Patent Application
20240174995
The Sequence Listing associated with the instant disclosure has been electronically submitted to the United States Patent and Trademark Office as a 177 kilobyte ASCII text file created on Jul. 27, 2021 and entitled “1547_35_PCT_US_ST25.txt”. The Sequence Listing submitted via EFS-Web is hereby incorporated by reference in its entirety.
The invention relates to the field of genetic engineering. In particular, the present invention relates to genome editing systems based on C2c1 nuclease and methods. The present invention also relates to artificial guide RNA which can be combined with C2c1 nuclease and used for genome editing
Precise genome editing is a promising area for its bright prospects in gene therapy, especially with the appearance of CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) systems. Up to now, three types of CRISPR-Cas systems, type II Cas9(Cong, L. et al. Science 339, 819-823 (2013); Mali, P. et al. Science 339, 823-826 (2013)), type V-A Cpf13 (Zetsche, B. et al. Cell 163, 759-771 (2015)) and type V-B C2c1, have been successfully harnessed to facilitate mammalian genome engineering. For type II and V CRISPR-Cas systems, the guide RNA and Cas effector protein are the two core components for target DNA recognition and cleavage (Wright, A. V., Nunez, J. K. & Doudna, J. A. Cell 164, 29-44 (2016); Shmakov, S. et al. Nat Rev Microbiol 15, 169-182 (2017)). Previous studies indicated that the RNA and protein components were interchangeable in closely related Cas9 systems (Fonfara, I. et al. Nucleic Acids Res 42, 2577-2590 (2014)) as well as Cpf1 systems (Zetsche, B. et al. Cell 163, 759-771 (2015)), and could be preliminarily optimized (Nishimasu, H. et al. Cell 156, 935-949 (2014); Zalatan, J. G. et al. Cell 160, 339-350 (2015)). Though a number of emerging CRISPR-Cas systems and boosting investigations promote the wide applications of CRISPR-Cas systems (Wright, A. V., Nunez, J. K. & Doudna, J. A. Cell 164, 29-44 (2016); Shmakov, S. et al. Nat Rev Microbiol 15, 169-182 (2017)), there is still little known about how to re-design or even de novo synthesize an enzymatic genome-engineering system.
The Type V-B CRISPR-C2c1 system is an emerging promising technology for genome engineering. However, C2c1, which can be used for mammalian genome editing, is rare, greatly limiting its application. There remains a need in the art for new C2c1 nuclease-based genome editing systems that can be used for mammalian genome editing.
In one aspect, the present invention provides a genome editing system for site-directed modification of a target sequence in the genome of a cell, comprising at least one of the following i) to v):
i) a C2c1 protein or variant thereof, and a guide RNA;
ii) an expression construct comprising a nucleotide sequence encoding a C2c1 protein or variant thereof, and a guide RNA;
iii) a C2c1 protein or variant thereof, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
iv) an expression construct comprising a nucleotide sequence encoding a C2c1 protein or variant thereof, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
v) an expression construct comprising a nucleotide sequence encoding a C2c1 protein or variant thereof and a nucleotide sequence encoding a guide RNA;
wherein the guide RNA is capable of forming complex with the C2c1 protein or variant thereof and targeting the C2c1 protein ortholog or variant thereof to the target sequence in the genome of the cell.
In some embodiments, the C2c1 protein is AaC2c1 protein derived from Alicyclobacillus acidiphilus, AkC2c1 protein derived from Alicyclobacillus kakegawensis, AmC2c1 protein derived from Alicyclobacillus macrosporangiidus, BhC2c1 protein derived from Bacillus hisashii, BsC2c1 protein derived from the genus Bacillus, Bs3C2c1 protein derived from the genus Bacillus, DiC2c1 protein derived from Desulfovibrio inopinatus, LsC2c1 protein derived from Laceyella sediminis, SbC2c1 protein derived from Spirochaetes bacterium, TcC2c1 protein derived from Tuberibacillus calidus. For example, the C2c1 protein is the AaC2c1 protein derived from Alicyclobacillus acidiphilus NBRC 100859, the AkC2c1 protein derived from Alicyclobacillus kakegawensis NBRC 103104, the AmC2c1 protein derived from Alicyclobacillus macrosporangiidus strain DSM 17980, the BhC2c1 protein derived from Bacillus hisashii strain C4, the BsC2c1 protein derived from genus Bacillus NSP2.1, the Bs3C2c1 protein derived from genus Bacillus V3-13 contig_40, the DiC2c1 protein derived from Desulfovibrio inopinatus DSM 10711, the LsC2c1 protein derived from Laceyella sediminis strain RHA1, the SbC2c1 protein derived from Spirochaetes bacterium GWB1_27_13, the TcC2c1 protein derived from Tuberibacillus calidus DSM 17572.
