a case where n is 1 represents Target 1 (DY2), a case where n is 2 represents Target 2 (DY10), and a case where n is 3 represents Target 3 (Intergenic-22).
The vector constructed in each example was transfected into HEK293 T cells according to Experimental Example 1.3, and indel generation efficiency was measured by Experimental Examples 1.4 to 1.5. The results were analyzed by Experimental Example 1.6 and are shown in
In order to measure indel efficiency of an engineered CRISPR/Cas12f1 system using an engineered Cas12f1 guide RNA, each of the examples was prepared by Experimental Examples 1.1 to 1.2. The target sequences used for the experiments are shown in Table 09 below.
The sequences of the engineered Cas12f1 guide RNAs for the respective examples are shown in Tables 10 to 13 below.
The vector constructed in each Example was transfected into HEK293T cells according to Experimental Example 1.3, and indel generation efficiency was measured by Experimental Examples 1.4 to 1.5. The results were analyzed by Experimental Example 1.6 and are shown in
In order to measure indel efficiency of an engineered CRISPR/Cas12f1 system using an engineered Cas12f1 guide RNA, each of the examples was prepared by Experimental Examples 1.1 to 1.2. The target sequences used for the experiments are as shown in Table 09.
The sequences of the engineered Cas12f1 guide RNAs used for the respective examples are shown in Tables 14 to 17 below.
The vector prepared in each example was transfected into HEK293 T cells according to Experimental Example 1.3, and indel generation efficiency was measured by Experimental Examples 1.4 to 1.5. The results were analyzed by Experimental Example 1.6 and are shown in
The above experimental results were compared with previous experimental data for the same targets (see
In order to measure indel efficiency of an engineered CRISPR/Cas12f1 system using an engineered Cas12f1 guide RNA, each of the examples was prepared by Experimental Examples 1.1 to 1.2. The target sequences used for the experiments are shown in Table 18 below.
The sequences of the engineered Cas12f1 guide RNAs used for the respective examples are shown in Table 19 below.
The vector constructed in each example was transfected into HEK293 T cells according to Experimental Example 1.3, and indel generation efficiency was measured by Experimental Examples 1.4 to 1.5. The results were analyzed by Experimental Example 1.6 and are shown in
From the above experimental results, it is possible to infer that the engineered CRISPR/Cas12f1 system including an engineered Cas12f1 guide RNA having a modified third region has higher gene editing efficiency than a CRISPR/Cas12f1 system having a naturally occurring scaffold region and a CRISPR/Cas12f1 system including a Cas12f1 single guide RNA in which a naturally occurring tracrRNA and a naturally occurring crRNA are linked by a linker.
Experimental Examples 2 to 4 show results obtained by measuring indel efficiency only for a few endogenous targets. To supplement the above results, experiments were conducted to see whether the engineered CRISPR/Cas12f1 system is capable of exerting gene editing activity on a wider range of targets.
1) Endogenous targets having 5′-TTTR-N20-NGG-3′ were searched in silico, and 88 targets were randomly selected (Tables 20 to 22). Each of the targets is a sequence that can be edited with any of Cas9, Cas12a, and Cas12f1, and thus can be used to compare gene editing efficiency of each CRISPR/Cas system.
2) Respective components of a CRISPR/SpCas9 system, a CRISPR/AsCas12a system, a naturally occurring CRISPR/Cas12f1 system, or an engineered CRISPR/Cas12f1 system were transfected into HEK293-T cells. Here, the engineered Cas12f1 guide RNAs used in the engineered CRISPR/Cas12f1 system are summarized in Table 23 below.
Here, the 5′-NNNNNNNNNNNNNNNNNNNN-3′ portion in the above sequences, which is a spacer sequence, was designed as a sequence corresponding to each of the protospacer sequences shown in Tables 20 to 22.
3) After transfection, gene editing efficiency of each system for the 88 targets is shown in
From the experimental results, it can be seen that the engineered CRISPR/Cas12f1 system disclosed herein 1) shows significantly higher gene editing efficiency than a naturally occurring CRISPR/Cas12f1 system, 2) shows gene editing efficiency, which is comparable to the CRISPR/SpCas9 system or the CRISPR/AsCas12a system, for any target in a eukaryotic cell, and 3) shows higher gene editing efficiency than the other CRISPR/Cas systems for some targets.
In order to supplementarily observe gene cleavage pattern of the engineered CRISPR/Cas12f1 system disclosed herein, an in vitro cleavage assay was performed. The target protospacer sequence used in Experimental Example 6 is 5′-TTTAAGAACACATACCCCTGGGCC-3′ (SEQ ID NO: 341, hereinafter Intergenic-22), and a PAM sequence of the target is 5′-TTTA-3′.
The (engineered) Cas12f1 guide RNA used in Experimental Example 6 is shown in Table 25 below.
The experimental method is as follows.
1) Recombinant Cas14 (2.5 μg) and each 2 g of canonical sgRNA, MS2/MS3/MS4 sgRNA, and MS2/MS3/MS4/MS5 sgRNA were incubated to form an RNA complex.
2) An in vitro cleavage assay was performed by allowing the resulting RNA complex to react with a plasmid vector (5 μg) including a target sequence (Intergenic-22) at 37° C. for 3 hours.
3) Cutting with Apa1 restriction enzyme was performed as a positive control.
4) DNA shearing was carried out on the cleavaged DNA under the following conditions using Covaris (M220) that is an ultrasonicator.
Peak incident power: 50 W; duty factor: 20%; cycles per Burst: 200 cpb; treatment time: 110 sec.
5) The fragmented DNA was purified using a DNA purification kit.
6) End-filling of the purified DNA was performed using a T4 ligase.
7) Then, a DNA library was constructed using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB, #E7103) and NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1) (NEB, #E7600) kit.
8) Quantitative polymerase chain reaction (qPCR) was performed to equally adjust a concentration of each sample.
9) 150-bp paired-end sequencing was performed on the constructed library using Illumina iSeq 100.
10) The analyzed sequences were aligned using the integrative genomics viewer (IGV).
The experimental results are shown in
From the experimental results, it was found that the engineered CRISPR/Cas12f1 system including the engineered guide RNA disclosed herein has higher cleavage activity for a non-target strand (NTS) than a naturally occurring CRISPR/Cas12f1 system. This is considered to be a factor affecting improved gene editing activity of the engineered CRISPR/Cas12f1 system disclosed herein.
The present disclosure provides a CRISPR/Cas12f1 system that can be used for gene editing techniques, in particular, an engineered CRISPR/Cas12f1 system with improved gene editing efficiency caused by introduction of a U-rich tail and an engineered scaffold region. When the engineered CRISPR/Cas12f1 system provided herein is used for gene editing, this system exhibits high gene editing efficiency as compared with when a naturally occurring CRISPR/Cas12f1 system is used, and thus can be used for editing a eukaryotic gene.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0129937 | Oct 2020 | KR | national |
| 10-2020-0185528 | Dec 2020 | KR | national |
| 10-2021-0050093 | Apr 2021 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/013898 | 10/8/2021 | WO |
