Patent Application
20240018580
This invention relates to compositions of matter, methods and instruments for improved nucleic acid-guided nickase editing of live cells, particularly mammalian cells.
In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the methods referenced herein do not constitute prior art under the applicable statutory provisions.
The ability to make precise, targeted changes to the genome of living cells has been a long-standing goal in biomedical research and development. Recently various nucleases have been identified that allow manipulation of gene sequence, and hence gene function. The nucleases include nucleic acid guided nucleases, which enable researchers to generate permanent edits in live cells. Of course, it is desirable to attain the highest editing rates possible in a cell population; however, in many instances the percentage of edited cells resulting from nucleic acid-guided nuclease editing can be in the single digits.
There is thus a need in the art of nucleic acid-guided nuclease editing for improved methods, compositions, modules and instruments for increasing the efficiency of editing. The present disclosure addresses this need.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.
The present disclosure relates to methods and compositions for stabilizing gRNAs during nucleic acid-guided nickase editing. With the present compositions and methods, editing efficiency is improved using nucleic acid-guided nickase/reverse transcriptase fusion proteins (e.g., nickase-RT fusion proteins) that retain certain characteristics of nucleic acid-directed nucleases (e.g., the binding specificity and ability to cleave one or more DNA strands in a targeted manner) combined with another enzymatic activity such as reverse transcriptase activity. The nickase-RT fusion enzyme is used with a CF editing cassette (“CREATE fusion editing cassette”) comprising a gRNA and repair template where the CF editing cassette is protected at the 3′ end of the repair template with an RNA stabilization moiety.
Thus, there is provided a CREATE fusion editing cassette for performing nucleic acid-guided nickase/reverse transcriptase fusion editing comprising from 3′ to 5′: 1) an RNA repair template comprising: an RNA stabilization moiety; a linker region; a primer binding region capable of binding to a nicked target DNA; a nick-to-edit region; and a region of post-edit homology; and 2) a gRNA comprising: a guide sequence; and a scaffold region.
In some aspects, the RNA stabilization moiety is a G quadraplex, an RNA hairpin, an RNA pseudoknot or an exoribonuclease resistant RNA. In some aspects, the RNA stabilization moiety is a G quadraplex, and in some aspects, the G quadraplex is selected from SEQ ID No: 1; SEQ ID No: 2; SEQ ID No: 3: SEQ ID No: 4; SEQ ID No: 5; SEQ ID No: 6; SEQ ID No: 7; SEQ ID No: 8; SEQ ID No: 9; SEQ ID No: 10; SEQ ID No: 11; SEQ ID No: 12; SEQ ID No: 13; SEQ ID No: 14; SEQ ID No: 15: SEQ ID No: 16; SEQ ID No: 17; SEQ ID No: 18: SEQ ID No: 19; SEQ ID No: 20; SEQ ID No: 21; SEQ ID No: 22; SEQ ID No: 23; SEQ ID No: 24; SEQ ID No: 25; SEQ ID No: 26; SEQ ID No: 27; SEQ ID No: 28; SEQ ID No: 29; SEQ ID No: 30; SEQ ID No: 31; SEQ ID No: 32; SEQ ID No: 33; SEQ ID No: 34; SEQ ID No: 35; SEQ ID No: 36; SEQ ID No: 37; SEQ ID No: 38; SEQ ID No: 39; SEQ ID No: 40; SEQ ID No: 41: SEQ ID No: 42; SEQ ID No: 43; SEQ ID No: 44; SEQ ID No: 45; SEQ ID No: 46; SEQ ID No: 47; SEQ ID No: 48; and SEQ ID No: 49. In some aspects, the RNA stabilization moiety is an RNA hairpin; and in some aspects, the RNA hairpin selected from SEQ ID No: 50; SEQ ID No: 51; SEQ ID No: 52: SEQ ID No: 53: SEQ ID No: 54: SEQ ID No: 55; SEQ ID No: 65; SEQ ID No: 66; SEQ ID No: 67; SEQ ID No: 68; SEQ ID No: 69; and SEQ ID No: 70. In some aspects, the RNA stabilization moiety is an RNA pseudoknot where the RNA pseudoknot is selected from SEQ ID No: 50; SEQ ID No: 56; SEQ ID No: 57; SEQ ID No: 58; SEQ ID No: 59; SEQ ID No: 60; SEQ ID No: 61; SEQ ID No: 62; SEQ ID No: 63; and SEQ ID No: 64. In some aspects, the RNA stabilization moiety is an exoribonuclease resistant RNA, and in some aspects, the exoribonuclease resistant RNA is selected from SEQ ID No: 71; SEQ ID No: 72; and SEQ ID No: 73.
In some aspects, the CREATE fusion editing cassette has a linker region from 0 to 20 nucleotides in length. In some aspects, the CREATE fusion editing cassette has a primer binding region from 0 to 20 nucleotides in length. In some aspects, the CREATE fusion editing cassette has a nick-to-edit region from 0 to 20 nucleotides in length. In some aspects, the CREATE fusion editing cassette has a region of post-edit homology 3 to 20 nucleotides in length. In some aspects, the CREATE fusion editing cassette has a guide sequence capable of hybridizing to a genomic target locus and a scaffold sequence is capable of interacting or complexing with a nucleic acid-guided nuclease.
These aspects and other features and advantages of the invention are described below in more detail.
The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:
It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.
All of the functionalities described in connection with one embodiment are intended to be applicable to the additional embodiments described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.
The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent procedures can, of course, also be used. Such techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual; Dieffenbach, Dveksler, Eds. (2003). PCR Primer: A Laboratory Manual; Mount (2004), Bioinformatics: Sequence and Genome Analysis; Sambrook and Russell (2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002), Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H. Freeman, New York N.Y.; Gait, “Oligonucleotide Synthesis: A Practical Approach” (1984), IRL Press, London; Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W. H. Freeman Pub., New York, N.Y.; Berg, et al. (2002) Biochemistry, 5th Ed., W.H. Freeman Pub., New York, N.Y.; all of which are herein incorporated in their entirety by reference for all purposes. CRISPR-specific techniques can be found in, e.g., Genome Editing and Engineering from TALENs and CRISPRs to Molecular Surgery, Appasani and Church (2018); and CRISPR: Methods and Protocols, Lindgren and Charpentier (2015); both of which are herein incorporated in their entirety by reference for all purposes.
Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an oligonucleotide” refers to one or more oligonucleotides, and reference to “an automated system” includes reference to equivalent steps and methods for use with the system known to those skilled in the art, and so forth. Additionally, it is to be understood that terms such as “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may be used herein merely describe points of reference and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, methods and cell populations that may be used in connection with the presently described invention.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.
The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′ is 100% complementary to a region of the nucleotide sequence 5′-TAGCTG-3.
The term DNA “control sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these types of control sequences need to be present so long as a selected coding sequence is capable of being replicated, transcribed and-for some components-translated in an appropriate host cell.