In the second aspect, the present invention provides a method of site-directed modifying a target sequence in the genome of a cell, comprising introducing the genome editing system of the invention into the cell.
In the third aspect, the invention provides a method of treating a disease in a subject in need thereof, comprising delivering to the subject an effective amount of a genome editing system of the invention to modify a gene related with the disease in the subject.
In the fourth aspect, the invention provides the use of a genome editing system of the invention for the preparation of a pharmaceutical composition for treating a disease in a subject in need thereof, wherein the genome editing system is for modifying a gene related with the disease in the subject.
In a fifth aspect, the invention provides a kit for use in the method of the invention, the kit comprising the genome editing system of the invention, and instructions for use.
In the sixth aspect, the invention provides a pharmaceutical composition for treating a disease in a subject in need thereof, comprising a genome editing system of the invention and a pharmaceutically acceptable carrier, wherein the genome editing system is for modifying a gene related with the disease in the subject.
T7EI assay results indicating the genome targeting activity of AaC2c1, DiC2c1 and TcC2c1 directed by AasgRNA (a), AksgRNA (b), AmsgRNA (c), Bs3sgRNA (d) and LssgRNA (e) in the human 293T cells. Red triangles indicate the cleaved bands.
In the present invention, the scientific and technical terms used herein have the meaning as commonly understood by a person skilled in the art unless otherwise specified. Also, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are terms and routine steps that are widely used in the corresponding field. For example, standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following document: Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as “Sambrook”). In the meantime, in order to better understand the present invention, definitions and explanations of related terms are provided below.
In one aspect, the present invention provides a genome editing system for site-directed modification of a target sequence in the genome of a cell, comprising at least one of the following i) to v):
i) a C2c1 protein or variant thereof, and a guide RNA;
ii) an expression construct comprising a nucleotide sequence encoding a C2c1 protein or variant thereof, and a guide RNA;
iii) a C2c1 protein or variant thereof, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
iv) an expression construct comprising a nucleotide sequence encoding a C2c1 protein or variant thereof, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
v) an expression construct comprising a nucleotide sequence encoding a C2c1 protein or variant thereof and a nucleotide sequence encoding a guide RNA;
wherein the guide RNA is capable of forming complex with the C2c1 protein or variant thereof and targeting the C2c1 protein or variant thereof to the target sequence in the genome of the cell. In some embodiments, the targeting results in substitution, deletion and/or addition of one or more nucleotides in the target sequence.
“Genome” as used herein encompasses not only chromosomal DNA present in the nucleus, but also organellar DNA present in the subcellular components (e.g., mitochondria, plastids) of the cell.
“C2c1 nuclease”, “C2c1 protein” and “C2c1” are used interchangeably herein and refer to an RNA-directed nuclease comprising a C2c1 protein or a fragment thereof. C2c1 has a guide RNA-mediated DNA binding activity and DNA cleavage activity, and can target and cleave DNA target sequences to form DNA double-strand breaks (DSBs) under the guidance of a guide RNA. DSB can activate the intracellular intrinsic repair mechanism, non-homologous end joining (NHEJ) and homologous recombination (HR), to repair DNA damage in cells. During the repair process, the specific DNA sequence is subjected to site-directed editing.
In some embodiments, the C2c1 protein is an AaC2c1 protein derived from Alicyclobacillus acidiphilus, an AkC2c1 protein derived from Alicyclobacillus kakegawensis, an AmC2c1 protein derived from Alicyclobacillus macrosporangiidus, a BhC2c1 protein derived from Bacillus hisashii, a BsC2c1 protein derived from the genus Bacillus, a Bs3C2c1 protein derived from the genus Bacillus, a DiC2c1 protein derived from Desulfovibrio inopinatus, a LsC2c1 protein derived from Laceyella sediminis, a SbC2c1 protein derived from Spirochaetes bacterium, a TcC2c1 protein derived from Tuberibacillus calidus.
