Crispr / cpf1 systems and methods

Abstract

To provide: recombinant Cpf1 nucleic acids and polypeptides for use in CRISPR / Cpf1 endonuclease systems; and mammalian cell lines encoding the recombinant Cpf1 polypeptides.SOLUTION: The invention includes utilizing the Cpf1 systems from Acidaminococcus sp., where CRISPR / Cpf1 endonuclease systems having a suitable AsCpf1 crRNA are selected from a length-truncated AsCpf1 crRNA, a chemically-modified AsCpf1 crRNA, and an AsCpf1 crRNA comprising both length truncations and chemical modifications. Methods of performing gene editing using these systems and reagents are also provided.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 62 / 425,307, filed November 22, 2016, and entitled "CPF1 CRISPR SYSTEMS AND METHODS," and U.S. Provisional Patent Application No. 62 / 482,896, filed April 7, 2017, and entitled "HEK293 CELL LINE WITH STABLE EXPRESSION OF ACIDAMINOCOCCUS SP.BV3L6 CPF1," the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on ___ is named IDT01-010-US_ST25.txt and is ___ bytes in size.

[0003] FIELD OF THEINVENTION The present invention relates to Cpf1-based CRISPR genes, polypeptides encoded thereby, mammalian cell lines stably expressing Cpf1, crRNA, and the use of these materials in compositions of CRISPR-Cpf1 systems and methods. [Background technology]

[0004] The use of clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas proteins (CRISPR-Cas systems) for site-specific DNA cleavage has shown great potential for many biological applications. CRISPR has been used for genome editing, genome-scale specific targeting of transcriptional repressors (CRISPRi) and activators (CRISPRa) to endogenous genes, and other applications of RNA-directed DNA targeting by Cas enzymes.

[0005] CRISPR-Cas systems are native to bacteria and archaea and provide adaptive immunity against viruses and plasmids. Three classes of CRISPR-Cas systems can potentially be adapted for research and therapeutic reagents. Type II CRISPR systems have desirable features in that they utilize a single CRISPR-associated (Cas) nuclease (specifically, Cas9) in complex with an appropriate guide RNA (gRNA). In bacteria or archaea, the Cas9 guide RNA comprises two separate RNA species. The target-specific CRISPR activating RNA (crRNA) binds to a specific DNA sequence and directs the Cas9 / gRNA complex to target it. The crRNA has two functional domains, a 5'-domain that is target-specific and a 3'-domain that directs the binding of the crRNA to the trans-activating crRNA (tracrRNA). The tracrRNA is a longer universal RNA that binds to the crRNA and mediates the binding of the gRNA complex to Cas9. Binding of tracrRNA induces a change in Cas9 structure, shifting it from an inactive to an active conformation. gRNA function can also be provided as an artificial single guide RNA (sgRNA) in which the crRNA and tracrRNA are fused into a single species (see Jinek, M. et al., Science 337, 816-21, 2012). The sgRNA format allows for transcription of a functional gRNA from a single transcription unit, which can be provided by a double-stranded DNA (dsDNA) cassette containing a transcription promoter and the sgRNA sequence. In mammalian systems, these RNAs have been introduced by transfection of an RNA Pol III promoter (such as U6 or H1) driving RNA transcription, viral vectors, and DNA cassettes containing single-stranded RNA after in vitro transcription (see Xu, T. et al., Appl Environ Microbiol, 2014.80(5):1544-52). In bacterial systems, these RNAs can be expressed as part of the primitive immune system or artificially expressed from plasmids introduced by transformation (see Fonfara, I. et al., Nature, 2016, 532(7600):517-21).

[0006] In the CRISPR-Cas system, using as an example the system present in Streptococcus pyogenes (S.py. or Spy), the native crRNA is about 42 bases long and contains a 5' region of about 20 bases long that is complementary to the target sequence (also referred to as the protospacer sequence or protospacer domain of the crRNA) and a 3' region, typically about 22 bases long, that is complementary to a region of the tracrRNA sequence and mediates binding of the crRNA to the tracrRNA. The crRNA:tracrRNA complex contains a functional gRNA that can direct Cas9 cleavage of a complementary target DNA. The native tracrRNA has a 5' region that is about 85-90 bases long and contains a region complementary to the crRNA. The remaining 3' region of the tracrRNA contains a secondary structure motif (referred to herein as the "tracrRNA 3' tail") that mediates binding of the crRNA:tracrRNA complex to Cas9.

[0007] Jinek et al. extensively investigated the physical domains of crRNA and tracrRNA required for proper functioning of the CRISPR-Cas system (Science 2012.337(6096):816-21). They devised truncated crRNA:tracrRNA fragments that could still function in CRISPR-Cas, where the crRNA was wild-type 42 nucleotides and the tracrRNA was truncated to 75 nucleotides. They also developed an embodiment where the crRNA and tracrRNA were linked with a linker loop to form a single guide RNA (sgRNA), which in different embodiments varies between 99 and 123 nucleotides.

[0008] At least three groups have solved the crystal structure of Streptococcus pyogenes Cas9 (SpyCas9). In Jinek, M. et al., the structure did not show the nuclease in complex with either the guide RNA or the target DNA. They performed molecular modeling experiments to reveal predicted interactions between the protein in complex with RNA and DNA (Science, 2014.343, 1215, DOI:10.1126 / science / 1247997).

[0009] In Nishimasu, H. et al., the crystal structure of Spy Cas9 has been shown at 2.5 angstrom resolution in complex with sgRNA and its target DNA (Cell, 2014.156(5):935-49, incorporated herein in its entirety). The crystal structure identified two lobes for the Cas9 enzyme: the recognition lobe (REC) and the nuclease lobe (NUC). The sgRNA:target DNA heteroduplex (negatively charged) lies in a positively charged groove between the two lobes. The REC lobe, which shows no structural similarity to known proteins and thus like the Cas9 specific functional domain, interacts with portions of the crRNA and tracrRNA that are complementary to each other.

[0010] Another group, Briner et al. (Mol Cell 2014.56(2):333-9, incorporated herein in its entirety) identified and characterized six conserved modules within the natural crRNA:tracrRNA duplex and sgRNA. Anders et al. (Nature 2014,513(7519),569-73) elucidated the structural basis for DNA sequence recognition of protospacer associated motif (PAM) sequences by Cas9 in association with sgRNA guides.

[0011] The CRISPR-Cas endonuclease system is utilized in genome engineering as follows: the gRNA complex (either the crRNA:tracrRNA complex or sgRNA) binds to Cas9, activating Cas9 and inducing a conformational change that opens the DNA-binding cleft, the protospacer domain of the crRNA (or sgRNA) aligns with the complementary target DNA, Cas9 binds to the PAM sequence and initiates unwinding of the target DNA, followed by annealing of the protospacer domain to the target, after which cleavage of the target DNA occurs. Cas9 contains two domains homologous to the endonucleases HNH and RuvC, respectively, the HNH domain cleaves the DNA strand complementary to the crRNA, and the RuvC-like domain cleaves the non-complementary strand. This results in a double-strand break in the genomic DNA. When repaired by non-homologous end joining (NHEJ), the break is typically repaired in an imprecise manner, resulting in a DNA sequence that is shifted by one or more bases, leading to the disruption of the native DNA sequence, and often leading to frameshift mutations when this event occurs within the coding exon of a gene that codes for a protein.Break can also be repaired by homology directed recombination (HDR), which allows the insertion of new genetic material based on exogenous DNA introduced into the cell with the Cas9 / gRNA complex, which is introduced into the break site created by Cas9 cleavage.

[0012] Although SpyCas9 is the most widely used protein, there are some barriers to its effectiveness. SpyCas9 recognizes targeting sequences in genomes that are immediately followed by GG dinucleotide sequences, and therefore this system is limited to GC-rich regions of genomes. Therefore, AT-rich species or genomic regions often cannot be targeted by the SpyCas9 system. Furthermore, the fact that the Cas9 system contains a gRNA with both crRNA and tracrRNA parts that contain more than 100 bases means that more RNA must be optimized and synthesized for sequence-specific targeting. Therefore, shorter and simpler gRNAs are desirable.

[0013] A second class 2 CRISPR system, assigned to type V, has been identified. This type V CRISPR-associated system contains Cpf1, an approximately 1300 amino acid protein that is slightly smaller than Cas9 from S. pyogenes. The PAM recognition sequence of Cpf1 from Acidaminococcus sp. BV3L6 or Lachnospiraceae bacterium ND2006 is TTTN in contrast to the NGG PAM recognition domain of S. pyogenes Cas9 (Figure 1). Having the ability to target AT-rich regions of the genome is highly beneficial for engineering and studying gene targets in regions lacking GG dinucleotide motifs. The Cpf1 system is also very simple in that it does not utilize a separate tracrRNA, requiring only a single short crRNA of 40-45 bases in length that both identifies the target DNA sequence and directs the binding of the RNA to the Cpf1 nuclease.

[0014] In contrast to Cas9, which produces blunt-end cleavage products, Cpf1 promotes double-stranded breaks with 4-5 nucleotide overhangs. The advantage of this is that it can ensure proper orientation and also provide microhomology during non-homologous end joining (NHEJ). This may also be advantageous in non-dividing cell types that tend to be resistant to homology-directed repair (HDR). Furthermore, when Cpf1 cuts, it does so at a position much farther away from the PAM and also from the target site than when Cas9 cuts. As a result, the protospacer, and especially the seed sequence of the protospacer, is less likely to be edited, thereby leaving open the possibility of a second round of cleavage if the desired repair event does not occur initially.

[0015] Cpf1 protein forms a complex with a single-stranded RNA oligonucleotide to mediate targeted DNA cleavage. The single-stranded guide RNA oligonucleotide consists of a 20 nt constant region and a 21-24 nt target region, with a total length of 41-44 nt. There are many known orthologues of Cpf1 from a variety of different bacterial and archaeal sources that differ in terms of activity and target selection and may be candidates for use in genome editing applications. For the purposes of the present invention, we primarily study As (Acidaminococcus sp. BV3L6) Cpf1 and Cpf1 nuclease from Lb (Lachnospiraceae bacterium ND2006) as representative examples, both of which have already been shown to be active in mammalian cells as a means for genome editing. Of note, the PAM recognition sequence is TTTN. The structure of Cpf1 crRNA and the relationship of RNA binding to the PAM site in genomic DNA are shown in Figure 1.

[0016] Since the discovery of Cpf1 as another CRISPR pathway with potential utility for genome editing in mammalian cells, several publications have confirmed that this system functions in mammals and can be used for embryo engineering, describing the crystal structure and mechanism of PAM site recognition. This system has also shown utility for screening purposes in genetically tractable bacterial species such as E. coli. Thus, this system has proven useful, and it would be beneficial to develop optimized reagents for performing genome editing using Cpf1.

