Novel mutations that increase DNA cleavage activity of amino acid coccus cpF1

Abstract

The present disclosure relates to polynucleotides and amino acids of the Aminococcus sp. Cas12a (Cpf1) and methods for their use in genome editing in eukaryotic cells.

Description

[0001] This application is a divisional application of the Chinese application with application number 201980066143.3, application date August 8, 2019, and invention name “Novel mutation for improving the DNA cleavage activity of Acidococcus CPF1”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application is based on 35 U.S.C.119 and claims U.S. Provisional Patent Application Serial No. 62 / 870,268, filed on July 3, 2019, entitled “OPTIMIZED CAS12A (CPF1) PROTEINS FOR EFFICIENT GENOME EDITING IN EUKARYOTIC CELLS”, U.S. Provisional Patent Application Serial No. 62 / 749,607, filed on October 23, 2018, entitled “DEEP-SCANNING MUTAGENESISUNCOVERS NOVEL MUTATIONS THAT ENHANCE THE DNA CLEAVAGE ACTIVITY OF ACIDAMINOCOCCUS SP.CAS12A / CAS12A AT NON-CANONICAL TTTT PAM SITES”, and U.S. Provisional Patent Application Serial No. 62 / 749,607, filed on August 8, 2018, entitled “NOVEL MUTATIONS THAT ENHANCE THE DNA CLEAVAGE ACTIVITY OF ACIDAMINOCOCCUS SP.CAS12A / CAS12A AT NON-CANONICAL TTTT PAM SITES” SP.CPF1", the contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0004] The present invention relates to the ability to cut (cleave) double-stranded DNA of living organisms at precise locations using the CRISPR / Cas12a (Cpf1) nuclease system. Specifically, a series of recombinant Cas12a proteins useful in eukaryotic cell environments are described. Background Art

[0007] Cas12a is an RNA-guided nuclease found in bacterial species including Acidaminococcus sp. and is part of the clustered regularly spaced short palindromic repeats (CRISPR) adaptive immune system. Through target site-specific 21-24-nt complementary RNA, Cas12a is directed to 21-24-nt DNA target sequences, or commonly referred to as pre-spacer sequences. Cas12a-gRNA ribonucleoprotein (RNP) complex mediates double-stranded DNA breaks (DSBs), which are then repaired by non-homologous end joining (NHEJ, usually introducing mutations or insertion-deletion mutations (indels) at the shear site) or by homology-directed repair (HDR) systems (for precise editing, if there is a suitable template nucleic acid).

[0008] The key to identifying the correct DNA target of Cas12a includes both crRNA and the canonical "TTTV" protospacer adjacent motif (PAM), which is a 4-bp sequence immediately upstream of the protospacer. Compared with the 2-bpNGG PAM of Cas9 from Streptococcus pyogenes, Cas12a expands the targetable loci in genome editing, especially on the rich AT sites that are difficult to access by the Cas9 system. However, due to its relatively low enzymatic activity, the possibility of achieving effective genome editing for a given site is much lower than that of the Cas9 system, which limits its wider application. Therefore, only when Cas9 cannot target a genomic site, the Cas12a system is often considered as an alternative.

[0009] Protein engineering via mutagenesis can alter the PAM sequence preferences of CRISPR systems. Through structure-guided mutagenesis screening of residues near the PAM sequence, previous studies have identified two AsCpf1 variants that are compatible with TYCV and TATV PAMs, respectively. Although these variants collectively expand the targetable sites of the Cpf1 system in the human genome coding region by 3-fold, the utility of each variant remains limited due to their mutually exclusive requirements for PAM sequences (TYCV vs. TATV vs. TTTV). It would be highly desirable to identify Cpf1 variants with shorter PAMs and greater sequence flexibility without sacrificing activity at the canonical PAM site.

[0010] Therefore, it is necessary to enhance the utility of Cas12a. One aspect of the present application is to improve the utility of Cpf1 by broadening its PAM compatibility. In this regard, certain novel AsCas12a variants with increased activity have been found. Another desired goal is to maximize the delivery of bacterial proteins to the nucleus of eukaryotic cells while avoiding destruction of basic Cas12a functions. Since Cas12a is a bacterial protein, certain molecular genetic barriers must first be overcome before the protein can be successfully delivered to eukaryotic cells. The present invention provides a unique solution to achieve these goals. Summary of the invention

[0011] In a first aspect, a CRISPR-associated protein is provided, comprising a polypeptide encoding an AsCpf1 variant. The AsCpf1 variant is selected from the group consisting of: M537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R / F870L (SEQ ID NO.: 465).

[0012] In a second aspect, a CRISPR ribonucleoprotein complex is provided. The CRISPR ribonucleoprotein complex comprises a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an AsCpf1 variant. The AsCpf1 variant is selected from the group consisting of: M537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R / F870L (SEQ ID NO.: 465).

[0013] In a third aspect, a method for increasing TTT in a cell using a CRISPR ribonucleoprotein complex is provided. N A method for improving gene editing efficiency at a PAM site. The method comprises contacting a cell with a CRISPR ribonucleoprotein complex. The CRISPR ribonucleoprotein complex comprises a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an AsCpf1 variant. The AsCpf1 variant is selected from the group consisting of: M537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R / F870L (SEQ ID NO.: 465).

[0014] In a fourth aspect, a kit is provided, comprising a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an AsCpf1 variant.