For example, the C2c1 protein is the AaC2c1 protein derived from Alicyclobacillus acidiphilus NBRC 100859, the AkC2c1 protein derived from Alicyclobacillus kakegawensis NBRC 103104, the AmC2c1 protein derived from Alicyclobacillus macrosporangiidus strain DSM 17980, the BhC2c1 protein derived from Bacillus hisashii strain C4, and the BsC2c1l protein derived from genus Bacillus NSP2.1, the Bs3C2c1 protein derived from Bacillus genus V3-13 contig_40, the DiC2c1 protein derived from Desulfovibrio inopinatus DSM 10711, the LsC2c1 protein derived from Laceyella sediminis strain RHA1, the SbC2c1 protein derived from Spirochaetes bacterium GWB1_27_13, the TcC2c1 protein derived from Tuberibacillus calidus DSM 17572.
In some embodiments of the invention, the C2c1 protein is a C2c1 protein whose natural locus does not have a CRISPR array. In some embodiments, the C2c1 protein whose natural locus does not have a CRISPR array is a DiC2c1 or TcC2c1 protein.
In some embodiments, the C2c1 protein comprises the amino acid sequence set forth in any one of SEQ ID NOs:1-10. For example, the AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, and TcC2c1 proteins comprise the amino acid sequences set forth in SEQ ID NOs: 1-10, respectively.
In some embodiments, the variant of the C2c1 protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with a wild-type C2c1 protein (e.g., wild-type AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, or TcC2c1 protein), respectively, and has genomic editing and/or targeting activity of a wild-type C2c1 protein (e.g., wild-type AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 protein), respectively.
In some embodiments, the variant of the C2c1 protein comprises an amino acid sequence in which one or more amino acid residues are substituted, deleted or added relative to a wild-type C2c1 protein (e.g., wild-type AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 protein), respectively, and has genome editing and/or targeting activity of wild-type C2c1 proteins (e.g., wild-type AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 protein). For example, the variant of the C2c1 protein comprises an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid residue(s) are substituted, deleted or added relative to a wild-type C2c1 protein (e.g., wild-type AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 proteins), respectively. In some embodiments, the amino acid substitution is a conservative substitution.
“Polypeptide,” “peptide,” and “protein” are used interchangeably in the present invention to refer to a polymer of amino acid residues. The terms apply to an amino acid polymer in which one or more amino acid residues is artificial chemical analogue of corresponding naturally occurring amino acid(s), as well as to a naturally occurring amino acid polymer. The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” may also include modified forms including, but not limited to, glycosylation, lipid ligation, sulfation, Y carboxylation of glutamic acid residues, and ADP-ribosylation.
Sequence “identity” has recognized meaning in the art, and the percentage of sequence identity between two nucleic acids or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. (See, for example, Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, 30) G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term “identity” is well known to the skilled person (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).
Suitable conservative amino acid substitution in peptides or proteins are known to those skilled in the art and can generally be carried out without altering the biological activity of the resulting molecule. In general, one skilled in the art recognizes that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (See, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p.224).
In some embodiments, the variant of the C2c1 protein encompasses a nuclease-dead C2c1 protein (dC2c1). The nuclease-dead C2c1 protein refers to a C2c1 protein that retains the RNA-mediated DNA-binding activity but does not have DNA cleavage activity. In some embodiments, the nuclease-dead C2c1 protein encompasses a C2c1 nickase that cleaves only one strand of double-stranded target DNA.
In some embodiments, the variant of the C2c1 protein is a fusion protein of dC2c1 and a deaminase. For example, dC2c1 and deaminase in the fusion protein can be linked by a linker such as a peptide linker.
As used herein, “deaminase” refers to an enzyme that catalyzes a deamination reaction. In some embodiments of the invention, the deaminase refers to a cytosine deaminase capable of accepting single-stranded DNA as a substrate and capable of catalyzing the deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments of the invention, the deaminase refers to adenine deaminase capable of accepting single-stranded DNA as a substrate and capable of catalyzing adenosine or deoxyadenosine (A) into inosine (I). Base editing in the target DNA sequence, such as C to T conversion or A to G conversion, can be achieved by using a fusion protein of a C2c1 variant and a deaminase.
In some embodiments of the present invention, the C2c1 protein or variant thereof of the present invention further comprises a nuclear localization sequence (NLS). In general, one or more NLSs in the C2c1 protein or variant thereof should be of sufficient strength to drive the C2c1 protein or variant thereof in the nucleus of a plant cell to achieve an amount accumulation of base editing function. In general, the intensity of nuclear localization activity is determined by the number, location, one or more specific NLSs used of the NLS in the C2c1 protein or variant thereof, or a combination of these factors.