[0017] Previous work performed on SpyCas9 crRNA and tracrRNA demonstrated that meaningful truncations of naturally occurring crRNA and tracrRNA species can be made to RNAs made by chemical synthesis, and that such truncated RNAs were 1) of higher quality, 2) less expensive to produce, and 3) showed improved performance in mammalian genome editing compared to wild-type (WT) RNA. See Collingwood, MA, Jacobi, AM, Rettig, GR, Schubert, MS, and Behlke, MA, CRISPR-BASED COMPOSITIONS AND METHOD OF USE, U.S. Patent Application Serial No. 14 / 975,709, filed December 18, 2015, published as U.S. Patent Application Publication No. US2016 / 0177304A1 on June 23, 2016, and issued as U.S. Patent No. 9,840,702 on December 12, 2017.

[0018] Previous studies have demonstrated that reducing the length of the FnCpf1 crRNA from 22 bases to 18 bases by deletion from the 3' end supports target DNA cleavage, but lengths of 17 or shorter show reduced activity. Deletions or mutations that disrupted base pairing in the universal loop domain abolished activity. See Zetsche, B., Gootenberg, JS, Abudayyeh, OO, Slaymaker, IM, Makarova, KS, Essletzbichler, P., Volz, SE, Joung, J., van der Oost, J., Regev, A., Koonin, EV, and Zhang, F. (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:1-13. However, FnCpf1 nuclease does not function to perform genome editing in mammalian cells. It is unclear whether the same length rule as observed for FnCpf1 crRNA applies to AsCpf1 crRNA. Herein, we establish the shortest form of AsCpf1 crRNA with full activity in mammalian genome editing applications. We also establish chemical modification patterns that maintain or improve the function of synthetic Cpf1 crRNA when used in mammalian or prokaryotic cells. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] US Patent Application Publication No. 2016 / 0177304 [Patent Document 2] U.S. Patent No. 9,840,702 [Patent Document 3] U.S. Patent Application Serial No. 14 / 975,709 [Non-patent literature]

[0020] [Non-Patent Document 1] Jinek, M. et al., Science 337, pp. 816 - 821, 2012 [Non - Patent Document 2] Xu, T. et al., Appl Environ Microbiol, 2014. 80(5): pp. 1544 - 1552 [Non - Patent Document 3] Fonfara, I. et al., Nature, 2016, 532(7600): pp. 517 - 521 [Non - Patent Document 4] Science, 2012. 337(6096): pp. 816 - 821 [Non - Patent Document 5] Science, 2014. 343, p. 1215, DOI: 10.1126 / science / 1247997 [Non - Patent Document 6] Cell, 2014. 156(5): pp. 935 - 949 [Non - Patent Document 7] Mol Cell, 2014. 56(2): pp. 333 - 339 [Non - Patent Document 8] Nature, 2014, 513(7519), pp. 569 - 573 [Non - Patent Document 9] Zetsche, B., Gootenberg, J. S., Abudayyeh, O. O., Slaymaker, I. M., Makarova, K. S., Essletzbichler, P., Volz, S. E., Joung, J., van der Oost, J., Regev, A., Koonin, E. V., and Zhang, F. (2015) Cpf1 is a single RNA - guided endonuclease of a class 2 CRISPR - Cas system. Cell 163: pp. 1 - 13 [Summary of the Invention] [Problems to be Solved by the Invention]

[0021] (Abstract of the Invention) The present invention relates to Cpf1-based CRISPR genes, polypeptides encoded thereby, mammalian cell lines stably expressing Cpf1, and chemically synthesized Cpf1 crRNA, and their use in compositions of CRISPR-Cpf1 systems and methods. The examples show the use of the Cpf1 systems from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006, but are not intended to be limiting in scope to cover Cpf1 homologs or orthologs isolated from other species. [Means for solving the problem]

[0022] In a first aspect, an isolated nucleic acid is provided. The isolated nucleic acid encodes an As Cpf1 polypeptide codon-optimized for expression in H. sapiens, as seen in SEQ ID NO:8, SEQ ID NO:15 and SEQ ID NO:22, including the use of a nuclear localization signal and an epitope tag. The isolated nucleic acid also encodes an As Cpf1 polypeptide codon-optimized for expression in E. coli, including SEQ ID NO:5, and can be fused or linked to a sequence encoding a nuclear localization signal, multiple nuclear localization signals or an epitope tag that allows detection by an antibody or other method, and / or an affinity tag that allows easy purification of recombinant proteins expressed from the nucleic acid, such as the His-Tag seen in SEQ ID NO:12 and SEQ ID NO:19.

[0023] In a second aspect, an isolated polypeptide is provided that encodes a wild-type As Cpf1 protein. In a first aspect, the isolated polypeptide comprises SEQ ID NO: 2. The protein can be fused or linked to a sequence encoding a nuclear localization signal, multiple nuclear localization signals, or an epitope tag that allows detection by an antibody or other method, and / or an affinity tag that allows easy purification of recombinant protein expressed from the nucleic acid, such as the His-Tag found in SEQ ID NO: 12, SEQ ID NO: 16, and SEQ ID NO: 19.

[0024] In a third aspect, an isolated nucleic acid is provided. The isolated nucleic acid encodes a codon-optimized Lb Cpf1 polypeptide for expression in humans, as seen in SEQ ID NO:9 and SEQ ID NO:17, including the use of a nuclear localization signal and an epitope tag. The isolated nucleic acid also encodes a codon-optimized Lb Cpf1 polypeptide for expression in E. coli, including SEQ ID NO:6, and can be fused or linked to a sequence encoding a nuclear localization signal, multiple nuclear localization signals, or an epitope tag that allows detection by an antibody or other method, and / or an affinity tag that allows easy purification of recombinant proteins expressed from the nucleic acid, such as the His-Tag seen in SEQ ID NO:13.

[0025] In a fourth aspect, there is provided an isolated polypeptide encoding a wild type Lb Cpf1 protein. In a first aspect, the isolated polypeptide comprises SEQ ID NO: 7 and SEQ ID NO: 10. The protein may be fused or linked to a sequence encoding a nuclear localization signal, multiple nuclear localization signals, or an epitope tag allowing detection by antibody or other methods and / or an affinity tag allowing easy purification of recombinant protein expressed from the nucleic acid, such as the His-Tag found in SEQ ID NO: 14.

[0026] In a fifth aspect, there is provided an isolated expression vector encoding SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17. The isolated expression vector comprises transcription initiation elements such as a promoter and an enhancer operably linked to SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17 to enable expression of a polypeptide encoded by SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16.

[0027] In a sixth aspect, there is provided a host cell comprising an isolated expression vector encoding SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17. The isolated expression vector encoding SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17 is operably linked to a suitable promoter and other genetic elements (as appropriate) to allow expression of a polypeptide comprising SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16.

[0028] In a seventh aspect, there is provided an isolated CRISPR / Cpf1 endonuclease system, the system comprising an AsCpf1 polypeptide and a suitable AsCpf1 crRNA.

[0029] In an eighth aspect, there is provided an isolated CRISPR / Cpf1 endonuclease system, the system comprising a human cell line expressing an AsCpf1 polypeptide and a suitable AsCpf1 crRNA.

[0030] In a ninth aspect, an isolated AsCpf1 crRNA is provided. The isolated AsCpf1 crRNA is active in the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein endonuclease system. Different variants of the crRNA are provided, including a species optimized for performance in mammalian cells and a species optimized for performance in bacteria.

[0031] In a tenth aspect, a method of performing gene editing is provided, comprising contacting a candidate editing target site locus with an active CRISPR / Cpf1 endonuclease system having a wild-type AsCpf1 polypeptide and a suitable AsCpf1 crRNA. [Brief description of the drawings]

[0032] [Figure 1]Diagrammatic representation of the Cpf1 PAM recognition site and alignment of guide crRNA to target DNA. The genomic DNA sequence of the human HPRT1 gene is shown in site "38595". The "TTTN" PAM site specifying the As Cpf1 site is highlighted and the sequence of the guide binding site is underlined. DNA is shown in uppercase and RNA in lowercase. In the Cpf1 crRNA, the protospacer target specific domain is underlined and contains the 3'-domain. The universal hairpin RNA sequence that mediates binding to the Cpf1 protein contains the 5'-domain. [Diagram 2] FIG. 1 shows a map of the plasmid vector designed to express recombinant, synthetic, codon-optimized AsCpf1. [Diagram 3] A schematic diagram showing the final plasmid constructs used to generate AsCpf1 stable cell lines is shown. [Figure 4] Figure 1 shows an exemplary Western blot showing expression of V5-tagged proteins. Cell extracts from a monoclonal HEK cell line stably expressing Cas9 with a V5 tag were run in lane 2. Cell extracts from a new polyclonal HEK cell culture expressing V5-tagged AsCpf1 were run in lane 3. Beta-actin is shown and represents a mass loading control. Lane 1 was run as a mass standard marker. [Diagram 5] FIG. 1 shows an exemplary expression profile of AsCpf1 mRNA normalized to the internal control HPRT1 mRNA in 10 clonal transgenic cell lines. The position of the RT-qPCR assay shifts according to the AsCpf1 mRNA. Negative control non-transgenic HEK1 cells are shown on the far right. [Figure 6] FIG. 1 shows an exemplary Western blot showing relative expression levels of AsCpf1 protein in 10 monoclonal transgenic cell lines based on detection of the V5 epitope, with a beta-actin loading control seen below the AsCpf1 band. [Figure 7]FIG. 1 shows modification tolerance maps of AsCpf1 crRNA at two sequence target sites, HPRT1-38351 (panel (i)) and HPRT1-38595 (panel (ii)). The sequence of the universal 5'-loop domain is shown (in the 5'-3' direction) for both the 24-nt protospacer domain (panels (ia) and (ii.a)) and the 21-nt protospacer domain (panels (ib) and (ii.b)). The sequence of the variable 3' target-specific protospacer domain is shown as an "N" base since this sequence varies from target to target. Positions that did not suffer loss of activity when modified as 2'OMe RNA residues in the single base walk are shown in uppercase, whereas positions that showed loss of activity upon modification are shown in lowercase. Above the lowercase residues are arrows indicating the relative magnitude of loss of activity, with large arrows representing a large loss of activity, medium sized arrows representing a moderate loss of activity, and small arrows representing a small loss of activity when the respective RNA residue is changed to 2'OMe RNA. [Figure 8] FIG. 1 shows an exemplary modified variant AsCpf1 crRNA that is active in genome editing applications in mammalian cells at multiple target sites and is therefore not site-specific. The sequence of the universal 5'-loop domain is shown (in the 5'-3' orientation) and underlined. The sequence of the variable 3' target-specific protospacer domain is shown as an "N" base since this sequence varies from target to target. 2'OMe RNA modifications are shown in uppercase and RNA residues in lowercase. "X" indicates a terminal non-basic modifier, such as a C3 spacer (propanediol) or a ZEN (naphthyl-azo) group. "*" indicates a phosphorothioate (PS) internucleotide linkage. [Figure 9]Figure 1 shows exemplary results comparing the target editing activity of LbCpf1 with that of AsCpf1 and SpyCas9 for 12 regions of the HPRT gene with low GC content by T7EI mismatch endonuclease assay. In this study, all enzymes and crRNA were delivered to HEK293 cells as RNP complexes (5 μM) by nucleofection using the Amaxa system from Lonza, and DNA was extracted after 48 hours. Percent editing was determined by T7E1 mismatch endonuclease assay. Error bars represent standard error of the mean. Of note, crRNA for LbCpf1 was tested at the natural 23mer nucleotide length and the previously optimized 21-mer length of AsCpf1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The methods and compositions of the invention described herein provide wild-type AsCpf1 nucleic acids and polypeptides for use in the CRISPR / Cpf1 system. The invention describes a HEK293 cell line that has stable low-level expression of AsCpf1 in HEK293 and can be used as a platform for investigating and optimizing the nucleic acid components of the system. AsCpf1 provides a useful complement to SpyCas9 by expanding the range of PAM sequences that can be targeted from GC-rich regions (Cas9) to AT-rich regions of the genome (Cpf1), thereby expanding the range of sequences that can be modified using CRISPR genome engineering methods. In addition to having T-rich PAM sites, another advantage of the AsCpf1 system compared to Cas9 is the use of a single short RNA molecule. However, unlike Cas9, which shows activity at most sites in the human genome, AsCpf1 shows little or no activity at half of the TTTN PAM sites. Thus, fully realizing the potential of the AsCpf1 CRISPR system is enhanced by the availability of appropriate predictive software that enriches for high activity sites based on sequence context. The use of stable constitutive Cpf1 expressing cell lines facilitates algorithm development with reduced effort and cost compared to using alternative methods such as electroporation of ribonucleoprotein (RNP) complexes. HEK293 cells are immortalized cell lines that are easily cultured, passaged, and stored at low temperatures. The inventors established clonal cell lines that constitutively express SpyCas9 and AsCpf1 as suitable test vehicles for algorithm development or rapid testing / optimization of chemical structures of guide RNAs. The present invention describes length and chemical modifications of AsCpf1 and LbCpf1 crRNA length-optimized variants that improve function in genome editing.