[0015] In a fifth aspect, a CRISPR-associated protein is provided, comprising a polypeptide encoding an AsCas12a variant, wherein the AsCas12a variant is selected from the group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, with the proviso that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

[0016] In a sixth aspect, a CRISPR ribonucleoprotein complex is provided. The CRISPR ribonucleoprotein complex comprises a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an AsCas12a variant, wherein the AsCas12a variant is selected from the group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

[0017] In a seventh aspect, a method for improving the efficiency of gene editing at a non-canonical TTTN PAM site in a cell using a CRISPR ribonucleoprotein complex is provided. The method comprises contacting a cell with a CRISPR ribonucleoprotein complex comprising a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an AsCas12a variant, wherein the AsCas12a variant is selected from a group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

[0018] In an eighth aspect, a kit is provided, comprising a guide RNA and a CRISPR-associated protein, the CRISPR-associated protein comprising a polypeptide encoding an AsCas12a variant. The AsCas12a variant is selected from the group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTTPAM site.

[0019] In a ninth aspect, a nucleic acid encoding a CRISPR-associated protein is provided, the CRISPR-associated protein comprising a polypeptide encoding an AsCas12a variant. The AsCas12a variant is selected from the group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

[0020] In the tenth aspect, a polynucleotide sequence encoding a Cas12a polypeptide is provided. The polynucleotide sequence comprises a member selected from the group consisting of SEQ ID NO.: 5-17.

[0021] In the eleventh aspect, an amino acid sequence encoding a Cas12a polypeptide is provided. The amino acid sequence comprises a member selected from the group consisting of SEQ ID NO.: 18-30.

[0022] In a twelfth aspect, a CAS endonuclease system is provided, comprising an expression cassette encoding a polynucleotide sequence encoding a Cas12a polypeptide, comprising a member selected from the group consisting of SEQ ID NO.: 5-17.

[0023] In the thirteenth aspect, a CAS endonuclease system is provided, comprising an amino acid sequence encoding a Cas12a polypeptide. The amino acid sequence comprises a member selected from the group consisting of SEQ ID NO.: 18-30.

[0024] In a fourteenth aspect, a method for genome editing in a eukaryotic cell is provided. The method comprises the following steps: introducing a CAS endonuclease system into a eukaryotic cell, wherein the CAS endonuclease system comprises an expression cassette encoding a polynucleotide sequence, wherein the polynucleotide sequence encodes a Cas12a polypeptide, and the polynucleotide sequence comprises a member selected from the group consisting of SEQ ID NO.: 5-17.

[0025] In a fifteenth aspect, a method for genome editing in a eukaryotic cell is provided. The method comprises the following steps: introducing a CAS endonuclease system into a eukaryotic cell, wherein the CAS endonuclease system comprises an amino acid sequence encoding a Cas12a polypeptide, wherein the amino acid sequence comprises a member selected from the group consisting of SEQ ID NO.: 18-30.

[0026] In the sixteenth aspect, a CRISPR-associated protein is provided, comprising a fusion polypeptide. The fusion polypeptide comprises an AsCas12a open reading frame, a nuclear localization signal, an optional amino acid linker, and an optional affinity tag.

[0027] In the seventeenth aspect, a method for genome editing in a eukaryotic cell is provided. The method comprises the following steps: introducing a CAS endonuclease system into a eukaryotic cell, wherein the CAS endonuclease system comprises a CRISPR-associated protein according to the sixteenth aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Describe multiple recombinant forms of Cas12a, which produce multiple editing efficiencies. A series of recombinant Cas12a proteins with different compositions and arrangements of NLS sequences, purification tags and joints (AM; corresponding to SEQ ID NO.: 18-30) were cloned and purified to homogeneity. The resulting purified Cas12a derivatives compounded with RNA guides targeting HPRT-38186 (SEQ ID NO.: 1) and HPRT-38228 loci (SEQ ID NO.: 2) were delivered to HEK293 cells by electroporation. DNA was isolated from the genome editing experiment 48 hours later, and HPRT-FWD (SEQ ID NO.: 3) and HPRT-REV (SEQ ID NO.: 4) primers and Alt-R genome editing detection kit (Integrated DNA Technologies) were used to measure the editing efficiency by PCR amplification of the edited loci. The following abbreviations apply to the constructs shown: V5 refers to the V5 epitope tag (SEQ ID NO.: 485); SV40 refers to the nuclear localization signal from the simian virus SV40 large tumor antigen (SEQ ID NO.: 475); Cas12a refers to the Cas12a coding sequence; HIS refers to the hexahistidine tag (SEQ ID NO.: 487); OpT refers to the optimized nuclear localization signal (SEQ ID NO.: 477); aNLS refers to the alternative nuclear localization signal (SEQ ID NO.: 479); BIP1 refers to the first two-part nuclear localization signal (SEQ ID NO.: 481); and BIP2 refers to the second two-part nuclear localization signal (SEQ ID NO.: 483). The arrows provided in the constructs depict the transcription start site of the mRNA transcript derived from the DNA.

[0029] Figure 2The exemplary results of M537R and F870L mutations with improved Cpf1 cleavage activity in bacterial-based activity assays are shown. The screened E. coli strains were transformed with a Cpf1 expression vector and crRNA targeting the HPRT-38346 site on a toxin expression plasmid. When equal amounts of plasmids were delivered, the apparent activity of Cpf1 on TTTT or TTTC PAMs could be estimated by the number of colonies that survived under arabinose selection. Apparently, both mutations increased the survival rate at the TTTC and TTTT PAM sites, indicating that their cleavage activity was superior to WT-Cpf1.

[0030] Figure 3A An exemplary SDS-PAGE analysis of AsCpf1 variants used in in vitro cleavage assays and subsequent genome editing in human cell lines is shown. The indicated amounts of protein were loaded and no differences were observed compared to WT-Cpf1. These results suggest that the M537R and F870L mutations can increase the in vitro cleavage activity of Cpf1 at non-canonical TTTT PAM sites while maintaining high activity at canonical TTTV sites.