In some embodiments of the present invention, the NLS of the C2c1 protein or variant thereof of the present invention may be located at the N-terminus and/or C-terminus. In some embodiments, the C2c1 protein or variant thereof comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the C2c1 protein or variant thereof comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the N-terminus. In some embodiments, the C2c1 protein or variant thereof comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the C-terminus. In some embodiments, the C2c1 protein or variant thereof comprises a combination of these, such as comprises one or more NLSs at the N-terminus and one or more NLSs at the C-terminus. When there is more than one NLS, each can be selected to be independent of other NLSs. In some preferred embodiments of the present invention, the C2c1 protein or variant thereof comprises two NLSs, for example, the two NLSs are located at the N-terminus and the C-terminus, respectively.
In general, NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the surface of the protein, but other types of NLS are also known. Non-limiting examples of NLS include: KKRKV (SEQ ID NO: 81), PKKKRKV (SEQ ID NO: 82), or SGGSPKKKRKV (SEQ ID NO: 83).
Furthermore, depending on the location of the DNA to be edited, the C2c1 protein or variant thereof of the present invention may also include other localization sequences, such as cytoplasmic localization sequences, chloroplast localization sequences, mitochondrial localization sequences, and the like.
In some embodiments of the invention, the target sequence is 18-35 nucleotides in length, preferably 20 nucleotides. In some embodiments of the invention, the 5′-end flanking of the target sequence is a protospacer adjacent motif (PAM) sequence selected from 5′TTTN-3′, 5′ATTN-3′, 5′GTTN-3′, 5′CTTN-3′, 5′TTC-3′, 5′TTG-340 , 5′TTA-3′, 5′TTT-3′, 5′TAN-3′, 5′TGN-3′, 5′TCN-3′ and 5′ATC-3′, wherein N is selected from A, G, C and T.
In the present invention, the target sequence to be modified may be located at any location in the genome, for example, in a functional gene such as a protein-encoding gene, or may be, for example, located in a gene expression regulatory region such as a promoter region or an enhancer region, thereby the gene functional modification or gene expression modification can be achieved. The substitution, deletion and/or addition in the target sequence of the genome can be detected by T7EI, PCR/RE or sequencing methods.
“guide RNA” and “gRNA” can be used interchangeably herein, typically composed of crRNA and tracrRNA molecules that are partially complementary to each other to form a complex, wherein the crRNA comprises a sequence that is sufficiently identical to the target sequence to hybridize to the complement of the target sequence and direct the CRISPR complex (C2c1+crRNA+tracrRNA) to sequence specifically bind to the target sequence. However, a single guide RNA (sgRNA) containing both crRNA and tracrRNA characteristics can be designed and used.
In some embodiments of the invention, the guide RNA is sgRNA. In some particular embodiments, the sgRNA is encoded by a nucleotide sequence selected from the group consisting of:
wherein Nx represents nucleotide sequence that consists of X consecutive nucleotides (spacer sequence), N is independently selected from A, G, C and T; X is an integer of 18X
35, preferably, X=20. In some embodiments, the sequence Nx (spacer sequence) is capable of specifically hybridizing to the complement of the target sequence. The sequence other than Nx in the sgRNA is a scaffold sequence of sgRNA. In some embodiments, the sgRNA comprises a scaffold sequence encoded by the nucleotide sequence of any one of SEQ ID NOs: 31-38.
The present inventors have surprisingly found that the C2c1 protein and the guide RNA in different C2c1 systems can be used interchangeably, thereby enabling the artificial design of universal guide RNAs.
Thus, in some embodiments, the invention provides an artificial sgRNA, which selected from the nucleotide sequence encoding:
wherein Nx represents nucleotide sequence that consists of X consecutive nucleotides(spacer sequence), N is independently selected from A, G, C and T; X is an integer of 18X
35, preferably, X=20. In some embodiments, the sequence Nx (spacer sequence) is capable of specifically hybridizing to the complement of the target sequence. The sequence other than Nx in the sgRNA is a scaffold sequence of sgRNA.
In some embodiments, the artificial sgRNA comprises a scaffold sequence encoded by the nucleotide sequence of any one of SEQ ID NOs:39-75.
In some embodiments, the guide RNA in the genome editing system of the invention is an artificial sgRNA of the invention.