[0034] Genes encoding AsCpf1, polypeptides, expression vectors and host cells The term "wild-type AsCpf1 protein" ("WT-AsCpf1" or "WT-AsCpf1 protein") encompasses a protein that has the same amino acid sequence of naturally occurring Acidaminococcus sp. BV3L6 Cpf1 (e.g., SEQ ID NO:2) and has biochemical and biological activity when combined with an appropriate crRNA to form an active CRISPR / Cpf1 endonuclease system.

[0035] The term "wild-type LbCpf1 protein" ("WT-LbCpf1" or "WT-LbCpf1 protein") encompasses a protein that has the same amino acid sequence of the naturally occurring Lachnospiraceae bacterium ND2006 Cpf1 (e.g., SEQ ID NO: 4) and has biochemical and biological activity when combined with an appropriate crRNA to form an active CRISPR / Cpf1 endonuclease system.

[0036] The term "wild-type CRISPR / Cpf1 endonuclease system" refers to a CRISPR / Cpf1 endonuclease system comprising a wild-type AsCpf1 protein and a suitable AsCpf1 crRNA as a guide RNA.

[0037] The term "polypeptide" refers to any linear or branched peptide containing more than one amino acid. Polypeptide includes proteins or fragments thereof or fusions thereof, provided that such proteins, fragments, or fusions retain useful biochemical or biological activity.

[0038] Fusion proteins typically contain additional amino acid information not native to a protein, which is covalently attached to the protein. Such additional amino acid information may include a tag that allows purification or identification of the fusion protein. Such additional amino acid information may include a peptide that allows the fusion protein to be transported into a cell and / or to a specific location within the cell. Examples of tags for these purposes include: AviTag (GLNDIFEAQKIEWHE), a peptide that allows for biotinylation by the enzyme BirA so that the protein can be isolated with streptavidin; Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL), a peptide to which the protein calmodulin binds; Polyglutamate tag (EEEEEE), a peptide that binds efficiently to anion exchange resins such as Mono-Q; E tag (GAPVPYPDPLEPR), a peptide that is recognized by an antibody; FLAG tag (DYKDDDDK), a peptide that is recognized by an antibody; HA tag (YPYDVPDYA), a peptide derived from hemagglutinin that is recognized by an antibody; typically 5-10 histidines that directly bind to a nickel or cobalt chelate. the His-tag (HHHHHH), which can be used to identify a target protein; the Myc-tag (EQKLISEEDL), which is a peptide derived from c-myc that is recognized by an antibody; the NE-tag (TKENPRSNQEESYDDNES), which is a novel 18 amino acid synthetic peptide recognized by a monoclonal IgG1 antibody, which is useful for a wide range of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation and affinity purification of recombinant proteins; the S-tag (KETAAAKFERQHMDS), which is a peptide derived from ribonuclease A; the SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP), which is a peptide that binds to streptavidin; Softag1 (SLAELLNAGLGGS), which is intended for mammalian expression; and Softag3 (TQDPSRVG), which is intended for prokaryotic expression.Strep tag (Strep tag II: WSHPQFEK), a peptide that binds to streptavidin or modified streptavidin called streptactin; TC tag (GKPIPNPLLGLDST), a tetracysteine ​​tag that is recognized by FlAsH and ReAsH biarsenical compounds (CCPGCC) V5 tag, a peptide that is recognized by antibodies; VSV tag (YTDIEMNRLGK), a peptide that is recognized by antibodies; Xpress tag (DLYDDDDK); Isopeptag (TDKDMTITFTNKKDAE), a peptide that is covalently bound to the pyrin-C protein; SpyTag (AHIVMVDAYKPTK), a peptide that is covalently bound to the SpyCatcher protein; and SnoopCatcher protein. SnoopTag (KLGDIEFIKVNK), a peptide; BCCP (biotin carboxyl carrier protein), a protein domain that is biotinylated by BirA to enable recognition by streptavidin; glutathione-S-transferase tag, a protein that binds immobilized glutathione; green fluorescent protein tag, a protein that is naturally fluorescent and can be bound by an antibody; HaloTag, a mutant bacterial haloalkane dehalogenase that is covalently attached to a reactive haloalkane substrate to enable attachment to a wide variety of substrates; maltose binding protein tag, a protein that binds amylose agarose; Nus tag; thioredoxin tag; and Fc tag, derived from an immunoglobulin Fc domain that allows dimerization and solubilization and can be used for purification on protein A sepharose.

[0039] A nuclear localization signal (NLS), such as that obtained from SV40, allows the protein to be transported to the nucleus immediately upon entry into the cell. Assuming that the native AsCpf1 protein is of bacterial origin and therefore does not naturally contain NLS motifs, the addition of one or more NLS motifs to the recombinant AsCpf1 protein is expected to show improved genome editing activity when used in eukaryotic cells where the target genomic DNA substrate is present in the nucleus. Functional testing in HEK293 cells revealed that using a bipartite NLS (nucleoplasmin) increases editing compared to the current commercial design (3 SV40 NLS) and the use of a single or double OpT NLS, which showed promise in the Cpf1 protein. Further combinations of NLS elements, including bipartites, are envisioned. Of note, nucleoplasmin works best in mammalian cells, while the SV40 NLS appears to function in almost all nucleated cells. The bipartite SV40 NLS is functional in both Cas9 and Cpf1. Having two different NLS domains can expand efficacy across a wide range of species.

[0040] Those of skill in the art understand how to make and use these various fusion tag technologies as well as fusion proteins containing them.

[0041] The term "isolated nucleic acid" includes DNA, RNA, cDNA, and vectors encoding them, free of other biological material from which they may be derived or associated, such as cellular components. Typically, isolated nucleic acids are purified from other biological material from which they may be derived or associated, such as cellular components.

[0042] The term "isolated wild-type AsCpf1 nucleic acid" refers to an isolated nucleic acid that encodes a wild-type AsCpf1 protein. Examples of isolated wild-type AsCpf1 nucleic acids include SEQ ID NO:1.

[0043] The term "isolated wild-type LbCpf1 nucleic acid" refers to an isolated nucleic acid that encodes a wild-type LbCpf1 protein. Examples of isolated wild-type LbCpf1 nucleic acids include SEQ ID NO:3.

[0044] In a first aspect, an isolated nucleic acid is provided. The isolated nucleic acid encodes an As Cpf1 polypeptide that is codon-optimized for expression in humans. In a first aspect, the isolated nucleic acid includes SEQ ID NO:8, SEQ ID NO:15, and SEQ ID NO:22, which includes the use of a nuclear localization signal and an epitope tag. The isolated nucleic acid also encodes an As Cpf1 polypeptide that is codon-optimized for expression in E. coli, including SEQ ID NO:5, and can be fused or linked to a sequence that encodes a nuclear localization signal, multiple nuclear localization signals, or an epitope tag that allows detection by an antibody or other method, and / or an affinity tag that allows easy purification of recombinant proteins expressed from the nucleic acid, such as the His-Tag found in SEQ ID NO:12 and SEQ ID NO:19.

[0045] In a second aspect, there is provided an isolated polypeptide encoding a wild-type As Cpf1 protein. In a first aspect, the isolated polypeptide comprises SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:16 or SEQ ID NO:19.

[0046] In a third aspect, an isolated expression vector is provided that encodes SEQ ID NO: 15. The isolated expression vector comprises transcription initiation elements, such as a promoter and an enhancer, operably linked to SEQ ID NO: 15 to allow expression of a polypeptide encoded by SEQ ID NO: 16. The isolated expression vector may further comprise transcription termination elements, post-transcriptional processing elements (e.g., splicing donor and acceptor sequences and / or polyadenylation signal sequences), mRNA stability elements and mRNA translation enhancer elements. Such genetic elements are understood and used by those skilled in the art.

[0047] In a fourth aspect, a host cell is provided comprising an isolated expression vector encoding SEQ ID NO: 15. The isolated expression vector encoding SEQ ID NO: 15 is operably linked to a suitable promoter and other genetic elements (if necessary) to allow expression of a polypeptide comprising SEQ ID NO: 16. In a first aspect, the host cell comprises a human cell. In a second aspect, the human cell comprises an immortalized cell line. In a third aspect, the immortalized cell line is a HEK293 cell line. As a further elaboration of this third aspect, the immortalized cell line comprises an isolated AsCpf1 crRNA capable of forming a ribonucleoprotein complex with a polypeptide comprising SEQ ID NO: 2 to form a wild-type CRISPR / Cpf1 endonuclease.