[0031] Figure 3B Exemplary DNA cleavage activities of Cpf1 variants at HPRT-38346 sites with TTTC or TTTT PAMs are shown. Both variants produced higher DNA cleavage percentages than WT-Cpf1 at the TTTT PAM site. These results suggest that the M537R and F870L mutations can increase the in vitro cleavage activity of Cpf1 at non-canonical TTTT PAM sites while maintaining high activity at the canonical TTTV site.

[0032] Figure 4 The summary of the results is shown, which show the exemplary results of M537R and F870L mutations, which have widely improved the genome targeting efficiency of AsCpf1 at TTTN PAM sites in human cell lines. The genome targeting efficiency of Cpf1 variants was tested in human cell line models using T7 endonuclease I assay (T7EI). Twenty-four crRNAs targeting the CTNNB1 gene with TTTN PAM were synthesized, assembled into RNP complexes together with Cpf1 variants, and delivered by nuclear transfection (Lonza). Genomic DNA was collected 48 hours after delivery to assess the formation of insertion-deletion mutations of T7EI. DNA cutting can be performed at all TTTT PAM sites (many of which cannot be targeted by WT-Cpf1), and not only that, the reported variants, particularly double mutants (M537R / F870L), also widely improved the targeting efficiency of Cpf1 at 22 of 24 test sites, regardless of PAM sequence.

[0033] Figure 5A An exemplary correlation of the relative survival of M537R / F870L-AsCas12a under condition 1 (X-axis) with the relative survival of M537R / F870L-AsCas12a under condition 2 (Y-axis) is shown. Consistent phenotypic measurements were obtained (ρ~0.7).

[0034] Figure 5B An exemplary correlation of the relative survival of M537R / F870L-AsCas12a under condition 1 (X axis) with the relative survival of WT-AsCas12a under condition 3 (Y axis) is shown. Consistent phenotypic measurements were obtained (ρ~0.7).

[0035] Figure 5C An exemplary correlation of the relative survival of M537R / F870L-AsCas12a under condition 2 (X axis) with the relative survival of WT-AsCas12a under condition 2 (Y axis) is shown. Consistent phenotypic measurements were obtained (ρ~0.7).

[0036] Figure 5D Exemplary phenotypic variant scores (relative survival) for a selected number of single point mutations of AsCas12a at positions 537 and 870 under conditions 1 and 2 are shown.

[0037] Figure 5E Exemplary phenotypic variant scores (relative survival) for a selected number of single point mutations of AsCas12a at positions 537 and 870 under conditions 1 and 2 are shown. Figure 5D and 5E M537R and F870L were shown to be the optimal substitutions at various positions, which was consistent with our previous screening and mutant characterization results.

[0038] Fig. 5F Exemplary phenotypic variant scores (relative survival) for a selected number of single point mutations of AsCas12a at positions 505, 510, 569, and 599 under conditions 1, 2, and 3 are shown.

[0039] Fig. 6A The survival rate of E. coli obtained by the DNA cleavage activity of WT-AsCas12a at TTTT PAM is shown. The cleavage activity of AsCas12a variants at the TTTT PAM site was measured by a bacteria-based activity assay. A culture dish with initial bacteria is shown on the left, while a culture dish with surviving bacterial colonies is shown on the right. The survival of E. coli under selection depends on the successful cleavage of the toxin expression plasmid containing the TTTT PAM site.

[0040] Figure 6BThe survival rate of E. coli obtained by DNA cleavage activity of L505K-AsCas12a variant at TTTT PAM is shown. The display of culture dishes and experimental details are as follows Fig. 6A shown.

[0041] Figure 6C The survival rate of E. coli obtained by DNA cleavage activity of S510L-AsCas12a variant at TTTT PAM is shown. The display of culture dishes and experimental details are as follows Fig. 6A shown.

[0042] Fig.6D The survival rate of E. coli obtained by the DNA cleavage activity of the M537R-AsCas12a variant at TTTT PAM is shown. The display of the culture dish and the experimental details are as follows Fig. 6A shown.

[0043] Fig. 6E The survival rate of E. coli obtained by DNA cleavage activity of P569D-AsCas12a variant at TTTT PAM is shown. The display of culture dishes and experimental details are as follows Fig. 6A shown.

[0044] Fig. 6F The survival rate of E. coli obtained by DNA cleavage activity of P599G-AsCas12a variant at TTTT PAM is shown. The display of culture dishes and experimental details are as follows Fig. 6A shown.

[0045] Fig. 7A The survival rate of E. coli obtained by the DNA cleavage activity of M537R / F870L-AsCas12a at TTTT PAM is shown. The cleavage activity of M537R / F870L-AsCas12a variants at the TTTT PAM site was measured by a bacterial-based activity assay. A culture dish with initial bacteria is shown on the left, while a culture dish with surviving bacterial colonies is shown on the right. The survival of E. coli under selection depends on the successful cleavage of the toxin expression plasmid containing the TTTT PAM site.

[0046] Figure 7B The survival rate of E. coli obtained by DNA cleavage activity of L505K / M537R / F870L-AsCas12a variant at TTTT PAM is shown. The display of culture dishes and experimental details are as follows Fig. 7A shown.

[0047] Figure 7CThe survival rate of E. coli obtained by the DNA cleavage activity of the M537R / F870L-AsCas12a variant at TTTT PAM is shown. The display of the culture dish and the experimental details are as follows Fig. 7A shown.

[0048] Fig.7D The survival rate of E. coli obtained by DNA cleavage activity of P569D / M537R / F870L-AsCas12a variant at TTTT PAM is shown. The display of culture dishes and experimental details are as follows Fig. 7A shown.