To achieve efficient expression in target cells, in some embodiments of the invention, the nucleotide sequence encoding a C2c1 protein or variant thereof is codon optimized for the organism from which the cell to be genome edited is derived.
Codon optimization refers to the replacement of at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of a native sequence by a codon that is used more frequently or most frequently in the gene of the host cell, modifying the nucleic acid sequence while maintaining the native amino acid sequence to enhance expression in the host cell of interest. Different species show specific preferences for certain codons of a particular amino acid. Codon preference (difference in codon usage between organisms) is often associated with the efficiency of translation of messenger RNA (mRNA), which is believed to depend on the nature of the translated codon and the availability of specific transfer RNA (tRNA) molecules. The advantages of selected tRNAs within cells generally reflect the most frequently used codons for peptide synthesis. Therefore, genes can be customized to be best gene expressed in a given organism based on codon optimization. The codon usage table can be easily obtained, for example, in the Kazusa Codon Usage Database available on the World Wide Web, and these tables can be adjusted in different ways. See, Nakamura Y. et. al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000 Nucl. Acids Res, 28: 292 (2000).
The organism from which the cell can be genome-edited by the system of the present invention preferably is eukaryote, including but not limited to mammals such as human, mouse, rat, monkey, dog, pig, sheep, cattle, cat; poultry such as chicken, duck, goose; plants including monocots and dicots, such as rice, corn, wheat, sorghum, barley, soybean, peanut and Arabidopsis thaliana and so on.
In some embodiments of the invention, the nucleotide sequence encoding a C2c1 protein or variant thereof is codon optimized for human. In some embodiments, the codon-optimized nucleotide sequence encoding AaC2c1, AkC2c1, AmC2c1, BhC2c1, BsC2c1, Bs3C2c1, DiC2c1, LsC2c1, SbC2c1, TcC2c1 protein is selected from SEQ ID NOs: 11-20, respectively.
In some embodiments of the present invention, the nucleotide sequence encoding the C2c1 protein or variant thereof and/or the nucleotide sequence encoding the guide RNA is operably linked to an expression regulatory element such as a promoter.
As used in the present invention, “expression construct” refers to a vector such as a recombinant vector that is suitable for expression of a nucleotide sequence of interest in an organism. “Expression” refers to the production of a functional product. For example, expression of a nucleotide sequence may refer to the transcription of a nucleotide sequence (e.g., transcription to produce mRNA or functional RNA) and/or the translation of RNA into a precursor or mature protein. The “expression construct” of the present invention may be a linear nucleic acid fragment, a circular plasmid, a viral vector or, in some embodiments, an RNA that is capable of translation (such as mRNA).
The “expression construct” of the present invention may comprise regulatory sequences and nucleotide sequences of interest from different origins, or regulatory sequences and nucleotide sequences of interest from the same source but arranged in a manner different from that normally occurring in nature.
“Regulatory sequence” and “regulatory element” are used interchangeably to refer to a nucleotide sequence that is located upstream (5′ non-coding sequence), middle or downstream (3′ non-coding sequence) of a coding sequence and affects the transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leaders, introns and polyadenylation recognition sequences.
“Promoter” refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present invention, the promoter is a promoter capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell. The promoter may be a constitutive promoter or tissue-specific promoter or developmentally-regulated promoter or inducible promoter.
“Constitutive promoter” refers to a promoter that may in general cause the gene to be expressed in most cases in most cell types. “Tissue-specific promoter” and “tissue-preferred promoter” are used interchangeably and mean that they are expressed primarily but not necessarily exclusively in one tissue or organ, but also in a specific cell or cell type. “Developmentally-regulated promoter” refers to a promoter whose activity is dictated by developmental events. “Inducible promoter” selectively expresses operably-linked DNA sequences in response to an endogenous or exogenous stimulus (environment, hormones, chemical signals, etc.).
As used herein, the term “operably linked” refers to the linkage of a regulatory element (e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.
Examples of promoters that can be used in the present invention include, but are not limited to, the polymerase (pol) I, pol II or pol III promoters. Examples of the pol I promoter include the gallus RNA pol I promoter. Examples of the pol II promoters include, but are not limited to, the immediate-early cytomegalovirus (CMV) promoter, the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter, and the immediate-early simian virus 40 (SV40) promoter. Examples of pol III promoters include the U6 and H1 promoters. An inducible promoter such as a metallothionein promoter can be used. Other examples of promoters include the T7 phage promoter, the T3 phage promoter, the β-galactosidase promoter, and the Sp6 phage promoter, and the like. Promoters that can be used in plants include, but are not limited to, cauliflower mosaic virus 35S promoter, maize Ubi-1 promoter, wheat U6 promoter, rice U3 promoter, maize U3 promoter, rice actin promoter.