[0048] Length and chemical structure optimized AsCpf1 crRNA The term "length-modified," as the term modifies RNA, refers to a shortened or truncated form of the reference RNA that lacks a nucleotide sequence or an extended form of the reference RNA that includes additional nucleotide sequences.

[0049] The term "chemically modified" refers to a form of reference RNA that contains chemically modified nucleotides or non-nucleotide chemical groups covalently linked to RNA when the term modifies RNA. Chemically modified RNA, as described herein, generally refers to synthetic RNA prepared using oligonucleotide synthesis procedures in which modified nucleotides are incorporated during synthesis of RNA oligonucleotides. However, chemically modified RNA also includes synthetic RNA oligonucleotides that are modified with appropriate modifying agents after synthesis.

[0050] The competent CRISPR / Cpf1 endonuclease system comprises a ribonucleoprotein (RNP) complex formed with a guide RNA consisting of an isolated AsCpf1 protein and an isolated AsCpf1 crRNA. In some embodiments, the length-modified and / or chemically modified isolated forms of the AsCpf1 crRNA are combined with a gene encoding the AsCpf1 protein in a purified AsCpf1 protein, an isolated mRNA encoding the AsCpf1 protein, or an expression vector. In certain assays, the length-modified and / or chemically modified isolated forms of the AsCpf1 crRNA can be introduced into a cell line that stably expresses the AsCpf1 protein from an endogenous expression cassette encoding the AsCpf1 gene.

[0051] For the synthesis of synthetic RNA, the sequence of unnecessary bases is shortened, but desirably not shortened to such an extent that loss of function occurs. The 5' constant region, which mediates the binding of crRNA to Cpf1 nuclease, shows loss of activity when truncated to less than 20 residues. The 3' variable domain, which includes the protospacer guide region that confers target sequence specificity to the crRNA, naturally occurs in lengths of around 25 bases. This domain can be shortened to about 20-21 bases without loss of functional activity. Thus, the optimized length of Cpf1 crRNA is 40-41 bases, including a 20-base 5' constant domain and a 20-21-base 3' variable domain.

[0052] The present invention provides suitable guide RNAs for inducing the DNA nuclease activity of AsCpf1 nuclease. These optimized reagents, both for length-modified and / or chemically modified forms of crRNA, provide improved genome editing in any application using AsCpf1. Applications of CRISPR-based tools include, but are not limited to, plant gene editing, yeast gene editing, rapid generation of knockout / knock-in animal systems, generation of animal models of disease states, correction of disease states, insertion of reporter genes, and whole genome functional screening. The "tool kit" can be further expanded by including nickase forms and dead mutants of AsCpf1 as fusion proteins with transcriptional activators (CRISPRa) and repressors (CRISPRi).

[0053] RNA-guided DNA cleavage by AsCpf1 is primarily useful due to its ability to target AT-rich gene regions (compared to GC-rich targeting by SpyCas9). The newly discovered AsCpf1 crRNA cleavage and modification variants are suitable for promoting AsCpf1-mediated staggered cleavage and providing advantages in gene silencing, homology-directed repair or exon excision. The present invention defines a truncated AsCpf1 guide RNA with sufficient potency to direct gene editing by CRISPR / Cpf1 endonuclease. This is useful in manufacturing to synthesize fully functional truncated compounds, resulting in higher quality, lower cost while maximizing functionality.

[0054] Unlike S.py.Cas9, which requires a complex of two RNAs to recognize and cleave a target DNA sequence (including a hybridized crRNA:tracrRNA pair) or a long synthetic single guide sgRNA, Cpf1 nuclease requires only a short single crRNA species to direct target recognition. This RNA contains two domains: a 5'-domain of 20 RNA residues that is universal and mediates binding of the RNA species to the Cpf1 protein, and a 3'-domain of 21-24 RNA residues that is target specific and mediates binding of the RNP complex to a precise DNA sequence. A functional nuclease complex contains a single crRNA (41-44 bases long) and isolated Cpf1 protein, which are combined in a 1:1 molar ratio to form an active complex. The guide crRNA species can be expressed in mammalian cells from an expression plasmid or viral vector. The crRNA can also be made as an in vitro transcript (IVT) and isolated as a pure enzymatic RNA species. More preferably, the crRNA can be produced as a synthetic chemical RNA oligonucleotide. Chemical production allows the use of modified residues which have a number of advantages, as outlined below.

[0055] Synthetic nucleic acids are attacked by cellular nucleases and are rapidly degraded in mammalian cells or serum. Chemical modifications can confer relative nuclease resistance to synthetic nucleic acids, extending their half-life and thereby dramatically improving functional performance and efficacy. To make matters even more complicated, synthetic nucleic acids are often recognized by antiviral surveillance mechanisms in mammalian cells that are part of the innate immune system and lead to interferon response pathway activation that can lead to cell death. Chemical modifications can reduce or eliminate undesirable immune responses to synthetic RNA. It is therefore useful to establish methods to chemically modify synthetic RNA oligonucleotides intended for use in living cells. However, nucleic acid species that have specific interactions with protein factors cannot be blindly modified because chemical modifications can change the tertiary structure of the nucleic acid and block important contact points between the nucleic acid and amino acid residues. For example, 2'-O-methyl RNA modification (2'OMe) can block the 2'-oxygen of RNA from interacting with amino acid residues, which in turn disrupts the functional interaction between the modified RNA and proteins. Similarly, phosphorothioate modifications can disrupt protein binding along the phosphate backbone of nucleic acids through replacement of non-bridging oxygen at the phosphate.

[0056] 2'OMe modification is particularly useful in this context, since it has been previously shown to increase the nuclease stability of antisense oligonucleotides (ASOs) and siRNAs, and in the same way can also reduce the risk of inducing an innate immune response when chemically synthesized RNA is introduced into mammalian cells. Specific modification patterns have been established that allow the incorporation of this modified residue into ASOs or siRNAs and retain function. Similarly, the inventors have recently developed chemical modification patterns that improved the stability of crRNAs and tracrRNAs that serve as guide RNAs in the SpyCas9 system. The use of 2'OMe modified residues in CRISPR guide RNAs improves RNA stability against nucleases, increasing the overall efficiency of editing in nuclease-rich environments, while simultaneously reducing cell death and toxicity (as seen with long unmodified RNAs) associated with immunogenic triggers.

[0057] The present invention relates to defining chemical modification patterns for the AsCpf1 crRNA that retain function in forming an active RNP complex that can be used in genome editing in mammalian cells. A modification "walk" was performed where a single 2'OMe residue was placed consecutively at all positions with the Cpf1 crRNA. Sites that reduced or eliminated the function of the RNP complex in genome editing were identified. A chemical modification pattern compatible with highly efficient genome editing was defined. The utility of 2'-fluoro (2'F) and locked nucleic acid (LNA) modifications at "modification competent" positions of the crRNA was also demonstrated. The use of phosphorothioate internucleotide linkages to modify select sites to reduce nuclease susceptibility as well as the successful use of non-basic modifiers as end blocks to reduce exonuclease attack on synthetic RNA was shown. Taken together, these studies provide a "map" of sites in the Cpf1 crRNA that are amenable to chemical modification, along with a set of modification chemistries that have been demonstrated to function in the intended application in mammalian cells.

[0058] Specific examples of modification patterns are shown in the Examples below. The 20 base 5' constant domain can be extensively modified and retain function. In particular, using a 20 base 5' constant region, and counting from the 5' end, the RNA residues at positions 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, and 19 can all be replaced by 2'OMe RNA residues without loss of activity. Such substitutions can modify single, multiple, or all 14 residues, thereby changing 14 / 20 residues in this domain from RNA to 2'OMe RNA. The maximum modification patterns tolerated in the 21 base 3' variable domain vary with the sequence of the domain. Within this domain, residues 21, 22, 23, 28, 29, 30, 32, 34, 35, 39, 40, and 41 (counting from the first base of the 5' constant region) can be replaced by 2'OMe residues without loss of activity.

[0059] Only select positions within the 21-24 base 3' target specific domain can be modified without compromising activity. Based on the crystal structure of Cpf1, there are many protein contact points within the constant and target regions. For constant region modifications, no clear correlation emerged when comparing the Cpf1 crystal structure contact points with the identified functional positions that can be modified, meaning that good modification patterns cannot be predicted from the crystal structure. Similarly, empirical testing was required to determine the target region modification patterns. Based on the initial 2'OMe modification studies, select regions within the Cpf1 crRNA were modified using 2'OMe in an attempt to narrow down the regions that tolerate modification. The location of single residues within the Cpf1 crRNA that are sensitive to 2'OMe modification is shown in Figure 7. The higher level modification patterns, which are a strong trigger for Cpf1-mediated genome editing, are shown in Figure 8. 2'F modifications can be located at any residue that is tolerant to 2'OMe modification. Furthermore, the 3' variable domain tolerates larger blocks of 2'F modifications than larger blocks of 2'OMe modifications. Thus, a highly modified version of Cpf1 crRNA contains 2'OMe modifications in the 3'-domain and 2'F modifications in the 5'-domain. For moderate or light modification patterns, either 2'OMe or 2'F modifications (or both) can be used in both domains. LNA residues can also be incorporated into the crRNA without compromising function, as defined in the examples below.

[0060] As an alternative to the widespread use of 2'OMe or other modified sugar approaches, blocking exonuclease attack with non-basic modifiers at the 3' and 5' ends is compatible with crRNA function and improves function in cells. Small C3 spacers (propanediol) or large ZEN groups work equally well for this approach. Furthermore, phosphorothioate internucleotide linkages can be placed at select sites, such as between the last 2-3 bases at each end of the crRNA, although complete PS modification of the crRNA or complete modification of either the loop domain or the protospacer domain show reduced activity.

[0061] Guide RNA is required for RNA-directed dsDNA cleavage by AsCpf1, which initiates the subsequent repair events involved in most CRISPR applications in mammalian cells. The use of modified synthetic AsCpf1 crRNA as a guide for AsCpf1 genome editing is provided. The usefulness of 2'OMe-modified AsCpf1 crRNA, 2'F-modified AsCpf1 crRNA, LNA-modified AsCpf1 crRNA and end-blocked AsCpf1 crRNA for CRISPR / Cpf1 applications in mammalian cells has been demonstrated. Those skilled in the art will recognize and understand that further chemical modifications are possible based on this disclosure. Many of these base modification groups are expected to function similarly according to the patterns taught in the present invention. To date, all crRNAs used with Cpf1 for genome editing have been unmodified RNA. In the present invention, functional modification patterns are provided that improve the properties of AsCpf1 crRNA and reduce the risk of toxicity.

[0062] AsCpf1 crRNA can be produced in cells from RNA transcription vectors as in vitro transcription products (IVT) or by chemical synthesis. Synthetic RNA oligonucleotides offer distinct advantages as they alone allow precise insertion of modified bases at specific sites in the molecule. The present invention provides a map of positions suitable for chemical modification that can be used to improve the performance of AsCpf1 crRNA in cells. For some applications, a "minimal modification" approach is sufficient. For use in higher nuclease environments or in cells with particularly high innate immune reactivity, a "high modification" approach may work well. The present invention provides methods for low, medium or high modification requirements.