The terms “CREATE fusion editing cassette” or “CF editing cassette” refer to a nucleic acid molecule comprising a coding sequence for transcription of a gRNA covalently linked to a coding sequence for transcription of a repair template for use with nickase-RT fusion enzymes. For additional information regarding traditional editing cassettes, e.g., comprising a gRNA and a repair template for use in nucleic acid-guided nuclease systems, see U.S. Pat. Nos. 9,982,278; 10,266,849; 10,240,167; 10,351,877; 10,364,442; 10,435,715; 10,465,207; 10,669,559; 10,771,284; 10,731,498; and 11,078,498, all of which are incorporated by reference herein.
The terms “CREATE fusion editing system” or “CF editing system” refer to the combination of a nucleic acid-guided nickase enzyme/reverse transcriptase fusion protein (“nickase-RT fusion”) and a CREATE fusion editing cassette (“CF editing cassette”) to effect editing in live cells.
The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a guide sequence capable of hybridizing to a genomic target locus, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease.
“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or, more often in the context of the present disclosure, between two nucleic acid molecules. The term “homologous region” refers to a region on the gRNA or repair template with a certain degree of homology with the target DNA sequence. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
As used herein, “nucleic acid-guided nickase/reverse transcriptase fusion” or “nickase-RT fusion” or “nickase-RT fusion enzyme” refer to a nucleic acid-guided nickase or nucleic acid-guided nuclease or CRISPR nuclease that has been engineered to act as a nickase rather than a nuclease that initiates double-stranded DNA breaks, and where the nucleic acid-guided nickase is fused to a reverse transcriptase, which is an enzyme used to generate cDNA from an RNA template. Utilization of a nickase-RT fusion enzyme along with a CF editing cassette incorporates an edit in the DNA target sequence at the RNA level through reverse transcription of the repair template rather than at the DNA level such as through homologous recombination. For information regarding nickase-RT fusions see, e.g., U.S. Pat. No. 10,689,669 and U.S. Ser. No. 16/740,421.
The term “nickase-RT editing components” refers to one or both of a nickase-RT fusion enzyme and a CF editing cassette, where the CF editing cassette may comprise an RNA stabilization moiety (“StCFEC”) or no RNA stabilization moiety.
“Operably linked” refers to an arrangement of elements where the components so described are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the transcription, and in some cases, the translation, of a coding sequence. The control sequences need not be contiguous with the coding sequence so long as they function to direct the expression of the coding sequence. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence. In fact, such sequences need not reside on the same contiguous DNA molecule (i.e. chromosome) and may still have interactions resulting in altered regulation.
A “PAM mutation” refers to one or more edits to a target sequence that removes, mutates, or otherwise renders inactive a PAM (i.e., protospacer adjacent motif) or spacer region in the target sequence.
A “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a polynucleotide or polypeptide coding sequence such as messenger RNA, ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind of RNA. Promoters may be constitutive or inducible. A “pol II promoter” is a regulatory sequence that is bound by RNA polymerase II to catalyze the transcription of DNA.
As used herein the term “repair template” in the context of a CREATE fusion editing system employing a nickase-RT fusion enzyme refers to a nucleic acid (here, a ribonucleic acid) that is designed to serve as a template (including a desired edit) to be incorporated into target DNA via reverse transcriptase.
The term “RNA stability moiety” refers to a moiety, such as those listed infra in Table 1, appended to the 3′ end of the repair template in a CF editing cassette. The term “stabilized CF editing cassette” or “StCFEC” refers to a CF editing cassette comprising an RNA stability moiety at the 3′ end of the repair template.
As used herein the term “selectable marker” refers to a gene introduced into a cell, which confers a trait suitable for artificial selection. General use selectable markers are well-known to those of ordinary skill in the art. Drug selectable markers such as ampicillin/carbenicillin, kanamycin, chloramphenicol, nourscothricin N-acetyl transferase, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and G418 may be employed. In other embodiments, selectable markers include, but are not limited to human nerve growth factor receptor (detected with a MAb, such as described in U.S. Pat. No. 6,365,373); truncated human growth factor receptor (detected with MAb); mutant human dihydrofolate reductase (DHFR; fluorescent MTX substrate available); secreted alkaline phosphatase (SEAP; fluorescent substrate available); human thymidylate synthase (TS; confers resistance to anti-cancer agent fluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1; conjugates glutathione to the stem cell selective alkylator busulfan; chemoprotective selectable marker in CD34+cells); CD24 cell surface antigen in hematopoietic stem cells; human CAD gene to confer resistance to N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1 (MDR-1; P-glycoprotein surface protein selectable by increased drug resistance or enriched by FACS); human CD25 (IL-2α; detectable by Mab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable by carmustine); rhamnose; and Cytidine deaminase (CD; selectable by Ara-C). “Selective medium” as used herein refers to cell growth medium to which has been added a chemical compound or biological moiety that selects for or against selectable markers.
The terms “target DNA sequence”, “target region”, “cellular target sequence”, or “genomic target locus” refer to any locus in vitro or in vivo, or in a nucleic acid (e.g., genome or episome) of a cell or population of cells, in which a change of at least one nucleotide is desired using a nucleic acid-guided nuclease editing system. The cellular target sequence can be a genomic locus or extrachromosomal locus. The target genomic DNA sequence comprises the edit region or edit locus.
A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, BACs, YACs, PACs, synthetic chromosomes, and the like. As used herein, the phrase “engine vector” comprises a coding sequence for a nickase-RT fusion enzyme to be used in the CREATE fusion editing systems and methods of the present disclosure. As used herein the phrase “editing vector” comprises a repair template—including an alteration to the cellular target sequence that prevents nuclease binding at a PAM or spacer in the cellular target sequence after editing has taken place—covalently linked to a coding sequence for a gRNA. The editing vector may also and preferably does comprise a selectable marker and/or a barcode, and/or, as described herein, an RNA stabilization moiety. In some embodiments, the engine vector and editing vector may be combined; that is, all nickase-RT editing components may be found on a single vector. Further, the engine and editing vectors comprise control sequences operably linked to, e.g., the nickase-RT fusion enzyme coding sequence and the CF editing cassette.
The compositions and methods described herein are a “twist on” or alternative to traditional nucleic acid-guided nuclease editing (i.e., RNA-guided nuclease editing or CRISPR editing) used to introduce desired edits to a population of cells; that is, the compositions and methods described herein employ a nucleic acid-guided nickase/reverse transcriptase fusion protein (“nickase-RT fusion”) as opposed to a nucleic acid-guided nuclease. The nickase-RT fusion employed herein differs from traditional CRISPR editing in that instead of initiating double-strand breaks in the target genome, the nickase initiates a nick in a single strand of the target genome. The fusion of the nickase to a reverse transcriptase eliminates the need for a repair template to be incorporated by homologous recombination; instead, the repair template is a nucleic acid—typically a ribonucleic acid—that serves as a template for the reverse transcription portion of the nickase-RT fusion. Utilization of a nickase-RT fusion incorporates the desired edit in the target genome at the RNA level rather than the DNA level. The nickase fused to a reverse transcriptase functions as the single-strand cutter (i.e., nickase)-having the specificity of a nucleic acid-guided nuclease—by first engaging the target DNA, then nicking a strand of the target DNA, followed by the annealing of the 3′ end of the CF editing cassette to the target DNA. The reverse transcriptase then copies the repair template to repair the target DNA thereby incorporating the desired edit into the target DNA. The present methods and compositions are drawn to stabilizing the 3′ end of the CF editing cassette with an RNA stabilization moiety, thereby creating a stabilized CF editing cassette or “StCFEC.”