The cell that can be edited by the method of the present invention preferably is an eukaryotic cell, including but not limited to a mammalian cell such as a cell of human, mouse, rat, monkey, dog, pig, sheep, cattle, cat; a cell of poultry such as chicken, duck, goose; a cell of plants including monocots and dicots, such as rice, corn, wheat, sorghum, barley, soybean, peanut and Arabidopsis thaliana and so on. In some embodiments of the invention, the cell is a eukaryotic cell, preferably a mammalian cell, more preferably a human cell.
In another aspect, the present invention provides a method of modifying the target sequence in cell genome, comprising introducing the genome editing system of the invention into the cell, whereby the guide RNA targets the C2c1 protein or variant thereof to a target sequence in the genome of the cell. In some embodiments, the targeting results in one or more nucleotides being substituted, deleted and/or added in the target sequence.
“Introduction” of a nucleic acid molecule (e.g., plasmid, linear nucleic acid fragment,
RNA, etc.) or protein of the invention into a cell means that the nucleic acid or protein is used to transform a cell such that the nucleic acid or protein is capable of functioning in the cell. As used in the present invention, “transformation” includes both stable and transient transformations. “Stable transformation” refers to the introduction of exogenous nucleotide sequence into the genome, resulting in the stable inheritance of foreign sequence. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any of its successive generations. “Transient transformation” refers to the introduction of a nucleic acid molecule or protein into a cell, executing its function without the stable inheritance of an exogenous sequence. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.
Methods that can be used to introduce the genome editing system of the present invention into a cell include, but are not limited to, calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, viral infection (e.g., baculovirus, vaccinia virus, adenovirus, adeno-associated virus, lentivirus and other viruses), gene gun method, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation.
In some embodiments, the method of the invention is performed in vitro. For example, the cell is an isolated cell. In some embodiments, the cell is a CAR-T cell. In some embodiments, the cell is an induced embryonic stem cell.
In other embodiments, the method of the invention may also be performed in vivo. For example, the cell is a cell within an organism, and the system of the present invention can be introduced into the cell in vivo by, for example, a virus-mediated method. For example, the cell can be a tumor cell within a patient.
In another aspect, the present invention provides a method of producing a genetically modified cell, comprising introducing the genome editing system of the present invention into a cell, whereby the guide RNA targets the C2c1 protein or variant thereof to a target sequence in the genome of the cell. In some embodiments, the targeting results in one or more nucleotides being substituted, deleted and/or added in the target sequence.
In another aspect, the invention also provides a genetically modified organism comprising a genetically modified cell produced by the methods of the invention or a progeny cell thereof.
As used herein, “organism” includes any organism that is suitable for genome editing, eukaryotes are preferred. Examples of the organism include, but are not limited to, mammals such as human, mouse, rat, monkey, dog, pig, sheep, cattle, cat; poultry such as chicken, duck, goose; plants including monocots and dicots such as rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis and the like. In some embodiments of the invention, the organism is eukaryote, preferably a mammal, and more preferably a human.
A “genetically modified organism” or “genetically modified cell” includes the organism or the cell which comprises within its genome an exogenous polynucleotide or a modified gene or modified expression regulatory sequence. For example, the exogenous polynucleotide is stably integrated within the genome of the organism or the cell such that the polynucleotide is passed on to successive generations. The exogenous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. The modified gene or modified expression regulatory sequence means that, in the genome of the organism or the cell, said sequence comprises one or more nucleotide substitution, deletion, or addition. “Exogenous” in reference to a sequence means a sequence from a foreign species, or refers to a sequence in which significant changes in composition and/or locus occur from its native form through deliberate human intervention if from the same species.
In another aspect, the invention provides a gene expression regulatory system based on a nuclease-dead C2c1 protein of the invention. This system, although not changing the sequence of the target gene, is also defined as a genome editing system within the scope herein.