[0049] Fig. 7E The survival rate of E. coli obtained by DNA cleavage activity of P599G / M537R / F870L-AsCas12a variant at TTTT PAM is shown. The display of culture dishes and experimental details are as follows Fig. 7A shown.

[0050] Figure 7F The survival rate of E. coli obtained by DNA cleavage activity of S510L / M537R / F870L-AsCas12a variant at TTTT PAM is shown. The display of culture dishes and experimental details are as follows Fig. 7A shown. DETAILED DESCRIPTION

[0051] The present invention relates to compositions of Cas12a variants and methods for enhancing the expression utility of Cas12a and its variants in eukaryotic cells.

[0052] definition

[0053] When introducing elements of aspects or specific embodiments of the present disclosure, the articles "a," "an," "the," and "said" are intended to indicate that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements in addition to the listed elements. Unless otherwise stated, the term "or" means any one member of a specific list and also includes any combination of members of that list.

[0054] As intended herein, the terms "substantially," "approximately," and "about," and similar terms are intended to have a broad meaning consistent with common and accepted usage in the art to which the subject matter of the present disclosure belongs. Those skilled in the art who review the present disclosure should understand that these terms are intended to allow for the description of certain features described and claimed without limiting the scope of these features to the precise numerical ranges provided. Therefore, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and claimed are considered to be within the scope of the present invention as described in the appended claims.

[0055] Definitions regarding certain terms and phrases applicable herein may be found in related U.S. patents and publications, such as U.S. patent application serial numbers 14 / 975,709, 15 / 299,549, 15 / 299,590, 15 / 299,593, 15 / 881,684, 15 / 729,491, 15 / 821,736, 15 / 964,041, 15 / 839,817, 15 / 839,820, 62 / 716,138 and U.S. Patent No. 9,840,702.

[0056] The term "substantially purified" when applied to a composition refers to compositions having at least 90% purity or greater, including compositions that are 90% pure, 95% pure, 99% pure, and greater than 99% pure.

[0057] The adjective "isolated", when modifying a composition, such as a polynucleotide, polypeptide or ribonucleoprotein complex, refers to a substantially purified composition, or in the case of a ribonucleoprotein complex, at least one component is a substantially purified component. With respect to isolated ribonucleoprotein complexes, it is further preferred that all components are substantially purified.

[0058] The terms "nucleic acid" and "polynucleotide" are interchangeable and have the same meaning.

[0059] The terms "amino acid," "polypeptide," and "protein" are interchangeable and have the same meaning.

[0060] The term "affinity tag" refers to a ligand that allows detection and / or selection of an oligonucleotide sequence linked to the ligand. For the purposes of this disclosure, a bait may include an affinity tag. Specifically, by using conventional chemical coupling techniques or recombinant DNA technology, the affinity tag is typically located at one or both of the N'-terminus and / or C'-terminus of the polypeptide. Exemplary affinity tags include biotin, digoxigenin, streptavidin, polyhistidine (e.g. (His6)), glutathione-S-transferase (GST), AviTag, calmodulin tag, polyglutamic acid tag, FLAG-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softag 3, V5 tag, Xpress tag, hapten, etc.

[0061] The term "eukaryotic cell" includes cells of a specific organism such as a plant or a mammal or derived from a specific organism such as a plant or a mammal, including but not limited to humans or non-human eukaryotic organisms or animals or mammals described herein, such as mice, rats, rabbits, dogs, livestock or non-human mammals or primates. In some embodiments, methods for modifying the germline genetic identity of humans and / or methods for modifying the genetic identity of animals (these genetic identities may cause humans or animals to suffer without any substantial medical benefit to them), as well as animals produced by such methods, may be excluded. Preferred human cells include cells derived from somatic cells and germline cells. Exemplary somatic cells include cells from each major organ and tissue system, including the immune system and the hematopoietic system.

[0062] As described herein, conditions 1, 2, and 3 refer to different combinations of background strains and the amount of gRNA introduced into the background strain before selecting a variant. Condition 1 is a M537R / F870L background, including the amount of gRNA introduced in which the variant is selected (100 pmol per 10 microliters of transformation / plating experiment). Condition 2 is a M537R / F870L background, including the amount of gRNA introduced in which the variant is selected (50 pmol per 10 microliters of transformation / plating experiment). Condition 3 is a wild-type AsCpf1 background, including the amount of gRNA introduced in which the variant is selected (200 pmol per 10 microliters of transformation / plating experiment).

[0063] Cas12a polypeptide with eukaryotic nuclear localization signal

[0064] Since Cas12a is a bacterial protein, it has no natural targeting mechanism to reach the eukaryotic nucleus where the target DNA resides.

[0065] In order to more effectively target proteins to the nucleus of eukaryotic cells, short protein sequences (called nuclear localization signals (NLS)) are usually added to the amino-terminus or carboxyl-terminus of a given open reading frame. NLS is recognized by the import protein on the nuclear membrane of eukaryotic cells, which first binds to the nuclear membrane and then allows the hole to be translocated into the nucleus through an energy-dependent process. Although recombinant protein tags like NLS can greatly improve positioning, any addition of exogenous amino acid sequences may disrupt protein function. Thus, it is very important and highly desirable to find that the recombinant Cas12a protein sequence that promotes the highest amount of nuclear delivery without negatively affecting its activity will eventually produce the most effective Cas12a genome editing solution.

[0066] In order to improve the nuclear delivery of Cas12a without interfering with its function, several different recombinant forms of Cas12a are constructed, in which the identity, position and quantity of protein tags (NLS, hexa-histidine tags (exemplary affinity tags)) are varied. Hexa-histidine and V5 tags are added to Cas12a constructs to help protein purification and / or detection, and NLS tags are added to assist in delivery to the nucleus of eukaryotic cells. The composition and position of the linker sequence of the destruction domain are also varied, to empirically determine the optimal arrangement and environment of the marker sequence. First, all constructs are expressed in Escherichia coli, and recombinant Cas12a proteins are purified by fixed metal affinity chromatography (IMAC) and previously described ion exchange chromatography.