Traditional nucleic acid-guided nuclease editing begins with a nucleic acid-guided nuclease complexing with an appropriate gRNA in a cell wherein the nucleic acid-guided nuclease can cut the genome of the cell at a desired location. The guide nucleic acid (i.e., gRNA) helps the nucleic acid-guided nuclease recognize and cut the DNA at a specific target sequence. By manipulating the nucleotide sequence of the guide nucleic acid, the nucleic acid-guided nuclease may be programmed to target any DNA sequence for cleavage as long as an appropriate protospacer adjacent motif (PAM) is nearby. In some CRISPR systems, the nucleic acid-guided nuclease editing system uses two separate guide nucleic acid molecules that combine to function as a guide nucleic acid, e.g., a CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In other CRISPR systems, the guide nucleic acid may be a single guide nucleic acid that includes both the crRNA and tracrRNA sequences. In general, a gRNA complexes with a compatible nucleic acid-guided nuclease that can then hybridize with a target sequence, thereby directing the nuclease to the target sequence. The nickase-RT fusions used in the present methods typically retain the PAM- and sequence-specificity of the nucleic acid-guided nucleases from which they are derived and, like nucleic acid-guided nucleases, complex with a gRNA.
A guide nucleic acid or gRNA comprises a guide sequence, where the guide sequence (as opposed to the scaffold sequence portion of the gRNA) is a polynucleotide sequence having sufficient complementarity with a target sequence to hybridize with the target sequence and direct sequence-specific binding of a complexed nucleic acid-guided nuclease to the target sequence. The degree of complementarity between a guide sequence and the corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%. 90%, 95%. 97.5%. 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences. In some embodiments, a guide sequence is about or more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is 10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20 nucleotides in length.
In the present methods and compositions, the gRNAs are provided as mRNAs or as sequences to be expressed from a CF editing cassette, optionally inserted into plasmid or vector and the gRNAs comprise both the guide sequence and the scaffold sequence as a single transcript. The gRNAs are engineered to target a desired target sequence by altering the guide sequence of the gRNA so that the guide sequence is complementary to a desired target DNA sequence, thereby allowing hybridization between the guide sequence and the target sequence. In general, to generate an edit in the target sequence, the gRNA/nickase-RT fusion complex binds to a target sequence as determined by the gRNA, and the nickase portion of the nickase-RT fusion recognizes a protospacer adjacent motif (PAM) sequence adjacent to the target DNA sequence. The target DNA sequence can be any polynucleotide endogenous or exogenous to a prokaryotic or eukaryotic cell, or in vitro. For example, the target DNA sequence can be a polynucleotide residing in the nucleus of a eukaryotic cell. A target DNA sequence can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide, an intron, a PAM, or “junk” DNA).
The gRNA is part of a CF editing cassette that also encodes the repair template which is copied by the reverse transcriptase portion of the nickase-RT fusion into the target DNA sequence.
The target DNA sequence is associated with a protospacer adjacent motif (PAM), which is a short nucleotide sequence recognized by the gRNA/nickase-RT fusion complex. The precise preferred PAM sequence and length requirements for different nucleic acid-guided nucleases vary; however, PAMs typically are 2-7 base-pair sequences adjacent or in proximity to the target sequence and, depending on the nuclease, can be 5′ or 3′ to the target sequence. Engineering of the PAM-interacting domain of a nickase-RT fusion may allow for alteration of PAM specificity, improve target site recognition fidelity, decrease target site recognition fidelity, or increase the versatility of a nickase-RT fusion enzyme.
The range of target DNA sequences that nickase-RT fusion enzymes can recognize is constrained by the need for a specific PAM to be located near the desired target sequence. As a result, it often can be difficult to target edits with the precision that is necessary for genome editing. It has been found that nickase-RT fusion enzymes can recognize some PAMs very well (e.g., canonical PAMs), and other PAMs less well or poorly (e.g., non-canonical PAMs). In certain embodiments and preferably, the editing of a target DNA sequence both introduces a desired DNA change to the cellular target sequence, e.g., the genomic DNA of a cell, and removes, mutates, or renders inactive a protospacer mutation (PAM) region in the cellular target sequence. Rendering the PAM at the cellular target sequence inactive precludes additional editing of the cell genome at that cellular target sequence, e.g., upon subsequent exposure to a nickase-RT fusion complexed with a gRNA in later rounds of editing.
As for the nickase-RT fusion component of the nickase-RT fusion editing system, a polynucleotide sequence encoding the nickase-RT fusion can be codon optimized for expression in particular cell types, such as archaeal, prokaryotic or eukaryotic cells. Eukaryotic cells can be yeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human mammals including non-human primates. The choice of nickase-RT fusion to be employed depends on many factors, such as what type of edit is to be made in the target sequence and whether an appropriate PAM is located close to the desired target sequence. For information of MADzyme nickases, see U.S. Pat. Nos. 10,883,077; 11,053,485; and 11,085,030; and U.S. Ser. No. 17/200,089 and Ser. No. 17/200,110 filed 12 Mar. 2021; Ser. No. 17/463,498, filed 23 Aug. 2021; and Ser. No. 17/463,581, filed 1 Sep. 2021.
In addition to the gRNA and repair template, an editing cassette may comprise and preferably does comprise one or more primer sites used to amplify the CF editing cassette by using oligonucleotide primers; for example, if the primer sites flank one or more of the other components of the CF editing cassette.
In addition, the CF editing cassette may comprise a barcode. A barcode is a unique DNA sequence that corresponds to the repair template sequence such that the barcode can identify the edit made to the corresponding cellular target sequence. The barcode typically comprises four or more nucleotides. In some embodiments, the CF editing cassettes comprise a collection or library of gRNAs and corresponding repair templates representing, e.g., gene-wide or genome-wide libraries of gRNAs and repair templates. The library of CF editing cassettes is cloned into vector backbones where, e.g., each different repair template is associated with a different barcode.
Improved Nucleic Acid-Guided Nickase/Reverse Transcriptase Fusion Editing using 3′ Stabilized Repair Templates
The present disclosure provides compositions of matter, methods and instruments for nucleic acid-guided nickase/reverse transcriptase fusion (“nickase-RT fusion”) editing of live cells using an RNA stabilization moiety at the 3′ end of a CF editing cassette (i.e., an “StCFEC”). With the present compositions and methods, editing efficiency is improved using fusion proteins (i.e., the nickase-RT fusions) that retain certain characteristics of nucleic acid-directed nucleases-the binding specificity and ability to cleave one or more DNA strands in a targeted manner combined with reverse transcriptase activity, which uses a repair template so that a desired edit is incorporated into the target DNA sequence at the RNA level.