[0063] Applications of AsCpf1-based tools are many and varied, including but not limited to bacterial gene editing, plant gene editing, yeast gene editing, mammalian gene editing, cell editing in living animal organs, embryo editing, rapid generation of knock-out / knock-in animal systems, generation of animal models of disease states, correction of disease states, insertion of reporter genes, and whole genome functional screening.

[0064] In a fifth aspect, an isolated CRISPR / Cpf1 endonuclease system is provided. The system comprises an AsCpf1 polypeptide and a suitable AsCpf1 crRNA. In a first aspect, the AsCpf1 polypeptide comprises SEQ ID NO: 2. In a second aspect, the suitable AsCpf1 crRNA is selected from a length-truncated AsCpf1 crRNA or a chemically modified AsCpf1 crRNA or an AsCpf1 crRNA containing both a length truncation and a chemical modification.

[0065] In a sixth aspect, an isolated CRISPR / Cpf1 endonuclease system is provided. The system comprises a human cell line expressing an AsCpf1 polypeptide and a suitable AsCpf1 crRNA. In a first aspect, the AsCpf1 polypeptide comprises at least one member selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:16 and SEQ ID NO:19. In a second aspect, the suitable AsCpf1 crRNA is selected from a length-truncated AsCpf1 crRNA or a chemically modified AsCpf1 crRNA or an AsCpf1 crRNA containing both a length truncation and a chemical modification.

[0066] In a seventh aspect, an isolated AsCpf1 crRNA is provided. The isolated AsCpf1 crRNA is active in the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein endonuclease system. In a first aspect, the isolated AsCpf1 crRNA is selected from a shortened AsCpf1 crRNA, a chemically modified AsCpf1 crRNA, or an AsCpf1 crRNA containing both a shortened length and a chemical modification.

[0067] In an eighth aspect, a method of performing gene editing is provided. The method comprises contacting a candidate editing target site locus with an active CRISPR / Cpf1 endonuclease system having a wild-type AsCpf1 polypeptide and a suitable AsCpf1 crRNA. In a first aspect, the wild-type AsCpf1 polypeptide comprises at least one member selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:16 and SEQ ID NO:19. In a second aspect, the suitable AsCpf1 crRNA is selected from a length-truncated AsCpf1 crRNA, a chemically modified AsCpf1 crRNA or an AsCpf1 crRNA containing both a length truncation and a chemical modification.

[0068] In another aspect, an isolated nucleic acid encoding an Lb Cpf1 polypeptide that is codon-optimized for expression in a human is provided. In a first aspect, the isolated nucleic acid comprises SEQ ID NO: 17 or SEQ ID NO: 396.

[0069] In another aspect, an isolated polypeptide encoding a wild-type Lp Cpf1 protein is provided. In a first aspect, the isolated polypeptide comprises SEQ ID NO:14 or SEQ ID NO:24.

[0070] In another aspect, an isolated expression vector encoding SEQ ID NO:17 or SEQ ID NO:396 is provided.

[0071] In another aspect, a host cell is provided comprising an isolated expression vector encoding SEQ ID NO: 17 or SEQ ID NO: 396. The isolated expression vector encoding SEQ ID NO: 17 or SEQ ID NO: 396 is operably linked to a suitable promoter to allow expression of a polypeptide comprising SEQ ID NO: 14 or SEQ ID NO: 24, respectively. In a first aspect, the host cell comprises a human cell. In a second aspect, the human cell comprises an immortalized cell line. In a third aspect, the immortalized cell line is a HEK293 cell line. In a further elaboration of this aspect, the host cell comprises an isolated Lb Cpf1 crRNA capable of forming a ribonucleoprotein complex with a polypeptide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 20 and SEQ ID NO: 24 to form a wild-type CRISPR / Cpf1 endonuclease.

[0072] In another aspect, an isolated CRISPR / Cpf1 endonuclease system is provided having an Lb Cpf1 polypeptide and a suitable Cpf1 crRNA. In a first aspect, the CRISPR / Cpf1 endonuclease system comprises an Lb Cpf1 polypeptide in the form of SEQ ID NO: 14. In a second aspect, the isolated CRISPR / Cpf1 endonuclease system comprises a suitable Cpf1 crRNA selected from a length- truncated Cpf1 crRNA or a chemically modified Cpf1 crRNA or a Cpf1 crRNA comprising both a length truncation and a chemical modification.

[0073] In another aspect, an isolated CRISPR / Cpf1 endonuclease system is provided having a human cell line expressing an Lb Cpf1 polypeptide and a suitable Cpf1 crRNA. In a first aspect, the Lb Cpf1 polypeptide is SEQ ID NO: 14 or SEQ ID NO: 24. In a second aspect, the suitable Cpf1 crRNA is selected from a length-truncated Cpf1 crRNA or a chemically modified Cpf1 crRNA or a Cpf1 crRNA comprising both a length truncation and a chemical modification.

[0074] In another aspect, a method for performing gene editing is provided. The method includes contacting a candidate editing target site locus with an active CRISPR / Cpf1 endonuclease system having a wild-type Lb Cpf1 polypeptide and a suitable Cpf1 crRNA. In a first aspect, the method includes a wild-type Lb Cpf1 polypeptide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 20 and SEQ ID NO: 24. In a second aspect, the suitable Cpf1 crRNA is selected from a length-truncated Cpf1 crRNA, a chemically modified Cpf1 crRNA or a Cpf1 crRNA that includes both length truncation and chemical modification.

[0075] In another aspect, a CRISPR endonuclease system is provided having a recombinant Cpf1 fusion protein and a suitable crRNA. In a first aspect, the recombinant Cpf1 fusion protein is an isolated and purified protein. In a second aspect, the recombinant Cpf1 fusion protein comprises an N-terminal NLS, a C-terminal NLS and multiple affinity tags located at either the N-terminal or C-terminal ends. In one preferred embodiment, the recombinant Cpf1 fusion protein comprises an N-terminal NLS, a C-terminal NLS and three N-terminal FLAG tags and a C-terminal 6xHis tag. In a third aspect, the recombinant Cpf1 fusion protein and a suitable crRNA are provided in a 1:1 stoichiometric ratio (i.e., equimolar amounts). EXAMPLES

[0076] [Example 1] DNA and Amino Acid Sequences of Wild-Type As Cpf1 Polypeptides Encoded in Isolated Nucleic Acid Vectors The following list shows wild type (WT) As Cpf1 nucleases expressed as polypeptide fusion proteins described in the present invention. It will be understood by those skilled in the art that in many cases, more than one codon can code for the same amino acid, and therefore many different DNA sequences can code for / express the same amino acid (AA) sequence. The DNA sequences shown below are given by way of example only, and other DNA sequences that code for the same protein (e.g., the same amino acid sequence) are contemplated. It will be further understood that additional features, elements, or tags, such as NLS domains, may be added to the sequence. An example is shown for WT AsCpf1, showing the amino acid and DNA sequences for these proteins as Cpf1 alone and Cpf1 fused with both C- and N-terminal SV40NLS domains and HIS tags. Amino acid sequences representing NLS sequences, domain linkers, or purification tags are shown in bold font.

[0077] [ka] TIFF2025011171000003.tif206160

[0078] [ka] TIFF2025011171000005.tif91160

[0079] [ka] TIFF2025011171000007.tif181162

[0080] [ka] TIFF2025011171000009.tif230161

[0081] [ka] TIFF2025011171000011.tif101157

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[0083] [ka] TIFF2025011171000014.tif90162

[0084] [ka]

[0085] [ka] TIFF2025011171000017.tif86158

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[0087] [ka] TIFF2025011171000020.tif244167TIFF2025011171000021.tif54160

[0088] [ka]

[0089] [Example 2] Preparation of isolated vectors expressing nucleic acids encoding human codon-optimized AsCpf1 polypeptide fusion proteins and human cell lines stably expressing AsCpf1 polypeptide fusion proteins.

[0090] The amino acid reference for AsCpf1 has been published. See Zetsche, B., Gootenberg, JS, Abudayyeh, OO, Slaymaker, IM, Makarova, KS, Essletzbichler, P., Volz, SE, Joung, J., van der Oost, J., Regev, A., Koonin, EV, and Zhang, F. (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:1-13. A plasmid encoding human codon-optimized AsCpf1, an adjacent nuclear localization signal (NLS) and a 5'-V5 epitope tag was generated by the Synthetic Biology department at Integrated DNA Technologies. Flanking the expression cassette were 5'XhoI and 3'EcoRI restriction enzyme sites (Figure 2). The Cpf1 plasmid was digested with XhoI and EcoRI (NEB), gel purified using a column-based purification system (Qiagen), and ligated into a pre-digested mammalian expression vector, pcDNA3.1- from Life Technologies, using T4 DNA ligase (NEB) (Figure 3). The resulting ligated construct was transformed into DH5α chemically competent E. coli cells. The resulting colonies were grown overnight at 37°C in LB medium and DNA isolation was performed using a Promega miniprep plasmid DNA kit. Flanking primers (T7 forward and BGH reverse) as well as 10 internal Cpf1-specific primers were used for sequence verification of the correct insertion using automated Sanger sequencing with BigDye Terminator reagent (ABI). The nucleic acid sequence of the Cpf1 clone utilized herein is shown in SEQ ID NO:15. The amino acid sequence of the expressed recombinant protein is shown in SEQ ID NO:16.

[0091] The AsCpf1-pcDNA3.1 vector was linearized with PvuI (NEB) located within the ampicillin resistance gene and transfected into HEK293 cells. Transfection utilized 500,000 HEK293 cells seeded in a 100 mm dish 24 hours prior to transfection. The linearized vector containing AsCpf1 and the neomycin resistance gene was complexed and transfected into adherent cells using the transfection reagent TransIT-X2 (Mirus). The transfection medium was removed after 24 hours and the cells were cultured in complete medium for 48 hours. Using a previously optimized method for the generation of stable transgenic HEK293 cells containing stably integrated pcDNA3.1(-) vector neomycin resistance, we cultured the transfected cells in the presence of the antibiotic geneticin (G418; Gibco), a neomycin analog, in complete medium to select for cells transfected with AsCpf1-pcDNA3.1(-) and therefore resistant to this antibiotic. The initial G418 administration was at 800 μg / ml with regular medium changes until viable cells began to recover and proliferate over 10 days. The parental HEK293 cell line was confirmed to be sensitive to the lowest dose of G418. The resulting polyclonal AsCpf1-pcDNA3.1(-) cell line, which showed G418 resistance, was split using limiting dilution. Cells were trypsinized, resuspended in complete medium, counted to determine concentration, and diluted in 96-well plates to a concentration of theoretically less than one cell per well.