[0067] AsCpf1 peptide variants with novel cleavage activity

[0068] The amino acid substitution in AsCpf1 that improves the cleavage activity at the canonical (TTTV) and non-canonical (TTTT) PAM sites using a bacterial screening method is described. This screening includes two parts: i) a toxin plasmid encoding an arabinose-induced cell proliferation toxin and a CRISPR / Cpf1 on-target cleavage site (HPRT-38346) with a TTTT PAM, and ii) a chloramphenicol resistance plasmid containing a random mutagenesis region within the AsCpf1 sequence (about 5 mutations per kb). The screening is performed as follows: in the absence of arabinose, Escherichia coli BW25141 (λDE3) is transformed with a toxin plasmid containing the HPRT-38346 target site, wherein no toxin is produced and the cells are allowed to survive. Subsequently, cells with a stably replicated toxin plasmid are transformed with an AsCpf1 expression plasmid and a crRNA targeting HPRT-38346, and the cells are then plated on a culture medium containing both chloramphenicol and arabinose. The bacteria that grew were: i) bacteria that were successfully transformed with the AsCpf1 expression plasmid, ii) bacteria that expressed enough AsCpf1 variants to cleave the toxin plasmid at the HPRT-38346 site using the TTTT PAM. The AsCpf1 expression plasmid in the surviving cells was recovered and used for the next round of selection. After multiple rounds of selection, the identity of the enriched AsCpf1 variants was determined by DNA sequencing and used for analysis in mammalian cells.

[0069] The present disclosure provides the following two novel point mutations and combinations in the AsCpf1 gene that improve cleavage activity: M537R and F870L. The cleavage activity of the single mutants was first tested in a bacterial-based activity assay. Next, the purified protein was further tested in vitro and in human cell lines. In summary, in all assays, both substitutions significantly improved the DNA cleavage activity of Cpf1 at the TTTT PAM site. In addition, the combination of M537R and F870L widely improved the targeting efficiency of AsCpf1 in human cell lines. In general, the present invention identifies novel amino acid positions in the AsCpf1 gene that can be mutated to improve its cleavage activity at all TTTN (N=A / G / C / T) PAM sites.

[0070] As described in the Background section, the prior art includes the use of wild-type Cpf1 protein or two variants compatible with TYCV and TATVPAMs. As previously mentioned, these variants have limited utility due to the complex and mutually exclusive requirements of the PAM sequence. In addition, none of the variants have been used in TTT T Unfortunately, TTT is not found throughout the human genome. T PAM occurs more frequently than other TTTV PAM sites (V=G / A / C). In contrast, the mutations reported in the present invention (M537R and F870L) are not only able to effectively cut at the TTTT PAM, but also widely improve the cleavage activity of Cpf1 at the canonical TTTV sites tested in human cell lines. In summary, the improved activity and broadened PAM flexibility (TTTN) of the present invention make it an excellent CRISPR enzyme that can directly replace the current wild-type Cpf1 in the genome editing of the present application.

[0071] High-throughput generation of AsCas12a variants with cleavage activity towards target sites containing TTTTPAM

[0072] The phenotypes of all point mutations in the following regions of AsCas12a in a bacterial screen measuring DNA cleavage activity at a non-canonical TTTT PAM are described: 499-640 and 840-913. Three sets of screens were performed to measure the phenotype of each point mutation in the context of both WT-AsCas12a and M537R / F870L-AsCas12a. Cross-comparison of the three data sets revealed consistent phenotypic measurements, which enabled the isolation of novel AsCas12a variants with improved activity (over M537R and F870L).

[0073] High-throughput characterization of Cas12a activity at the TTTT PAM site provides the functional consequences of every possible single amino acid change in the targeted region. The unbiased strategy of the present invention enables the identification of a large number of mutants to further improve the cleavage activity of AsCas12a compared to the previous invention (M537R / F870L).

[0074] In order to improve the coverage and efficiency of screening, we generated an AsCas12a deep scanning mutagenesis library containing all possible point mutations at the protein level in the targeted region (490-640 and 840-913), most of which contained only one mutation. This type of library allows the phenotype of each point mutation to be directly evaluated by measuring the relative survival rate relative to the reference protein in bacterial screening. In brief, the screening strain carrying the toxic plasmid was transformed with the AsCas12a library, wherein the target site containing TTTT PAM was on the toxic plasmid. After transformation, the cells were plated on a selective medium. The AsCas12a expression plasmid carried by the surviving Escherichia coli cells was extracted and purified. Both the input and selected plasmid libraries were PCR amplified and sequenced on Illumina MiSeq, with each library having 1 to 2 million reads. The occurrence rate of each AsCas12a variant in the two libraries was determined using Enrich 2 and normalized to the reference protein (WT or M537R / F870L). The relative survival rate of each point mutation relative to the reference was calculated as the ratio of the normalized occurrence rate between the selected library and the input library. Since the degree of cell survival indicates the DNA cleavage activity of each AsCas12a variant, any variant with a higher survival rate than the reference protein is a variant with improved activity at the TTTT PAM.