A variety of delivery systems may be used to introduce (e.g., transform or transfect) nucleic acid-guided nickase fusion editing system components into a host cell 108. These delivery systems include the use of yeast systems, lipofection systems, microinjection systems, biolistic systems, virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acid conjugates, virions, artificial virions, viral vectors, electroporation, cell permeable peptides, nanoparticles, nanowires, exosomes. Alternatively, molecular trojan horse liposomes may be used to deliver nucleic acid-guided nuclease components across the blood brain barrier. Of particular interest is the use of electroporation, particularly flow-through electroporation (either as a stand-alone instrument or as a module in an automated multi-module system) as described in, e.g., U.S. Pat. Nos. 10,253,316; 10,329,559; 10,323,242; 10,421,959; 10,465,185; 10,519,437; and U.S. Ser. No. 16/666,964. filed 29 Oct. 2019. and Ser. No. 16/680,643, filed 12 Nov. 2019 all of which are herein incorporated by reference in their entirety.
Once transformed 110, the next step in method 100a is to provide conditions for nickase-RT fusion editing 112. “Providing conditions” includes incubation of the cells in appropriate medium and may also include providing conditions to induce transcription of an inducible promoter (e.g., adding antibiotics, increasing temperature) for transcription of one or both of the CF editing cassette and the nickase-RT fusion. Once editing is complete, the cells are allowed to recover and are preferably enriched for cells that have edited 114. Enrichment can be performed directly, such as via cells from the population that express a selectable marker, or by using surrogates, e.g., cell surface handles co-introduced with one or more components of the editing components and using cell sorting, e.g., using FACs (fluorescent activated cell sorting). At this point in method 100a, the cells can be characterized phenotypically or genotypically or optionally steps 110-114 may be repeated to make additional edits 116.
Following transformation 120, the next step in method 100b is to provide conditions for nucleic acid-guided nuclease editing 112. Again, “providing conditions” includes incubating the cells in an appropriate medium and may also include providing conditions to induce transcription of an inducible promoter (e.g., adding antibiotics, increasing temperature) for transcription of one or both of the CF editing cassette and the nickase-RT fusion. Once editing is complete, the cells are allowed to recover and are preferably enriched for cells that have edited 114. Again, enrichment can be performed directly, such as via cells from the population that express a selectable marker, or by using surrogates, e.g., cell surface handles co-introduced with one or more components of the editing components. At this point in method 100b, the cells can be characterized phenotypically or genotypically or optionally steps 118, 120, 112 and 114 may be repeated to make additional edits 122.
The linker region between the RNA stabilization moiety and the primer binding region can vary from 0 to 20 nucleotides, or from 2 to 15 nucleotides, or from 4-10 nucleotides. 5′ of the linker region is the a primer binding region (PBR) which anneals to the genomic target region that is nicked, followed by a nick-to-edit distance of 0 to 10 nucleotides in length and preferably 0 to 5 nucleotides in length. The edit region (edit) is the region of the StCFEC comprising the desired edit, as well as the one or more edits to the target sequence that removes, mutates, or otherwise renders inactive a PAM or spacer region in the target sequence. Following the region comprising the desired edit and the edit to the PAM is the post-edit homology region (PEH), which typically is from 3 to 20 nucleotides in length, or from 3 to 10 nucleotides in length. The post-edit homology region of the repair template optionally is contiguous or nearly contiguous with the guide sequence portion of the gRNA.
In some implementations, the reagent cartridges 210 are disposable kits comprising reagents and cells for use in the automated multi-module cell processing/editing instrument 200. For example, a user may open and position each of the reagent cartridges 210 comprising various desired inserts and reagents within the chassis of the automated multi-module cell editing instrument 200 prior to activating cell processing. Further, each of the reagent cartridges 210 may be inserted into receptacles in the chassis having different temperature zones appropriate for the reagents contained therein.
Also illustrated in
Inserts or components of the reagent cartridges 210, in some implementations, are marked with machine-readable indicia (not shown), such as bar codes, for recognition by the robotic handling system 258. For example, the robotic liquid handling system 258 may scan one or more inserts within each of the reagent cartridges 210 to confirm contents. In other implementations, machine-readable indicia may be marked upon each reagent cartridge 210, and a processing system (not shown, but see element 237 of
Inside the chassis 290, in some implementations, will be most or all of the components described in relation to
A bioreactor may be used to grow cells off-instrument or to allow for cell growth, editing and recovery on-instrument; e.g., as one module of a multi-module fully-automated closed instrument. Further, the bioreactor supports cell selection/enrichment, via expressed antibiotic markers in the growth process or via expressed antibodies coupled to magnetic beads and a magnet associated with the bioreactor. There are many bioreactors known in the art, including those described in, e.g., WO2019/046766; U.S. Pat. Nos. 10,699,519; 10,633,625; 10,577,576; 10,294,447; 10,240,117; 10,179,898; 10,370,629; and 9,175,259; and those available from Lonza Group Ltd. (Basel, Switzerland); Miltenyi Biotec (Bergisch Gladbach, Germany), Terumo BCT (Lakewood, CO, USA) and Sartorius GmbH (Gottingen, Germany).
Bioreactor assembly 300 further comprises bioreactor stand assembly 303 comprising a main body 312 and growth vessel holder 314 comprising a heat jacket or other heating means (not shown) into which the main body 304 of growth vessel 301 is disposed in operation. The main body 304 of growth vessel 301 is biocompatible and preferably transparent—in some embodiments, in the UV and IR range as well as the visible spectrum—so that the growing cells can be visualized by, e.g., cameras or sensors integrated into lid assembly 302 or through viewing apertures or slots 346 in the main body 312 of bioreactor stand assembly 303. Camera mounts are shown at 344.
Bioreactor assembly 300 supports growth of cells from a 500,000 cell input to a 10 billion cell output, or from a 1 million cell input to a 25 billion cell output, or from a 5 million cell input to a 50 billion cell output or combinations of these ranges depending on, e.g., the size of main body 304 of growth vessel 301, the medium used to grow the cells, the type and size and number of microcarriers used for growth (if microcarriers are used), and whether the cells are adherent or non-adherent. The bioreactor that comprises assembly 300 supports growth of both adherent and non-adherent cells, wherein adherent cells are typically grown of microcarriers as described in detail in U.S. Ser. No. 17/237,747, filed 24 Apr. 2021. Alternatively, another option for growing mammalian cells in the bioreactor described herein is growing single cells in suspension using a specialized medium such as that developed by ACCELLTA™ (Haifa, Israel). Cells grown in this medium must be adapted to this process over many cell passages; however, once adapted the cells can be grown to a density of >40 million cells/ml and expanded 50-100× in approximately a week, depending on cell type.