[0092] At this time, an aliquot of cells was taken and lysed with protein lysis buffer (RIPA) to determine whether AsCpf1 was expressed by Western blot. Cellular protein was quantified using Bio-Rad Protein Assay (Bio-Rad) and 15 μg of total protein was loaded onto an SDS-PAGE Stainfree 4-20% gradient gel (Bio-Rad). As a positive control, protein from the previous cell line, SpyCas9-pcDNA3.1(-), was run in parallel for size and expression comparison. Gels were run at 180 volts for 45 minutes and transferred to PVDF membranes using a Bio-Rad TransBlot for 7 minutes. Blots were then blocked in SuperBlock T20 Blocking Buffer (Thermo) followed by blocking with a 1:1000 dilution of V5 primary antibody (Abcam) and 1:5000 of β-actin primary antibody (Abcam) for 1 hour at room temperature. The blots were then washed three times for 15 min each in Tris-buffered saline with Tween-20 (TBST). A goat anti-mouse HRP secondary antibody was used at a 1:3000 dilution along with the ladder-specific StrepTactin secondary antibody and incubated for 1 h at room temperature. The blots were then washed three times for 15 min in TBST. Luminescence detection was performed using Pierce West-Femto ECL (Thermo) substrate and the results are shown in Figure 4, confirming expression of recombinant proteins of the expected size.

[0093] Cells were grown continuously under selection in G418-containing medium to expand individual cells (monoclonal colonies). Surviving colonies were characterized for the presence of AsCpf1 by RT-qPCR, Western blotting and functional testing of crRNA-guided dsDNA cleavage. Four RT-qPCR assays were designed to detect different positions within the large AsCpf1 mRNA. The sequences are shown in Table 1 below.

[0094] [Table 1]

[0095] Monoclonal cell lines resistant to G418 were seeded in 6-well plates and cultured for 24 hours. Cells were lysed with GITC-containing buffer and RNA was isolated using Wizard 96-well RNA isolation binding plates (Promega) on a Corbett liquid-handling robot. A liquid-handling robot (Perkin Elmer) was used to synthesize complementary DNA (cDNA) using SuperScriptII (Invitrogen) and qPCR assays were set up using Immolase (Bioline) with 500 nmol primers and 250 nmol probe (IDT). qPCR plates were run on an AB7900-HT and analyzed using associated software (Applied Biosystems). Figure 5 shows the relative levels of AsCpf1 mRNA expression normalized to HPRT1 expression for a range of clonal lines. Not surprisingly, different clones showed different levels of AsCpf1 mRNA expression.

[0096] Total protein was isolated from the same AsCpf1-expressing monoclonal cell line in culture grown in parallel. Cells were lysed in RIPA buffer in the presence of proteinase inhibitors. Protein concentration in each lysate was determined by BCA assay (Pierce). Fifteen micrograms of total protein from each sample was loaded onto an SDS-PAGE stainfree 4-20% gradient gel (Bio-Rad) and run for 45 min at 180 V in 1× Tris / glycine running buffer along with a broad range molecular weight marker (Bio-Rad). Proteins were transferred to PDVF membranes using a Bio-Rad TransBlot transfer unit for 7 min. Blots were blocked in SuperBlock T20 Blocking Buffer (Thermo) and subsequently incubated with a 1:1000 dilution of V5 primary antibody (Abcam) and a 1:5000 dilution of β-actin primary antibody (Abcam) for 1 h at room temperature. Blots were washed three times for 15 min each in Tris-buffered saline with Tween-20 (TBST). Goat anti-mouse HRP secondary antibody was used at 1:3000 dilution with ladder-specific StrepTactin secondary antibody and incubated for 1 h at room temperature. Blots were then washed three times for 15 min in TBST. Luminescence detection was performed using Pierce West-Femto ECL (Thermo) substrate. Figure 6 shows detection of V5-tagged AsCpf1 recombinant protein expression levels in 10 monoclonal cell lines. There is good agreement between the observed protein levels seen in Figure 6 and the corresponding mRNA levels from the same cell lines shown in Figure 5.

[0097] Three monoclonal AsCpf1 stable cell lines (1A1, 2A2 and 2B1) were expanded and tested for their ability to support AsCpf1-directed genome editing. Based on previously determined AsCpf1 mRNA and protein levels, 1A1 is a "high" expresser, 2A2 is a "medium" expresser, and 2B1 is a "low" expresser. The cell lines were transfected with six different crRNAs targeting different sites within the exons of the human HRPT1 gene, as shown in Table 2 below. The crRNAs contain a universal 20-base Cpf1-binding domain at the 5' end and a 24-base target-specific protospacer domain at the 3' end.

[0098] [Table 2]

[0099] In the reverse transfection format, anti-HPRT1 crRNA was mixed individually with Lipofectamine RNAiMAX (Life Technologies) and transfected into each of the three HEK-Cpf1 cell lines. Transfections were performed with 40,000 cells per well in a 96-well plate format. RNA was introduced at a final concentration of 30 nM in 0.75 μl of lipid reagent. Cells were incubated at 37° C. for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified using KAPA HiFi DNA polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO: 394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO: 395). PCR products were melted and reannealed in NEB buffer 2 (New England Biolabs) to allow heteroduplex formation, followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37° C. Digested products were visualized in a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of cleavage products / (average molar concentration of cleavage products+molar concentration of uncut band)×100. Cleavage efficiencies observed in the three cell lines are shown in Table 3 below.

[0100] [Table 3]

[0101] As expected, different crRNAs targeting different sites in HPRT1 showed different levels of gene editing activity. In cell line 1A1, this ranged from 18% to 73%. The "high" and "medium" Cpf1 expressing clones 1A1 and 2A2 showed nearly identical gene editing activity, indicating that both clones expressed Cpf1 at a sufficient level to reach maximum gene editing activity at each site. Clone 2B1, a "low" expressing clone, showed reduced editing activity. Thus, both clones 1A1 and 2A2 are suitable for Cpf1 crRNA optimization and site screening.

[0102] [Example 3] Length optimization of crRNA: testing truncation of the 5'-20 base universal loop domain.

[0103] A set of six sites in the human HPRT1 gene was selected to study the length optimization of AsCpf1 crRNA. A set of all crRNAs were synthesized with a 3'-24 base target specific protospacer domain and with 20, 19, 18 and 17 base 5'-loop domains, representing a set of series of 1 base deletions from the 5' end. A second set of all crRNAs were synthesized at the same sites with a 3'-21 base target specific protospacer domain and with 20, 19, 18 and 17 base 5'-loop domains as well.

[0104] A HEK cell line stably expressing AsCpf1 endonuclease was utilized in these studies (Example 2). In the reverse transfection format, anti-HPRT1 crRNA was individually mixed with Lipofectamine RNAiMAX (Life Technologies) and transfected into HEK-Cpf1 cell line. Transfection was performed with 40,000 cells per well in a 96-well plate format. RNA was introduced at a final concentration of 30 nM in 0.75 μl lipid reagent. Cells were incubated at 37° C. for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified using KAPA HiFi DNA polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO: 394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO: 395). PCR products were melted and reannealed in NEB buffer 2 (New England Biolabs) to allow heteroduplex formation, followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. Digested products were analyzed using a Fragment Analyzer (Advanced Analytical The cleavage of targeted DNA was visualized on a 3D RT-PCR machine (Microfluidics Technology). The percent cleavage of targeted DNA was calculated as the average molar concentration of cleavage products / (average molar concentration of cleavage products+molar concentration of uncleaved band)×100. The results are shown in Table 4 below and demonstrate that 20 and 19 base long 5'-universal loop domains function well, but when 18 or 17 base loop domains are utilized, there is a significant loss of activity. The observations are nearly identical whether 24 base or 21 base protospacer domains are utilized.

[0105] [Table 4] TIFF2025011171000027.tif243163TIFF2025011171000028.tif120168

[0106] [Example 4] Length optimization of crRNA: testing truncation of the 3'-24 base target-specific protospacer domain.

[0107] The same set of six sites in the human HPRT1 gene was used to study the effect of truncations in the 3'-protospacer (target-specific) domain. A series of AsCpf1 crRNAs were synthesized all with the same 5'-20 base universal loop domain. These were paired with 21, 19, 18 or 17 base 3' target-specific protospacer domains with a series of deletions from the 3' end.

[0108] A HEK cell line stably expressing AsCpf1 endonuclease was utilized in these studies (Example 2). In the reverse transfection format, anti-HPRT1 AsCpf1 crRNA was individually mixed with Lipofectamine RNAiMAX (Life Technologies) and transfected into HEK-Cpf1 cell line. Transfection was performed with 40,000 cells per well in a 96-well plate format. RNA was introduced at a final concentration of 30 nM in 0.75 μl lipid reagent. Cells were incubated at 37° C. for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified using KAPA HiFi DNA polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO: 394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO: 395). PCR products were melted and reannealed in NEB buffer 2 (New England Biolabs) to allow heteroduplex formation, followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. Digested products were analyzed using a Fragment Analyzer (Advanced Analytical The results were visualized in a 3D Immunofluorescence Spectroscopy (3D Imaging Technologies). The percent cleavage of targeted DNA was calculated as the average molar concentration of cleavage products / (average molar concentration of cleavage products+molar concentration of uncleaved band)×100. The results are shown in Table 5 below and demonstrate that a 21-base long 3'-protospacer (target-specific) domain functions well, but when this domain is shortened, loss of activity is observed in a sequence / site-dependent manner. Although some highly active sites (such as 38351) maintain recognized activity even when truncated to 17 bases, a 21-base protospacer is recommended to maintain the highest probability of functionality at all sites.Therefore, a consistent minimal length AsCpf1 crRNA is 41 bases, containing a 20 base 5'-universal loop domain and a 21 base 3'-protospacer target specific domain.

[0109] [Table 5] TIFF2025011171000030.tif213167

[0110] [Example 5] Walk through two AsCpf1 crRNAs with single base 2'OMe modification.

[0111] Two sites in the human HPRT1 gene were chosen to study the effect of replacing a single RNA residue with a 2'OMe-RNA residue at all possible positions in the AsCpf1 crRNA (38351 and 38595). Given the possibility of sequence-specific tolerance to modifications, it was necessary to perform this screening at two sites. In a single-base step, a series of crRNAs with a single 2'OMe residue at all possible positions were synthesized. The crRNAs were either 44 bases long or 41 bases long. All had a 5'-terminal 20-base universal loop domain followed by a 3'-terminal 21- or 24-base protospacer target-specific domain.