In some embodiments, the gene expression regulation system is a gene suppressing or silencing system, comprising one of the following:
i) a nuclease-dead C2c1 protein or a fusion protein of the nuclease-dead C2c1 protein and a transcriptional repressor protein, and a guide RNA;
ii) an expression construct comprising a nucleotide sequence encoding a nuclease-dead C2c1 protein or a fusion protein of the nuclease-dead C2c1 protein and a transcriptional repressor protein, and a guide RNA;
iii) a nuclease-dead C2c1 protein or a fusion protein of the nuclease-dead C2c1 protein and a transcriptional repressor protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
iv) an expression construct comprising a nucleotide sequence encoding a nuclease-dead C2c1 protein or a fusion protein of the nuclease-dead C2c1 protein and a transcriptional repressor protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA; or
v) an expression construct comprising a nucleotide sequence encoding a nuclease-dead C2c1 protein or a fusion protein of the nuclease-dead C2c1 protein and a transcriptional repressor protein and a nucleotide sequence encoding a guide RNA.
The definition of the nuclease-dead C2c1 protein or the guide RNA is as described above. Selection of the transcriptional repressor protein is within the skill of those ordinary people in the art.
As used herein, gene suppression or silencing refers to the down-regulation or elimination of gene expression, preferably at the transcriptional level.
However, the gene expression regulatory system of the present invention can also use a fusion protein of a nuclease-dead C2c1 protein and a transcriptional activator protein. In this case, the gene expression regulatory system is a gene expression activation system. For example, the gene expression activation system of the present invention may comprise one of the following:
i) a fusion protein of a nuclease-dead C2c1 protein and a transcriptional activator protein, and a guide RNA;
ii) an expression construct comprising a nucleotide sequence encoding a fusion protein of a nuclease-dead C2c1 protein and a transcriptional activator protein, and a guide RNA;
iii) a fusion protein of a nuclease-dead C2c1 protein and a transcriptional activator protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA;
iv) an expression construct comprising a nucleotide sequence encoding a fusion protein of a nuclease-dead C2c1 protein and a transcriptional activator protein, and an expression construct comprising a nucleotide sequence encoding a guide RNA; or
v) an expression construct comprising a nucleotide sequence encoding a fusion protein of a nuclease-dead C2c1 protein and a transcriptional activator protein and a nucleotide sequence encoding a guide RNA.
The definition of the nuclease-dead C2c1 protein or the guide RNA is as described above. Selection of the transcriptional activator protein is within the skill of those ordinary people in the art.
As used herein, gene activation refers to up-regulation of gene expression levels, preferably at the transcriptional level.
In another aspect, the invention also encompasses the use of the genome editing system of the invention in the treatment of diseases.
By modifying a disease-related gene by the genome editing system of the present invention, it is possible to achieve up-regulation, down-regulation, inactivation, activation, or mutation correction of the disease-related gene, thereby achieving prevention and/or treatment of the disease. For example, in the present invention, the target sequence may be located in the protein coding region of the disease-related gene, or may be, for example, located in a gene expression regulatory region such as a promoter region or an enhancer region, thereby enabling functional modification of the disease-related gene or modification of the expression of the disease-related gene.
A “disease-related” gene refers to any gene that produces a transcriptional or translational product at an abnormal level or in an abnormal form in a cell derived from a disease-affected tissue as compared to a tissue or cell of non-disease control. When altered expression is related with the appearance and/or progression of a disease, it may be a gene that is expressed at an abnormally high level or it may be a gene that is expressed at an abnormally low level. A disease-related gene also refers to a gene having one or more mutations or a genetic variation that is directly responsible for or has genetic linkage with one or more genes responsible for the etiology of the disease. The transcribed or translated product may be known or unknown and may be at normal or abnormal levels.
Accordingly, in another aspect, the invention also provides a method of treating a disease in a subject in need thereof, comprising delivering to the subject an effective amount of a genome editing system of the invention to modify a gene related to the disease.
In another aspect, the invention also provides the use of a genome editing system of the invention for the preparation of a pharmaceutical composition for treating a disease in a subject in need thereof, wherein the genome editing system is for modifying a gene related to the disease.
In another aspect, the invention also provides a pharmaceutical composition for treating a disease in a subject in need thereof, comprising a genome editing system of the invention and a pharmaceutically acceptable carrier, wherein the genome editing system is for modifying a gene related to the disease.
In some embodiments, the subject is a mammal, such as a human.
Examples of such diseases include, but are not limited to, tumors, inflammation, Parkinson's disease, cardiovascular disease, Alzheimer's disease, autism, drug addiction, age-related macular degeneration, schizophrenia, hereditary diseases, and the like.