Main body 304 of growth vessel 301 preferably is manufactured by injection molding, as is, in some embodiments, impeller 306 and the impeller shaft 352. Impeller 306 also may be fabricated from stainless steel, metal, plastics or the polymers listed infra. Injection molding allows for flexibility in size and configuration and also allows for, e.g., volume markings to be added to the main body 304 of growth vessel 301. Additionally, material from which the main body 304 of growth vessel 301 is fabricated should be able to be cooled to about 4° C. or lower and heated to about 55° C. or higher to accommodate cell growth. Further, the material that is used to fabricate the vial preferably is able to withstand temperatures up to 55° C. without deformation. Suitable materials for main body 304 of growth vessel 301 include cyclic olefin copolymer (COC), glass, polyvinyl chloride, polyethylene, polyetheretherketone (PEEK), polypropylene, polycarbonate, poly(methyl) methacrylate (PMMA), polysulfone, poly(dimethylsiloxane), cyclo-olefin polymer (COP), and co-polymers of these and other polymers. Preferred materials include polypropylene, polycarbonate, or polystyrene. The material used for fabrication may depend on the cell type to be grown, transfected and edited, and be conducive to growth of both adherent and non-adherent cells and workflows involving microcarrier-based transfection. The main body 304 of growth vessel 301 may be reusable or, alternatively, may be manufactured and configured for a single use. In one embodiment, main body 304 of growth vessel 301 may support cell culture volumes of 25 ml to 500 ml, but may be scaled up to support cell culture volumes of up to 3 L.
The bioreactor stand assembly comprises a stand or frame 350, a main body 312 which holds the growth vessel 301 during operation. The stand/frame 350 and main body 312 are fabricated from stainless steel, other metals, or polymer/plastics. The bioreactor stand assembly main body further comprises a heat jacket (not seen in
The ports shown in vessel lid assembly 302 in this
Additional sensors include those that detect dissolved O2 concentration, dissolved CO2 concentration, culture pH, lactate concentration, glucose concentration, biomass, and optical density. The sensors may use optical (e.g., fluorescence detection), electrochemical, or capacitance sensing and either be reusable or configured and fabricated for single-use. Sensors appropriate for use in the bioreactor are available from Omega Engineering (Norwalk, CT, USA); PreSens Precision Sensing (Regensburg, Germany); C-CIT Sensors AG (Waedenswil, Switzerland), and ABER Instruments Ltd. (Alexandria, VA, USA). In one embodiment, optical density is measured using a reflective optical density sensor to facilitate sterilization, improve dynamic range and simplify mechanical assembly. The rupture disc, if present, provides safety in a pressurized environment, and is programmed to rupture if a threshold pressure is exceeded in growth vessel. If the cell culture in the growth vessel is a culture of adherent cells, microcarriers may be used as described in U.S. Ser. No. 17/237,747, filed 24 Apr. 2021. In such an instance, the liquid-out port may comprise a filter such as a stainless steel or plastic (e.g., polyvinylidene difluoride (PVDF), nylon, polypropylene, polybutylene, acetal, polyethylene, or polyamide) filter or frit to prevent microcarriers from being drawn out of the culture during, e.g., medium exchange, but to allow dead cells to be withdrawn from the vessel. Additionally, a liquid port may comprise a filter sipper to allow cells that have been dissociated from microcarriers to be drawn into the cell corral while leaving spent microcarriers in main body of the growth vessel. The microcarriers used for initial cell growth can be nanoporous (where pore sizes are typically <20 nm in size), microporous (with pores between >20 nm to <1 μm in size), or macroporous (with pores between >1 μm in size, e.g. 20 μm) and the microcarriers are typically 50-200 μm in diameter; thus the pore size of the filter or frit in the liquid-out port will differ depending on microcarrier size.
The microcarriers used for cell growth depend on cell type and desired cell numbers, and typically include a coating of a natural or synthetic extracellular matrix or cell adhesion promoters (e.g., antibodies to cell surface proteins or poly-L-lysine) to promote cell growth and adherence. Microcarriers for cell culture are widely commercially available from, e.g., Millipore Sigma, (St. Louis, MO, USA); ThermoFisher Scientific (Waltham, MA, USA); Pall Corp. (Port Washington, NY, USA); GE Life Sciences (Marlborough, MA, USA); and Corning Life Sciences (Tewkesbury, MA, USA). As for the extracellular matrix, natural matrices include collagen, fibrin and vitronectin (available, e.g., from ESBio, Alameda, CA, USA), and synthetic matrices include MATRIGEL® (Corning Life Sciences, Tewkesbury, MA, USA). GELTREX™ (ThermoFisher Scientific, Waltham, MA, USA), CULTREX® (Trevigen, Gaithersburg, MD, USA), biomemetic hydrogels available from Cellendes (Tubingen, Germany); and tissue-specific extracellular matrices available from Xylyx (Brooklyn, NY, USA); further, denovoMatrix (Dresden, Germany) offers screenMATRIX™, a tool that facilitates rapid testing of a large variety of cell microenvironments (e.g., extracellular matrices) for optimizing growth of the cells of interest.
The cell corral 361, like the main body 304 of growth vessel is fabricated from any biocompatible material such as polycarbonate, cyclic olefin copolymer (COC), glass, polyvinyl chloride, polyethylene, polyetheretherketone (PEEK), polypropylene, poly(methyl methacrylate (PMMA)), polysulfone, poly(dimethylsiloxane), cyclo-olefin polymer (COP), and co-polymers of these and other polymers. Likewise, the end caps are fabricated from a biocompatible material such as polycarbonate, cyclic olefin copolymer (COC), glass, polyvinyl chloride, polyethylene, polyetheretherketone (PEEK), polypropylene, poly(methyl methacrylate (PMMA)), polysulfone, poly(dimethylsiloxane), cyclo-olefin polymer (COP), and co-polymers of these and other polymers. The cell corral may be coupled to or integrated with one or more devices, such as a flow cell where an aliquot of the cell culture can be counted. Additionally, the cell corral may comprise additional liquid ports for adding medium, other reagents, and/or fresh microcarriers to the cells in the cell corral. The volume of the main body 364 of the cell corral 361 may be from 25 to 3000 mL, or from 250 to 1000 mL, or from 450 to 500 mL.
In operation, the bioreactor/cell corral assembly 360 comprising the bioreactor assembly 300 (not shown in this
Once again, the now-spent microcarriers are allowed to settle to the bottom of the growth vessel and the cells are aspirated through a filter sipper into the cell corral 361. The growth vessel is configured to allow for a “dead volume” of 2 mL to 200 mL, or 6 mL to 50 mL, or 8 mL to 12 mL below which the filter sipper does not aspirate medium to ensure the settled spent microcarriers are not transported to the filter sipper during fluid exchanges. Once the cells are aspirated from the bioreactor vessel leaving the “dead volume” of medium and spent microcarriers, the spent microcarriers are aspirated through a non-filter sipper into waste. The spent microcarriers (and the bioreactor vessel) are diluted in phosphobuffered saline or other buffer one or more times, wherein the wash agent and spent microcarriers continue to be aspirated via the non-filter sipper leaving a clean bioreactor vessel. After washing, fresh microcarriers or RBMCs and fresh medium are dispensed into the bioreactor vessel and the cells in the cell corral are dispensed back into the bioreactor vessel for another round of passaging or for transfection and editing, respectively.