[0075] As described herein, codon-optimized Cas12a polypeptides are provided, including codon-optimized Cas12a polypeptides for CRISPR ribonucleoprotein complexes. An example of a codon-optimized sequence is a sequence optimized for expression in a eukaryotic organism such as a human (i.e., optimized for expression in a human) in this case or a sequence of another eukaryotic organism, animal, or mammal as discussed herein. Although this is preferred, it should be understood that other examples are also possible, and codon optimization for host species other than humans or codon optimization for specific organs is known. In some embodiments, the enzyme coding sequence encoding the CRISPR Cas12a polypeptide is a codon optimized for expression in a specific cell such as a eukaryotic cell. Eukaryotic cells may be specific organisms such as plants or mammals or cells derived from specific organisms such as plants or mammals, including but not limited to humans or non-human eukaryotic organisms or animals or mammals as described herein, such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates. In some embodiments, methods for modifying the germline genetic identity of humans and / or methods for modifying the genetic identity of animals that may cause suffering to humans or animals without any substantial medical benefit to them, as well as animals produced by such methods, may be excluded.

[0076] Preferred human cells include cells derived from somatic cells and germline cells. Exemplary somatic cells include cells from every major organ and tissue system, including the immune system and the hematopoietic system.

[0077] In general, codon optimization refers to a method of modifying a nucleic acid sequence to increase expression in a host cell by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of a native sequence with a codon that is more frequently or most frequently used in the genes of a host cell of interest while maintaining the native amino acid sequence. Various species exhibit special biases for certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) is generally associated with the translation efficiency of messenger RNA (mRNA), which is believed to be particularly dependent on the properties of the codons translated and the utilization of specific transfer RNA (tRNA) molecules. The advantage of selected tRNA in cells generally reflects the most frequently used codons in peptide synthesis. Therefore, genes can be customized based on codon optimization to achieve optimal gene expression in a given organism. Codon usage tables are readily available. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28: 292 (2000). Computer algorithms such as Gene Forge (Aptagen; Jacobus, Pa.) for codon optimization of specific sequences for expression in specific host cells are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more or all codons) in the sequence encoding Cas12a correspond to the most frequently used codons for a specific amino acid.

[0078] In addition, codon-optimized Cas12a polypeptides are provided, including codon-optimized Cas12a polypeptides for use in CRISPR ribonucleoprotein complexes, wherein the Cas12a polypeptide sequence is optimized for expression in prokaryotes such as bacteria (e.g., E. coli).

[0079] application

[0080] The present invention is applicable to any CRISPR / Cas12a DNA cutting and / or gene editing experiment or treatment of basic research or therapeutic fields. The excellent activity of these recombinant variants can be applied to Cas12a from any species or potentially any CRISPR enzyme.

[0081] In a first aspect, a CRISPR-associated protein is provided, comprising a polypeptide encoding an AsCpf1 variant. The AsCpf1 variant is selected from the group consisting of: M537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R / F870L (SEQ ID NO.: 465). In the first case, the CRISPR-associated protein corresponds to the AsCpf1 variant, and the AsCpf1 variant is M537R. In the second case, the CRISPR-associated protein corresponds to the AsCpf1 variant, and the AsCpf1 variant is F870L (SEQ ID NO.: 473). In the third case, the CRISPR-associated protein corresponds to the AsCpf1 variant, and the AsCpf1 variant is M537R / F870L (SEQ ID NO.: 465).

[0082] In a second aspect, a CRISPR ribonucleoprotein complex is provided. The CRISPR ribonucleoprotein complex comprises a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an AsCpf1 variant. The AsCpf1 variant is selected from the group consisting of: M537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R / F870L (SEQ ID NO.: 465). In the first case, the CRISPR ribonucleoprotein complex comprises an AsCpf1 variant, and the AsCpf1 variant is M537R. In the second case, the CRISPR ribonucleoprotein complex comprises an AsCpf1 variant, and the AsCpf1 variant is F870L (SEQ ID NO.: 473). In the third instance, the CRISPR ribonucleoprotein complex comprises an AsCpf1 variant, which is M537R / F870L (SEQ ID NO.: 465).

[0083] In a third aspect, a method for increasing TTT in a cell using a CRISPR ribonucleoprotein complex is provided. NA method for improving gene editing efficiency at a PAM site. The method comprises contacting a cell with a CRISPR ribonucleoprotein complex. The CRISPR ribonucleoprotein complex comprises a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an AsCpf1 variant. The AsCpf1 variant is selected from the group consisting of M537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R / F870L (SEQ ID NO.: 465). In the first case, the TTTN PAM site is composed of one selected from the group consisting of TTTA, TTTT and TTTC PAM sites.

[0084] In a fourth aspect, a kit is provided, comprising a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an AsCpf1 variant. The AsCpf1 variant is selected from the group consisting of: M537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R / F870L (SEQ ID NO.: 465). In the first case, the AsCpf1 variant is M537R. In the second case, the AsCpf1 variant is F870L (SEQ ID NO.: 473). In the third case, the AsCpf1 variant is M537R / F870L (SEQ ID NO.: 465).

[0085] In a fifth aspect, a CRISPR-associated protein is provided, comprising a polypeptide encoding an AsCas12a variant, wherein the AsCas12a variant is selected from a group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-related nuclease activity at a non-canonical TTTT PAM site. In the first case, the AsCas12a variant is selected from a group consisting of SEQ ID NO.: 59-245. In the second case, the AsCas12a variant as described in the first aspect or the first aspect above the first case further comprises a mutation of M537R / F870L (SEQ ID NO.: 465).

[0086] In a sixth aspect, a CRISPR ribonucleoprotein complex is provided. The CRISPR ribonucleoprotein complex comprises a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an AsCas12a variant, wherein the AsCas12a variant is selected from a group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-related nuclease activity at a non-canonical TTTT PAM site. In the first case, the AsCas12a variant is selected from a group consisting of SEQ ID NO.: 59-245. In the second case, the AsCas12a variant as described in the second aspect or the first case above the second aspect further comprises a mutation of M537R / F870L (SEQ ID NO.: 465).