[0112] A HEK cell line stably expressing AsCpf1 endonuclease was utilized in these studies (HEK-Cpf1) (Example 2). In the reverse transfection format, anti-HPRT1 crRNA was individually mixed with Lipofectamine RNAiMAX (Life Technologies) and transfected into HEK-Cpf1 cell line. Transfection was performed with 40,000 cells per well in a 96-well plate format. RNA was introduced at a final concentration of 30 nM in 0.75 μl lipid reagent. Cells were incubated at 37° C. for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified using KAPA HiFi DNA polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO: 394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO: 395). PCR products were melted and reannealed in NEB buffer 2 (New England Biolabs) to allow heteroduplex formation, followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. Digested products were analyzed using a Fragment Analyzer (Advanced Analytical The results were visualized in a 3D Imaging Technologies system. The percent cleavage of targeted DNA was calculated as the average molar concentration of cleavage products / (average molar concentration of cleavage products+molar concentration of uncleaved band)×100. The results for HPRT1 site 38351 are shown in Table 6 below, and the results for HRPT1 site 38595 are shown in Table 7 below. The results demonstrate the location of sites that, when replaced with RNA residues by 2'OMe modifications, reduce or completely abolish the activity of Cpf1 to cleave dsDNA. The results are nearly identical whether a 24-base or 21-base protospacer domain is utilized.

[0113] Sites where substitution of 2'OMe RNA residues for RNA residues showed loss of activity in genome editing assays were mapped to positions within the 5'-universal loop domain or the 3' target-specific protospacer domain. The results are summarized in Figure 7. Modification of residues A2, A3, U4, U11, G15 and U20 within the universal loop domain resulted in loss of activity; the same sites were identified for all four crRNA classes studied (site 38351 44mer, site 38351 41mer, site 38595 44mer and site 38595 41mer). In contrast, the exact pattern of modification effects varied for sites within the protospacer domain, which is expected since modification tolerance varies with sequence context and it is common for protospacer domains to have different sequences for all target sites. For the sequences studied, positions 5, 6, 13, 16 and 18 showed loss of activity upon modification for all four crRNA classes, thus identifying positions to avoid 2'OMe RNA chemical modifications.

[0114] [Table 6] TIFF2025011171000032.tif246164TIFF2025011171000033.tif244156TIFF20250111710 00034.tif243155TIFF2025011171000035.tif244157TIFF2025011171000036.tif206169

[0115] [Table 7] TIFF2025011171000038.tif239155TIFF2025011171000039.tif243157TIFF20250111710 00040.tif243155TIFF2025011171000041.tif244157TIFF2025011171000042.tif102163

[0116] [Example 6] Modification of blocks of sequences in AsCpf1 crRNA.

[0117] Three sites in the human HPRT1 gene were selected to study the effect of replacing blocks of RNA residues in the AsCpf1 crRNA with 2'OMe-RNA, 2'F RNA or LNA residues (38351, 38595 and 38104). Modification of internucleotide linkages with phosphorothioate linkages (PS) and non-nucleotide terminal modifiers were also tested. The crRNAs were either 44 or 41 bases long. All had a 5'-terminal 20-base universal loop domain followed by a 3'-terminal 21 or 24-base protospacer target specific domain.

[0118] A HEK cell line stably expressing AsCpf1 endonuclease was utilized in these studies (HEK-Cpf1) (Example 2). In the reverse transfection format, anti-HPRT1 crRNA was individually mixed with Lipofectamine RNAiMAX (Life Technologies) and transfected into HEK-Cpf1 cell line. Transfection was performed with 40,000 cells per well in a 96-well plate format. RNA was introduced at a final concentration of 30 nM in 0.75 μl lipid reagent. Cells were incubated at 37° C. for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified using KAPA HiFi DNA polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO: 394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO: 395). PCR products were melted and reannealed in NEB buffer 2 (New England Biolabs) to allow heteroduplex formation, followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37° C. Digested products were visualized in a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of cleaved products / (average molar concentration of cleaved products+molar concentration of uncut band)×100. The results are shown in Table 8 below.

[0119] Large blocks of the universal 5-loop domain can be modified and retain activity (14 / 20 bases). However, the target-specific 3'-protospacer domain shows a significant loss of activity when 2-3 consecutive 2'OMe residues are replaced with RNA residues, even though these positions showed no loss of activity in single base walks (Example 5). The modification patterns in the protospacer domain are expected to be influenced by the sequence context, such that in many cases one modification pattern works well for one sequence but not for another. The modification map shown in Figure 7 shows modification patterns that range from minimal to high levels of modification, showing high performance at some sites and likely usable regardless of sequence context.

[0120] 2'F residues could be placed at any position that is tolerant to 2'OMe modification. LNA residues could also be placed within the AsCpf1 crRNA, and the use of terminal modifiers is shown in Table 8 below. Phosphorothioate (PS) internucleotide linkages confer nuclease resistance and can be placed at the ends of the crRNA to block exonuclease attack or in the central region to block endonuclease attack. Modification of large blocks of the crRNA with PS linkages (such as full modification of the loop domain or protospacer domain) is not compatible with crRNA function, and significant loss of activity is seen when this modification pattern is utilized. Limited use such as 2-3 internucleotide linkages at each end can be effectively utilized, and such patterns are useful for blocking exonuclease attack. Non-basic modifiers (such as the C3 spacer propanediol group or the ZEN modifier naphthyl-azo group) can be placed at one or both ends of the crRNA without loss of activity, and also block exonuclease attack.

[0121] [Table 8] TIFF2025011171000044.tif238157TIFF2025011171000045.tif241156TIFF2025011171000046.tif123167

[0122] [Example 7] Use of modified crRNA with AsCpf1 protein delivered as an RNP complex.

[0123] A site in the human HPRT1 gene (38104) was chosen to study the ability to use chemically modified crRNA with AsCpf1 protein to perform genome editing in HEK-293 cells using electroporation to deliver the ribonucleoprotein (RNP) complex into the cells.

[0124] Purified recombinant AsCpf1 protein, isolated from E. coli using standard techniques, was utilized in this example. The amino acid sequence of the recombinant protein is shown in SEQ ID NO:12.

[0125] AsCpf1 crRNA was heated to 95°C for 5 min and then cooled to room temperature. crRNA was mixed with AsCpf1 protein at a 1.2:1 RNA:protein molar ratio in phosphate-buffered saline (PBS) (for a single transfection, 202 pmoles of RNA with 168 pmoles of protein in a volume of 6 μL). RNP complexes were allowed to form for 15 min at room temperature. HEK293 cells were resuspended after trypsinization, washed in medium, and washed twice in PBS before use. Cells were diluted to 3.5 × 10 in 20 μL of Nucleofection solution. 5Cells were resuspended at a final concentration of 10 ... Genomic DNA was amplified using KAPA HiFi DNA polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO: 394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO: 395). PCR products were melted and reannealed in NEB buffer 2 (New England Biolabs) to allow heteroduplex formation, followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. Digested products were visualized in a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of cleaved products / (average molar concentration of cleaved products + molar concentration of uncleaved band) x 100. The results are shown in Table 9 below. As shown below, AsCpf1 with low or high levels of modifications was The crRNA is adapted for delivery by electroporation as an RNP complex to mediate genome editing in mammalian cells.

[0126] [Table 9]

[0127] [Example 8] Use of modified crRNA with AsCpf1 expression plasmid in E. coli A site in the human HPRT1 gene (38346) was cloned into an E. coli plasmid and used to study the ability to use chemically modified crRNA to perform site-specific cleavage in E. coli cells. AsCpf1 was expressed from a plasmid. Electroporation was used to deliver both the AsCpf1 expression plasmid and the chemically synthesized crRNA.

[0128] The AsCpf1 protein was expressed from a plasmid in this example using the phage T7 promoter and standard E. coli translation elements. The amino acid sequence of the expression construct is shown in SEQ ID NO: 16).

[0129] AsCpf1 crRNA was heated to 95°C for 5 min and then cooled to room temperature. The crRNA and AsCpf1 plasmid were mixed in TE (60 femtomoles of AsCpf1 plasmid with 400 picomoles of RNA in a volume of 5 μL for a single transformation) and added directly to 20 μL of competent E. coli cells. Bacterial strains in which survival was linked to successful cleavage by Cpf1 were made competent by growing cells to mid-log phase and washing three times in ice-cold 10% glycerol to a final suspension in 1:100 volume of 10% glycerol. Electroporation was performed by adding 25 μL of the transformation mixture to a pre-chilled 0.1 cm electroporation cuvette and pulsing with a 1.8 kV exponential decay. After electroporation, 980 μL of SOB medium was added to the electroporation cuvette with mixing, and the resulting cell suspension was transferred to a sterile 15 ml culture tube. The cells were incubated with shaking (250 rpm) at 37° C. for 1.5 h, at which time IPTG (1 mM) was added, followed by an additional incubation with shaking at 37° C. After incubation, the cells were plated onto selection medium to assess survival.

[0130] This example demonstrates that chemically modified synthetic crRNA can be used with Cpf1 for gene editing in bacteria. However, high efficiency is only seen when using more extensively modified RNA with exonuclease-blocking PS internucleotide linkages. The modification patterns that work best in bacterial cells work poorly in mammalian cells (Table 10).

[0131] [Table 10] TIFF2025011171000049.tif160163

[0132] [Example 9] DNA and Amino Acid Sequences of Wild-Type Lb Cpf1 Polypeptides Encoded in Isolated Nucleic Acid Vectors The following list shows wild-type (WT) Lb Cpf1 nuclease expressed as a polypeptide fusion protein described in the present invention. It will be understood by those skilled in the art that in many cases, more than one codon can code for the same amino acid, and therefore many different DNA sequences can code for / express the same amino acid (AA) sequence. The DNA sequences shown below serve only as examples, and other DNA sequences that code for the same protein (e.g., the same amino acid sequence) are contemplated. It will be further understood that additional features, elements, or tags, such as an NLS domain, may be added to the sequence.

[0133] Examples are shown for WT LbCpf1 showing the amino acid and DNA sequences for these proteins as LbCpf1 alone and as LbCpf1 fused with an N-terminal V5 tag, an N-terminal SV40 NLS domain, a C-terminal SV40 NLS domain and a C-terminal 6xHis tag.

[0134] [ka] TIFF2025011171000051.tif94160

[0135] [ka] TIFF2025011171000053.tif42156

[0136] [ka] TIFF2025011171000055.tif109157

[0137] [ka]

[0138] [ka] TIFF2025011171000058.tif57160

[0139]

change

[0140]

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[0141]

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[0142]

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[0143]

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[0144]

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[0145]

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[0146]

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[0147] [Example 10] Use of modified crRNA with LbCpf1 protein delivered as an RNP complex.