In parallel with the off-instrument cell growth, reagent bundle microcarriers (RBMCs) are manufactured, also off-instrument. The present description provides depictions of two exemplary methods where several steps involve manufacturing RBMCs (see
The cells are grown in 3D culture on microcarriers in the bioreactor for, e.g., three to four days or until a desired number of cells, e.g., 1e8, cells are present. Note that all processes in this
In another alternative, the cells may express a fluorescent protein and fluorescence in the cell culture is measured or fluorescent dye may be used to stain cells, particularly live cells. This microcarrier-based workflow can be performed in the bioreactor and cell corral with most if not all steps performed in the same device; thus, several bioreactors and cell corrals may be deployed in parallel for two to many samples simultaneously. In yet another alternative, permittivity or capacitance is used to monitor cell coverage on the microcarriers. In yet another embodiment, an aliquot of cells may be removed from the bioreactor or cell corral and transported out of the instrument and manually counted on a commercial cell counter (i.e., Thermofisher Countess, Waltham, MA, USA).
The microcarriers used for initial cell growth can be nonporous (where pore sizes are typically <20 nm in size), microporous (with pores between >20 nm to <1 μm in size), or macroporous (with pores between >1 μm in size, e.g. 20 μm). In microcarrier culture, cells grow as monolayers on the surface of nonporous or microporous microcarriers, which are typically spherical in morphology; alternatively, the cells grow on the surface and as multilayers in the pores of macroporous microcarriers. The microcarriers preferably have a density slightly greater than that of the culture medium to facilitate easy separation of cells and medium for, e.g., medium exchange and imaging and passaging; yet the density of the microcarriers is also sufficiently low to allow complete suspension of the microcarriers at a minimum stirring or babbling rate. Maintaining a low stirring or bubbling rate is preferred so as to avoid hydrodynamic damage to the cells.
The microcarriers used for cell growth depend on cell type and desired cell numbers, and typically include a coating of a natural or synthetic extracellular matrix or cell adhesion promoters (e.g., antibodies to cell surface proteins or poly-L-lysine) to promote cell growth and adherence. Microcarriers for cell culture are widely commercially available from, e.g., Millipore Sigma, (St. Louis, MO, USA); Thermo Fisher (Waltham, MA, USA); Pall Corp. (Port Washington, NY, USA); GE Life Sciences (Marlborough, MA, USA); and Corning Life Sciences (Tewkesbury, MA, USA). As for the extracellular matrix, natural matrices include collagen, fibrin and vitronectin (available, e.g., from ESBio, Alameda, CA, USA), and synthetic matrices include Matrigel® (Corning Life Sciences, Tewkesbury, MA, USA). Geltrex™ (Thermo Fisher Scientific, Waltham, MA, USA), Cultrex® (Trevigen, Gaithersburg, MD, USA), biomemetic hydrogels available from Cellendes (Tubingen, Germany); and tissue-specific extracellular matrices available from Xylyx (Brooklyn, NY, USA); further, denovoMatrix (Dresden, Germany) offers screenMATRIX™, a tool that facilitates rapid testing of a large variety of cell microenvironments (e.g., extracellular matrices) for optimizing growth of the cells of interest.
Following cell growth, passaging is performed by, e.g., stopping the impeller rotation or bubbling action in the bioreactor and allowing the microcarriers to settle. In one method, the cells are removed from the microcarriers using enzymes such as collagenase, trypsin or pronase, or by non-enzymatic methods including EDTA or other chelating chemicals, and once removed from the carriers, medium is added to dilute the enzyme to inhibit enzymatic action. The dissociation procedures relating to the cell corral are described in detail infra. Once medium is added, then the cells are separated from the microcarriers by allowing the microcarriers to settle and aspirating the cells via a filtered sipper into the cell corral. The cells then may be optionally dissociated from one another via a filter, sieve or by bubbling or other agitation in the cell corral. Next, microcarriers comprising the manufactured reagent bundles (reagent bundle microcarrier microcarriers or RBMCs) and the dissociated cells are combined in an appropriate medium in the growth vessel. Alternatively, instead of removing cells from the cell growth microcarriers and re-seeding on RBMCs, the cells may be transferred from the cell growth microcarriers to RBMCs via microcarrier bridge passaging either in the growth vessel in a reduced volume or in the cell corral. Bridge passaging involves allowing a new microcarrier (e.g. an RBMC) to come into physical contact with a cell-laden microcarrier, such that cells on the latter microcarrier can migrate to the RBMC.
RBMCs are not prepared on-instrument but are pre-manufactured. The microcarriers used for reagent bundles may be microporous microcarriers, which, due to the plethora of micropores, can carry a larger reagent payload per carrier diameter than nonporous or macroporous microcarriers. Preferred microcarriers are microporous, to provide increased surface area for reagent delivery, and functionalized on the surface so as to be able to bind reagents. Preferred microcarriers for RBMCs include Pierce™ Streptavidin UltraLink™ Resin, a cross-linked polyacrylamide carrier functionalized with streptavidin comprising a pore size of 50 to 100 nm; Pierce™ NeutrAvidin™ Plus UltraLink™ Resin, cross-linked polyacrylamide carrier functionalized with avidin comprising a pore size of 50 to 100 nm; and UltraLink™ Hydrazide Resin, a cross-linked polyacrylamide carrier functionalized with hydrazine comprising a pore size of 50 to 100 nm, all available from Thermo Fisher (Waltham, MA, USA); cross-linked agarose resins with alkyne, azide, photo-cleavable azide and disulfide surface functional groups available from Click Chemistry Tools (Scottsdale, AZ, USA); Sepharose™ Resin, cross-linked agarose with amine, carboxyl, carbodiimide, N-hydroxysuccinimide (NHS), and epoxy surface functional groups available from GE Health (Chicago, IL, USA).
The microcarriers are loaded with amplified CF editing cassettes or amplified CF editing plasmids, engine plasmids, nickase-RT fusion enzyme, nickase-RT fusion mRNAs or ribonucleoproteins (RNPs) depending on, e.g., the functionalized group, via, e.g., via chemical or photo linkage or depending on a surface coating on the microcarrier, if present. RBMCs are prepared by 1) partitioning and amplifying a single copy of an editing cassette to produce clonal copies in an RBMC, or by 2) pooling and amplifying editing cassettes, followed by dividing the editing cassettes into sub-pools and “pulling down” the amplified editing cassettes with microcarriers comprising nucleic acids specific to and complementary to unique sequences on the editing cassettes. The step of sub-pooling acts to “de-multiplex” the editing cassette pool, thereby increasing the efficiency and specificity of the “pull down” process. De-multiplexing thus allows for amplification and error correction of the editing cassettes to be performed in bulk followed by efficient loading of clonal copies of the editing cassettes onto a microcarrier.