[0087] In the seventh aspect, a method for improving the gene editing efficiency at a non-canonical TTTN PAM site in a cell using a CRISPR ribonucleoprotein complex is provided. The method includes contacting a cell with a CRISPR ribonucleoprotein complex comprising a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an AsCas12a variant, wherein the AsCas12a variant is selected from a group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-related nuclease activity at a non-canonical TTTT PAM site. In the first case, the AsCas12a variant is selected from a group consisting of SEQ ID NO.: 59-245. In the second case, the AsCas12a variant as described in the first case above the third aspect or the third aspect further comprises a mutation of M537R / F870L (SEQ ID NO.: 465).

[0088] In an eighth aspect, a kit is provided, comprising a guide RNA and a CRISPR-associated protein, wherein the CRISPR-associated protein comprises a polypeptide encoding an AsCas12a variant. The AsCas12a variant is selected from a group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-related nuclease activity at a non-canonical TTTTPAM site. In the first case, the AsCas12a variant is selected from a group consisting of SEQ ID NO.: 59-245. In the second case, the AsCas12a variant as described in the first case above the fourth aspect or the fourth aspect further comprises a mutation of M537R / F870L (SEQ ID NO.: 465).

[0089] In the ninth aspect, there is provided a nucleic acid encoding a CRISPR-associated protein, the CRISPR-associated protein comprising a polypeptide encoding an AsCas12a variant. The AsCas12a variant is selected from a group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site. Highly preferred nucleic acids encoding CRISPR-associated proteins include isolated nucleic acids encoding CRISPR-associated proteins. In the first case, the AsCas12a variant is selected from a group consisting of SEQ ID NO.: 59-245. In the second case, the AsCas12a variant also includes a mutation of M537R / F870L (SEQ ID NO.: 465). In the third case, the nucleic acid is operably connected to a suitable transcription element to express the nucleic acid. In the fourth case, the nucleic acid is DNA or RNA.

[0090] In the tenth aspect, a polynucleotide sequence encoding a Cas12a polypeptide is provided. The polynucleotide sequence comprises a member selected from the group consisting of SEQ ID NO.: 5-17.

[0091] In the eleventh aspect, an amino acid sequence encoding a Cas12a polypeptide is provided. The amino acid sequence comprises a member selected from the group consisting of SEQ ID NO.: 18-30.

[0092] In the twelfth aspect, a CAS endonuclease system is provided, comprising an expression cassette encoding a polynucleotide sequence encoding a Cas12a polypeptide. The polynucleotide sequence comprises a member selected from the group consisting of SEQ ID NO.: 5-17.

[0093] In the thirteenth aspect, a CAS endonuclease system is provided, comprising an amino acid sequence encoding a Cas12a polypeptide. The amino acid sequence comprises a member selected from the group consisting of SEQ ID NO.: 18-30.

[0094] In a fourteenth aspect, a method for genome editing in a eukaryotic cell is provided. The method comprises the following steps: introducing a CAS endonuclease system into a eukaryotic cell, wherein the CAS endonuclease system comprises an expression cassette encoding a polynucleotide sequence, wherein the polynucleotide sequence encodes a Cas12a polypeptide, and the polynucleotide sequence comprises a member selected from the group consisting of SEQ ID NO.: 5-17.

[0095] In a fifteenth aspect, a method for genome editing in a eukaryotic cell is provided. The method comprises the following steps: introducing a CAS endonuclease system into a eukaryotic cell, wherein the CAS endonuclease system comprises an amino acid sequence encoding a Cas12a polypeptide, wherein the amino acid sequence comprises a member selected from the group consisting of SEQ ID NO.: 18-30.

[0096] In the sixteenth aspect, a CRISPR-associated protein is provided, comprising a fusion polypeptide. The fusion polypeptide comprises an AsCas12a open reading frame, a nuclear localization signal, an optional amino acid linker, and an optional affinity tag. Highly preferred CRISPR-associated proteins include isolated CRISPR-associated proteins. In the first case, the AsCas12a open reading frame is selected from the group consisting of SEQ ID NO.: 59-245. In the second case, the nuclear localization signal is selected from SEQ ID NO.: 475, 477, 479, 481, and 483. In the third case, the CRISPR-associated protein is encoded by SEQ ID NO.: 488-491. In the fourth case, the CRISPR-associated protein is selected from SEQ ID NO.: 492 and 493.

[0097] In a seventeenth aspect, a method for genome editing in a eukaryotic cell is provided. The method comprises the following steps: introducing a CAS endonuclease system into a eukaryotic cell, the CAS endonuclease system comprising a CRISPR-associated protein according to the sixteenth aspect. Highly preferred CRISPR-associated proteins include isolated CRISPR-associated proteins.

[0098] Example

[0099] Example 1. Recombinant Cas12a proteins with different tag / linker sequences produce a variety of editing efficiencies

[0100] The following examples show that the recombinant Cas12a protein has only moderate changes in the tag sequence at the amino and carboxyl termini, resulting in a wide range of editing efficiencies in human cells ( Figure 1 ).

[0101] Briefly, site-directed mutagenesis (SDM) was used to create expression constructs with AsCas12a coding sequences containing different nuclear localization signals (NLS's). Site-directed mutagenesis was performed by designing complementary primers containing the desired nucleotide base changes and flanking plasmid vector sequences, where the melting temperature (T m ) is at least 60 ° C. Then the polymerase chain reaction (PCR) operation is performed using standard cycling conditions for a total of 16 cycles. The restriction enzyme DPN I is added to digest the starting plasmid vector material, so only the new product containing the base change is retained. After DPN I treatment, a small amount of PCR product is transformed into competent Escherichia coli cells, recovered in SOC medium, and plated on kanamycin-resistant Luria broth (LuriaBroth) (LB) agar plates. Colonies are screened using Sanger sequencing to verify the correct base changes in the selected clones.