[0148] Twelve sites in the human HPRT1 gene, 38094-S (SEQ ID NO: 358), 38104-S (SEQ ID NO: 361), 38115-AS (SEQ ID NO: 364), 38146-AS (SEQ ID NO: 367), 38164-AS (SEQ ID NO: 370), 38164-S (SEQ ID NO: 372), 38186-S (SEQ ID NO: 376), 38228-S (SEQ ID NO: 379), 38330-AS (SEQ ID NO: 382), 38343-S (SEQ ID NO: 385), 38455-S (SEQ ID NO: 388), and 38486-S (SEQ ID NO: 391), (where A and AS represent the sense and antisense strands, respectively), were selected to study the target editing activity of LbCpf1 in comparison with the target editing activity of AsCpf1 and SpyCas9. A study was performed to compare the ability to use chemically modified crRNA with LbCpf1 protein to perform genome editing in HEK-293 cells using electroporation to deliver the ribonucleoprotein (RNP) complex to the cells.

[0149] Purified recombinant LbCpf1 protein, isolated from E. coli using standard techniques, was utilized in this example. The amino acid sequence of the recombinant protein is shown in SEQ ID NO:14.

[0150] LbCpf1 crRNA and AsCpf1 control crRNA were heated to 95°C for 5 min and then cooled to room temperature. crRNA was mixed with LbCpf1 or AsCpf1 at a 1:1 RNA:protein molar ratio in PBS (5 μM RNP complex in a volume of 10 μL for a single transfection). RNP complexes were allowed to form for 15 min at room temperature. HEK293 cells were resuspended after trypsinization, washed in medium, and washed a second time in PBS before use. Cells were diluted to 3.5 × 10 in 20 μL of Nucleofection solution. 5Cells were resuspended at a final concentration of 1000 x 1000 cells. 20 μL of cell suspension was placed in a V-bottom 96-well plate and 5 μL of Cpf1 RNP complex was added to each well (5 μM final concentration) and 3 μM of Cpf1 electroporation enhancer solution was added to each well (Integrated DNA Technologies). 25 μL of the final mixture was transferred to each well of a 96-well Nucleocuvette electroporation module. Cells were electroporated using the Amaxa 96-well shuttle protocol, program 96-DS-150. After electroporation, 75 μL of medium was added to each well and 25 μL of the final cell mixture was transferred to 175 μL of pre-warmed medium in a 96-well incubation plate (final volume 200 μL). Cells were incubated at 37° C. for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified using KAPA HiFi DNA polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO: 394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO: 395)). PCR products were melted and reannealed in NEB buffer 2 (New England Biolabs) to allow heteroduplex formation, followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. Digested products were visualized in a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of cleaved products / (average molar concentration of cleaved products + molar concentration of uncut band) x 100. Sequences are shown in Table 10 and results are graphically depicted in Figure 9.

[0151] [Table 11] TIFF2025011171000074.tif131156

[0152] Biological material deposit information The cell lines described herein (e.g., 1A1, 2A2, and 2B1) have been deposited at the American Type Culture Collection (ATCC), located at 10801 University Blvd, Manassas, VA 20110, and assigned the following accession number to _____.

[0153] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0154] The use of the terms "a," "an," and "the," and similar referents in the context of describing the present invention (particularly in the context of the claims below) shall be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" shall be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The recitation of ranges of values ​​herein is only intended to serve as a shorthand method of individually referring to each separate value contained within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the invention, and does not pose a limitation on the scope of the invention, unless otherwise required. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0155] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors expect that those of skill in the art will employ such variations as appropriate, and the inventors intend for the invention to be carried out in ways other than those specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited within the scope of the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. An isolated nucleic acid encoding an As Cpf1 polypeptide that is codon-optimized for expression in H. sapiens.

2. The isolated nucleic acid of claim 1 comprising SEQ ID NO:

15.

3. An isolated polypeptide encoding a wild-type As Cpf1 protein.

4. 4. The isolated polypeptide of claim 3 comprising SEQ ID NO:

12.

5. An isolated expression vector encoding SEQ ID NO:

15.

6. A host cell comprising an isolated expression vector encoding SEQ ID NO:15, wherein the isolated expression vector encoding SEQ ID NO:15 is operably linked to a suitable promoter to enable expression of a polypeptide comprising SEQ ID NO:

12.

7. The host cell of claim 6 , comprising a human cell.

8. The host cell of claim 7 , wherein the human cell comprises an immortalized cell line.

9. The host cell of claim 8, wherein the immortalized cell line is a HEK293 cell line.

10. 7. The host cell line of claim 6, further comprising an isolated AsCpf1 crRNA capable of forming a ribonucleoprotein complex with a polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:16 and SEQ ID NO:19 to form a wild-type CRISPR / Cpf1 endonuclease.

11. AsCpf1 polypeptide, and Appropriate AsCpf1 crRNA An isolated CRISPR / Cpf1 endonuclease system comprising:

12. 12. The isolated CRISPR / Cpf1 endonuclease system of claim 11, wherein the AsCpf1 polypeptide comprises SEQ ID NO:

12.

13. The isolated CRISPR / Cpf1 endonuclease system of claim 11, wherein the suitable AsCpf1 crRNA is selected from a length-truncated AsCpf1 crRNA, or a chemically modified AsCpf1 crRNA, or an AsCpf1 crRNA that includes both length truncation and chemical modification.

14. An isolated CRISPR / Cpf1 endonuclease system comprising a human cell line expressing an AsCpf1 polypeptide and an appropriate AsCpf1 crRNA.

15. 15. The isolated CRISPR / Cpf1 endonuclease system of claim 14, wherein the AsCpf1 polypeptide comprises SEQ ID NO:

12.

16. The isolated CRISPR / Cpf1 endonuclease system of claim 14, wherein the suitable AsCpf1 crRNA is selected from a length-truncated AsCpf1 crRNA, or a chemically modified AsCpf1 crRNA, or an AsCpf1 crRNA that includes both length truncation and chemical modification.

17. An isolated AsCpf1 crRNA that is active in the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein endonuclease system.

18. 18. The isolated AsCpf1 crRNA of claim 17, selected from a length-truncated AsCpf1 crRNA, a chemically modified AsCpf1 crRNA, or an AsCpf1 crRNA that contains both a length truncation and a chemical modification.

19. The isolated AsCpf1 crRNA of claim 17, which is a shortened AsCpf1 crRNA comprising a 5'-universal loop domain of 19-20 nucleotides in length and a 3' target-specific protospacer domain of 19-21 nucleotides in length.

20. 18. The isolated AsCpf1 crRNA of claim 17, comprising both length truncation and chemical modification.

21. 21. The isolated AsCpf1 crRNA of claim 20, wherein the chemical modification is selected from the group consisting of an end group modification (e.g., a C3 spacer), a 2'OMe modification, a 2'-fluoro modification, and an LNA modification.

22. A method of performing gene editing comprising contacting a candidate editing target site locus with an active CRISPR / Cpf1 endonuclease system having a wild-type AsCpf1 polypeptide and an appropriate AsCpf1 crRNA.

23. 23. The method of claim 22, wherein the wild-type AsCpf1 polypeptide is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:16 and SEQ ID NO:

19.

24. 23. The method of claim 22, wherein the suitable AsCpf1 crRNA is selected from a length-truncated AsCpf1 crRNA, a chemically modified AsCpf1 crRNA, or an AsCpf1 crRNA that includes both a length truncation and a chemical modification.

25. An isolated nucleic acid encoding an Lb Cpf1 polypeptide that is codon-optimized for expression in humans.

26. 26. The isolated nucleic acid of claim 25, comprising SEQ ID NO: 17 or SEQ ID NO:

396.

27. An isolated polypeptide encoding a wild-type Lp Cpf1 protein.

28. 28. The isolated polypeptide of claim 27 comprising SEQ ID NO: 14 or SEQ ID NO:

24.

29. An isolated expression vector encoding SEQ ID NO:17 or SEQ ID NO:

396.

30. A host cell comprising an isolated expression vector encoding SEQ ID NO:17 or SEQ ID NO:396, wherein the isolated expression vector encoding SEQ ID NO:17 or SEQ ID NO:396 is operably linked to a suitable promoter to enable expression of a polypeptide comprising SEQ ID NO:14 or SEQ ID NO:24, respectively.

31. The host cell of claim 30 , comprising a human cell.

32. The host cell of claim 31 , wherein the human cell comprises an immortalized cell line.

33. 33. The host cell of claim 32, wherein the immortalized cell line is a HEK293 cell line.

34. 31. The host cell line of claim 30, further comprising an isolated Lb Cpf1 crRNA capable of forming a ribonucleoprotein complex with a polypeptide selected from the group consisting of SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:20 and SEQ ID NO:24 to form a wild-type CRISPR / Cpf1 endonuclease.

35. Lb Cpf1 polypeptide and Appropriate Cpf1 crRNA An isolated CRISPR / Cpf1 endonuclease system comprising:

36. 36. The isolated CRISPR / Cpf1 endonuclease system of claim 35, wherein the Lb Cpf1 polypeptide comprises SEQ ID NO:

14.

37. 36. The isolated CRISPR / Cpf1 endonuclease system of claim 35, wherein the suitable Cpf1 crRNA is selected from a length-truncated Cpf1 crRNA, or a chemically modified Cpf1 crRNA, or a Cpf1 crRNA that includes both length truncation and chemical modification.

38. An isolated CRISPR / Cpf1 endonuclease system comprising a human cell line expressing an Lb Cpf1 polypeptide and an appropriate Cpf1 crRNA.

39. 39. The isolated CRISPR / Cpf1 endonuclease system of claim 38, wherein the Lb Cpf1 polypeptide comprises SEQ ID NO:14 or SEQ ID NO:

24.

40. 39. The isolated CRISPR / Cpf1 endonuclease system of claim 38, wherein the suitable Cpf1 crRNA is selected from a length-truncated Cpf1 crRNA, or a chemically modified Cpf1 crRNA, or a Cpf1 crRNA that includes both length truncation and chemical modification.

41. A method of performing gene editing comprising contacting a candidate editing target site locus with an active CRISPR / Cpf1 endonuclease system having a wild-type Lb Cpf1 polypeptide and an appropriate Cpf1 crRNA.

42. 42. The method of claim 41, wherein the wild-type Lb Cpf1 polypeptide is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:20 and SEQ ID NO:

24.

43. 42. The method of claim 41 , wherein the suitable Cpf1 crRNA is selected from a length-truncated Cpf1 crRNA, a chemically modified Cpf1 crRNA, or a Cpf1 crRNA that includes both length truncation and chemical modification.

44. The method of claim 41, wherein the suitable Cpf1 crRNA is a truncated Cpf1 crRNA comprising a 5'-universal loop domain of 19-20 nucleotides in length and a 3' target-specific protospacer domain of 19-21 nucleotides in length.

45. The method of claim 41 , wherein the suitable Cpf1 crRNA comprises both a length truncation and a chemical modification.

46. 46. ​​The method of claim 45, wherein the chemical modification is selected from the group consisting of an end group modification (eg, a C3 spacer), a 2'OMe modification, a 2'-fluoro modification, and an LNA modification.

Images