An alternative exemplary option for the method shown in
As an alternative to the method 400a shown in
At this point, the fully-loaded microcarriers 424 comprising the editing LNPs 406 and the nickase-RT fusion LNPs 420 are added to medium in the bioreactor comprising the mammalian cells 414 to be transfected, optionally with additional lipofect reagent 402. The mammalian cells 414 have been grown and passaged in the bioreactor and cell corral one to many times. The cells 414 populate the fully-loaded RBMCs 424, where the cells 414 then take op (i.e., are transfected by) the editing LNPs 406 and the nickase-RT fusion LNPs 420, a process that may take several hours up to several days. At the end of the transfection process, transfected mammalian cells reside on the surface of the fully-loaded microcarriers 424. In these exemplary methods, nickase-RT fusion mRNAs are used to form the nickase-RT fusion LNPs; however, the nickase-RT enzymes may be loaded on to form LNPs, or CF editing cassettes and nickase-RT fusion enzymes may be loaded in the form of ribonucleoproteins (RNPs) on the LNPs. For additional details on microcarriers and RBMCs, please see U.S. Ser. No. 17/239,540, filed 24 Apr. 2021.
After recovery, the cells may be transferred to a storage module 512, where the cells can be stored at, e.g., 4° C. or −20° C. for later processing, or the cells may be diluted and transferred to a selection/singulation/growth/induction/editing/normalization (or, e.g., SWIIN) module 520. In the SWIIN 520, the cells are arrayed such that there is an average of one to twenty or fifty or so cells per microwell. The arrayed cells may be in selection medium to select for cells that have been transformed or transfected with the editing vector(s). Once singulated, the cells grow through 2-50 doublings and establish colonies. Once colonies are established, editing is induced by providing conditions (e.g., temperature, addition of an inducing or repressing chemical) to induce editing. Editing is then initiated and allowed to proceed, the cells are allowed to grow to terminal size (e.g., normalization of the colonies) in the microwells and then are treated to conditions that care the editing vector from this round. Once cured, the cells can be flushed out of the microwells and pooled, then transferred to the storage (or recovery) unit 512 or can be transferred back to the growth module 504 for another round of editing. In between pooling and transfer to a growth module, there typically is one or more additional steps, such as cell recovery, medium exchange (rendering the cells electrocompetent), cell concentration (typically concurrently with medium exchange by, e.g., filtration).
Note that the selection/singulation/growth/induction/editing/normalization and curing modules may be the same module, where all processes are performed in, e.g., a solid wall device, or selection and/or dilution may take place in a separate vessel before the cells are transferred to the solid wall singulation/growth/induction/editing/normalization/editing module (or e.g., SWIIN) 520. Similarly, the cells may be pooled after normalization, transferred to a separate vessel, and cured in the separate vessel. As an alternative to singulation in, e.g., a solid wall device, the transformed cells may be grown in—and editing can be induced in—bulk liquid (see, e.g., U.S. Ser. No. 16/540,767, filed 14 Aug. 2019 and Ser. No. 16/545,097, filed 20 Aug. 2019). Once the putatively-edited cells are pooled, they may be subjected to another round of editing, beginning with growth, cell concentration and treatment to render electrocompetent, and transformation by yet another repair template in another editing cassette via the electroporation module 508.
In electroporation device 508, the cells selected from the first round of editing are transformed by a second set of editing vectors and the cycle is repeated until the cells have been transformed and edited by a desired number of, e.g., CF editing cassettes. The multi-module cell processing instrument exemplified in
It should be apparent to one of ordinary skill in the art given the present disclosure that the process described may be recursive and multiplexed; that is, cells may go through the workflow described in relation to
In any recursive process, it is advantageous to “cure” the editing vectors comprising the CF editing cassette. “Curing” is a process in which one or more CF editing vectors used in the prior round of editing is eliminated from the transformed cells. Curing can be accomplished by, e.g., cleaving the editing vector(s) using a curing plasmid thereby rendering the editing vectors nonfunctional; diluting the editing vector(s) in the cell population via cell growth (that is, the more growth cycles the cells go through, the fewer daughter cells will retain the editing vector(s)), or by, e.g., utilizing a heat-sensitive origin of replication on the editing vector. The conditions for curing will depend on the mechanism used for curing; that is, in this example, how the curing plasmid cleaves the editing vector. For additional information on curing, see, e.g., U.S. Pat. Nos. 10,837,021 and 11,053,507; and U.S. Ser. No. 17,353,282, filed 21 Jun. 2021; and Ser. No. 17/300,518, filed 27 Jul. 2021.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventor regards as his invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.
A GFP to BFP reporter cell line was created using mammalian cells with a stably integrated genomic copy of the GFP gene (HEK293T-GFP). These cell lines enabled phenotypic detection of genomic edits of different classes (NHEJ, HDR, no edit) by various different mechanisms, including flow cytometry, fluorescent cell imaging, and genotypic detection by sequencing of the genome-integrated GFP gene. Lack of editing, or perfect repair of cut events in the GFP gene, result in cells that remain GFP-positive. Cut events that are repaired by the Non-Homologous End-Joining (NHEJ) pathway often result in nucleotide insertion or deletion events (indels), resulting in frame-shift mutations in the coding sequence that cause loss of GFP gene expression and fluorescence. Cut events that are repaired by the Homology-Directed Repair (HDR) pathway, using the GFP to BFP HDR donor as a repair template, result in conversion of the cell fluorescence profile from that of GFP to that of BFP.
The CREATE fusion editing system is a live cell editing system that uses a nickase-RT fusion protein (e.g., MAD2007 nickase and others, see U.S. Pat. Nos, 10,883,077; 11,053,485; and 11,085,030; and U.S. Ser. No. 17/200,089 and Ser. No. 17/200,110 filed 12 Mar. 2021; Ser. No. 17/463,498. filed 23 Aug. 2021; and Ser. No. 17/463,581. filed 1 Sep. 2021) fused to a peptide with reverse transcriptase activity along with a nucleic acid encoding a gRNA/repair template (i.e., CF editing cassette) comprising a region complementary to a target region of a nucleic acid in one or more cells, which comprises a mutation of at least one nucleotide relative to the target region in the one or more cells and a protospacer adjacent motif (PAM) mutation.
In a first design, a nickase enzyme derived from the MAD2007 nuclease (see U.S. Pat. Nos. 9,982,279 and 10,337,028), e.g., MAD7 nickase (see U.S. Pat. No. 10,883,077), was fused to an engineered reverse transcriptase (RT) on the C-terminus and cloned downstream of a CMV promoter. In this instance, the RT used was derived from Moloney Murine Leukemia Virus (MMLV).
gRNAs and repair templates (CF editing cassettes) were designed that were complementary to a single region proximal to the EGFP-to-BFP editing site. The repair template on the 3′ end included a region of 13 bp comprising the TY-to-SH edit and a second region of 13 bp that was complementary to the nicked EGFP DNA sequence. This allowed the nicked genomic DNA to anneal to the 3′ end of the repair template which can then be extended by the reverse transcriptase to incorporate the edit in the genome. A second gRNA and repair template (CF editing cassette) targeted a region in the EGFP DNA sequence that is 86 bp upstream of the edit site. This CF editing cassette was designed such that it enables the nickase to cut the opposite strand relative to the other CF editing cassette. Both of these CF editing cassettes were cloned downstream of a U6 promoter. A poly-T sequence was also included that terminates the transcription of the CF editing cassette.