In another aspect, the invention also includes a kit for use in the methods of the invention, the kit comprising the genome editing system of the invention, and an instruction. The kits generally include a label indicating the intended use and/or method of use of the contents in the kit. The term label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
In order to facilitate the understanding of the present invention, the present invention will be described more fully hereinafter with reference to the specific examples and the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the present disclosure will be more fully understood.
DNA manipulation including DNA preparation, digestion, ligation, amplification, synthesis, purification, agarose gel electrophoresis, etc. were conducted according to Molecular Cloning: A Laboratory Manual with some modifications. Briefly, targeting chimeric single guide RNA (sgRNA) scaffolds for cell transfection assay were constructed by ligation annealed oligonucleotides (oligos) (Table 1) into BsaI-digested pUC19-U6-sgRNA (SEQ ID NO:23-30) vectors.
PSI-BLAST program (Altschul, S. F. et al. Nucleic Acids Res 25, 3389-3402 (1997)) was adopted to identify new CRISPR-C2c1 orthologs. Their coding sequences were humanized (Grote, A. et al., Nucleic Acids Res 33, W526-531 (2005)) and oligos for C2c1 gene and sgRNA synthesis were designed using GeneDesign program (Richardson, S. M. et al., Genome Res 16, 550-556 (2006)). Each C2c1 gene was synthesized according to article (Li, G. et al., Methods Mol Biol 1073, 9-17 (2013)). Purified products were assembled into expression vectors via homologous recombination in vitro using NEBuilder® HiFi DNA Assembly Master Mix (NEB). The pCAG-2AeGFP (SEQ ID NO:21) and pUC19-U6 vectors (SEQ ID NO:22) were applied for mammalian expression of C2c1 proteins and sgRNAs, respectively.
Human embryonic kidney 293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% Antibiotic-Antimycotic (Gibco) at 37° C. with 5% CO2 incubation. 293T cells were transfected using Lipofectamine LTX (Invitrogen) following the manufacturer's recommended protocol. For each well of a 48-well plate, a total of 400 ng plasmid (C2c1: sgRNA=2:1) were used. Then 48 h following transfection, cells were harvested directly for genomic DNA extraction.
Harvested cells were lysed directly with Buffer L (Bimake) supplemented with Protease K and incubated at 55° C. for 3 h and inactivated at 95° C. for 10 min. Genomic region surrounding the C2c1 target site for each gene was PCR-amplified (Supplementary Table 5). 200˜400 ng PCR products were mixed with ddH2O to a final volume of 10 μL, and subjected to re-annealing process to enable heteroduplex formation according to previous methods. After re-annealing, products were treated with 1/10 volume of NEBuffer™M 2.1 and 0.2 μL T7EI (NEB) at 37° C.for 30 min, and analyzed on 3% agarose gels. Indels were quantitated based on relative band intensities (Cong, L. et al., Science 339, 819-823 (2013)). T7EI assay identified mutated products were subjected to be cloned into TA cloning vector and transformed into competent E. coli strain (Transgen Biotech). After overnight culture, colonies were randomly picked out and sequenced.
Six representative C2c1-family proteins from diverse bacteria as well as four previously reported C2c1 orthologs were selected, and de novo synthesized to conduct genome editing in human embryonic kidney 293T cells (
To conduct mammalian genome editing, we co-transfected 293T cells with individual C2c1 enzymes and their cognate chimeric single guide RNAs (sgRNAs) targeting human endogenous loci containing appropriate PAMs (
To investigate the interchangeability between the dual-RNA and protein components in C2c1 systems, we first analyze the conservation of both C2c1 proteins and dual-RNAs. Besides the conserved amino acid sequences of C2c1 orthologs (
To further demonstrate our hypothesis, we chose two C2c1 orthologs (DiC2c1 and TcC2c1) whose loci harbored no CRISPR array (
The exchangeability between C2c1 and dual-RNA in different C2c1 systems further promoted us to design novel artificial sgRNA (artsgRNA) scaffolds to facilitate C2c1-mediated genome editing. Considering the conservation of DNA sequences and secondary structure among C2c1 orthologs (
| Number | Date | Country | Kind |
|---|---|---|---|
| 201811300251.6 | Nov 2018 | CN | national |
This application is a U.S. National Phase Application of PCT International Patent Application PCT/CN2018/118458, filed Nov. 30, 2018, which claims the benefit of Chinese Patent Application Serial No. 201811300251.6, filed Nov. 2, 2018, the disclosure of each of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2018/118458 | 11/30/2018 | WO |