The plasmids were transformed into NEB stable E. coli (Ipswich, NY, USA) and grown overnight in 25 mL LB cultures. The following day the plasmids were purified from E. coli using the Qiagen Midi Prep kit (Venlo, Netherlands). The purified plasmid was then RNase A (ThermoFisher, Waltham, MA, USA) treated and re-purified using the DNA Clean and Concentrator kit (Zymo, Irvine, CA, USA).
HEK293T cells were cultured in DMEM medium which was supplemented with 10% FBS and 1× Penicillin and Streptomycin. 100 ng of total DNA (50 ng of gRNA plasmid and 50 ng of CFE plasmids) was mixed with 1 μl of PolyFect (Qiagen, Venlo, Netherlands) in 25 μl of OptiMEM in a 96 well plate. The complex was incubated for 10 minutes and then 20,000 HEK293T cells resuspended in 100 μl of DMEM were added to the mixture. The resulting mixture was then incubated for 80 hours at 37 C and 5% CO2.
The cells were harvested from flat bottom 96 well plates using TrypLE Express reagent (ThermoFisher, Waltham, MA, USA) and transferred to v-bottom 96-well plate. The plate was then spun down at 500 g for 5 minutes. The TrypLE solution was then aspirated and the cell pellet was resuspended in FACS buffer (1×PBS. 1% FBS, 1 mM EDTA and 0.5% BSA). The GFP+, BFP+and RFP+ cells were then analyzed on the Attune NxT flow cytometer and the data was analyzed on FlowJo software.
The RFP+BFP+ cells that were identified were indicative of the proportion of enriched cells that have undergone a precise or imprecise editing process. BFP+ cells indicate cells that have undergone successful editing process and express BFP. The GFP− cells indicate cells that have been imprecisely edited, leading to disruption of the GFP open reading frame and loss of expression.
In this exemplary experiment, the edit is positioned roughly 5′ in the repair template and 3′ of the edit is a region complementary to the nicked genome, although the intended edit could also be present further within the region homologous to the nicked genome. A nickase-RT fusion enzyme (MAD2007 nickase) created a nick in the target site and the nicked DNA annealed to its complementary sequence on the 3′ end of the repair template. The reverse transcriptase portion of the nickase-RT fusion then extended the DNA, thereby incorporating the intended edit directly in the genome.
The effectiveness of the CREATE fusion editing system in GFP+HEK293T cells was then tested. In the assay system devised, a successful precise edit resulted in a BFP+ cell whereas imprecisely edited cells turned the cell both BFP and GFP negative. A CF editing cassette in combination with CFE2.1 or CFE2.2 gave ˜40-45% BFP+ cells indicating that almost half the cell population has undergone successful editing (data not shown), The GFP− cells are ˜10% of the population. The use of a second nicking editing cassette, as described in Liu, et al., Nature, 576 (7785): 149-157 (2019) did not increase the precision edit rate any further; in fact, it significantly increased the imprecisely edited, GFP-negative cell population and the editing rate was lower.
Previous literature has shown that double nicks on opposite strands (<90 bp away) do result in a double strand break which tend to be repaired via NHEJ resulting in imprecise insertions or deletions. Overall, the results indicated that CREATE fusion editing predominantly yielded precisely edited cells and the imprecisely edited cells proportion is much lower (data not shown).
An enrichment handle, specifically a fluorescent reporter (in this case, red fluorescent protein or RFP) linked to nuclease expression was included in this experimentation as a proxy for cells receiving the editing machinery. When only the RFP-positive cells were analyzed (computational enrichment) after 3-4 cell divisions, up to 75% of the cells were BFP+ when tested with CF editing cassettes (data not shown), indicating uptake or expression-linked reporters can be used to enrich for a population of cells with higher rates of CREATE fusion editing system-mediated gene editing. In fact, the combined use of CREATE fusion editing and the described enrichment methods resulted in a significantly improved rate of intended edits (data not shown).
CREATE fusion editing was carried out in mammalian cells using a CF editing cassette having an intended edit to the native sequence and an edit that disrupts nuclease cleavage at this site. Briefly, lentiviral vectors were produced using the following protocol: 1000 ng of Lentiviral transfer plasmid containing the editing cassettes along with 1500 ng of Lentiviral Packaging plasmids (ViraSafe Lentivirus Packaging System Cell BioLabs) were transfected into HEK293T cells using Lipofectamine LTX in 6-well plates. Media containing the lentivirus was collected 72 hrs post transfection. Two clones of a lentiviral CF editing cassette design were chosen, and an empty lentiviral backbone was included as negative control.
The day before the transduction, 200,000 HEK293T cells were seeded in six well plates. Different volumes of CF editing cassette lentivirus (10 to 1000 μl) were added to HEK293T cells in 6-well plates along with 10 μg/ml of Polybrene. 48 hours after transduction, media with 15 μg/ml of Blasticidin was added to the wells. Cells were maintained in selection for one week. Following selection, the well with lowest number of surviving cells was selected for future experiments (<5% cells).
The experimental constructs or wild-type SpCas9 were electroporated into HEK293T cells using the Neon Transfection System (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, 400 ng of total plasmid DNA was mixed with 100,000 cells in Buffer R in a total of 15 μl volume. The 10 μl Neon tip was used to electroporate cells using 2 pulses of 20 ms and 1150 v. Cells were analyzed on the flow cytometer 80 hrs post electroporation. Unenriched editing rates of up to 15% were achieved from single copy delivery of an editing cassette (data not shown).
When the editing was combined with computational selection of RFP+ cells, however, enriched editing rates of up to 30% were achieved from a single copy delivery CF editing cassette. This enrichment via selection of cells receiving the editing machinery was shown to result in a 2-fold increase in precise, complete intended edits (data not shown). Two or more enrichment/delivery steps can also be used to achieve higher editing rates of CREATE fusion editing in an automated instrument, e.g., use of a module for cell handle enrichment and identification of cells having BFP expression. When the method enriched for cells that have higher CF editing cassette expression levels, the editing rate was even further increased, and thus a growth and/or enrichment module of the instrument may include editing cassette enrichment.
While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. § 112, ¶6.
This application claims priority to U.S. Ser. No. 63/122,339 filed 7 Dec. 2020, entitled “gRNA STABILIZATION IN NUCLEIC ACID-GUIDED NICKASE EDITING” which is incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63122339 | Dec 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17538166 | Nov 2021 | US |
| Child | 18460058 | US |