[0102] The results show that the ideal sequence and placement of NLS sequences on Cas12a is not obvious, and efficient Cas12a genome editing solutions must be determined empirically, as was done in this study. Guides targeting HPRT-38186 (SEQ ID NO.: 1) and HPRT-38228 (SEQ ID NO.: 2) loci were used to test proteins in human cells. SEQ ID NO.: 1-4 are shown in Table 1.

[0103] Table 1. Oligonucleotide sequences used as crRNA or PCR primers.

[0104]

[0105] The following nucleotide sequence encoding the preferred Cas12a polypeptide of this embodiment is depicted as follows:

[0106] SEQ ID NO.:5

[0107]

[0108]

[0109] SEQ ID NO.:6

[0110]

[0111]

[0112] SEQ ID NO.:7

[0113]

[0114]

[0115] SEQ ID NO.:8

[0116]

[0117]

[0118] SEQ ID NO.:9

[0119]

[0120]

[0121]

[0122] SEQ ID NO.:10

[0123]

[0124]

[0125] SEQ ID NO.:11

[0126]

[0127]

[0128] SEQ ID NO.:12

[0129]

[0130]

[0131]

[0132] SEQ ID NO.:13

[0133]

[0134]

[0135] SEQ ID NO.:14

[0136]

[0137]

[0138] SEQ ID NO.:15

[0139]

[0140]

[0141]

[0142] SEQ ID NO.:16

[0143]

[0144]

[0145] SEQ ID NO.:17

[0146]

[0147]

[0148] The following amino acid sequences of preferred Cas12a polypeptides are described below.

[0149] SEO ID NO.:18

[0150]

[0151]

[0152] SEQ ID NO.:19

[0153]

[0154] SEQ ID NO.:20

[0155]

[0156]

[0157] SEQ ID NO.:21

[0158]

[0159] SEQ ID NO.:22

[0160]

[0161]

[0162] SEQ ID NO.:23

[0163]

[0164] SEQ ID NO.:24

[0165]

[0166]

[0167] SEQ ID NO.:25

[0168]

[0169] SEQ ID NO.:26

[0170]

[0171]

[0172] SEQ ID NO.:27

[0173]

[0174] SEQ ID NO.:28

[0175]

[0176]

[0177] SEQ ID NO.:29

[0178]

[0179] SEQ ID NO.:30

[0180]

[0181] Example 2. Novel Cpf1 mutants have increased DNA cleavage activity at the TTTT PAM site in a bacteria-based activity assay.

[0182] The following examples demonstrate the activity enhancement of the present invention at the TTTT and TTTC PAM sites in a bacterial-based activity assay ( Figure 2). The screening strains containing the toxic plasmid were transformed with WT, M537R or F870LAsCpf1 expression plasmids. After recovery and IPTG induction, the cells were plated on LB-chloramphenicol medium with or without arabinose. The degree of cell survival under arabinose selection compared to the transformed input control (without arabinose) indicated the cleavage activity of the Cpf1 variants at the HPRT-38346 protospacer sequence on the toxic plasmid in the context of TTTT or TTTC PAM.

[0183] For WT-Cpf1, the survival rate of E. coli transformed at TTTC was significantly higher than that at TTTT PAM, which is highly consistent with the existing knowledge that TTTT is a low-activity PAM site. [6] In contrast, both M537R and F870L improved the survival rate at the TTTT PAM, indicating that these mutants broadened the PAM compatibility of AsCpf1 at this alternative PAM site. More importantly, the survival rate of both mutants at the canonical TTTC PAM was even higher than that of WT-Cpf1, suggesting that these mutants generally also improve the performance of AsCpf1 proteins at other TTTV sites. Given these positive results, single AsCpf1 variants and the double mutant (M537R / F870L) were expressed and purified to determine their intrinsic cleavage activity in vitro.

[0184] Example 3. Novel Cpf1 mutants increase intrinsic DNA cleavage activity at the in vitro TTTT PAM site

[0185] The intrinsic DNA cleavage activity of AsCpf1 variants (M537R, F870L, and M537R / F870L) was compared with that of the wild-type protein using an in vitro cleavage assay. Figure 3A ) was incubated with HPRT-38346crRNA in 1X cleavage buffer (20mM HEPES, pH 7.5, 150mM KCl, 5mM MgCl2, 10% glycerol and 1mM DTT) at 37°C for 15 minutes to assemble the Cpf1-crRNA ribonucleoprotein (RNP) complex. In the context of TTTC or TTTT PAM, the cleavage reaction was initiated by titrating the RNP complex (8-500nM) in 10nM dsDNA substrate containing the HPRT-38346 pre-spacer sequence. The cleavage reaction was sampled at different time points and quenched by 50mM EDTA. After removing the AsCpf1 protein by ProteaniseK treatment (56°C, 30 minutes), the reaction was analyzed using capillary electrophoresis (Fragment Analyzer, AATI). The relative concentrations of cleavage products and uncut dsDNA were quantified to calculate the percentage of DNA cleavage.

[0186] exist Figure 3B The intrinsic DNA cleavage activities of WT and Cpf1 variants at TTTT and TTTC PAM sites were compared in Figure 2. For simplicity, only a single RNP concentration (31 nM) at the 20 s time point is shown. As expected, the PAM sequence changes from TTT C To TTT T The single nucleotide change of 100 reduced the cleavage activity of WT-Cpf1 from ∼95% to ∼40%. Consistent with the observations in the bacteria-based activity assay, both mutants significantly increased DNA cleavage at TTTTPAM while maintaining high activity at TTTC PAM ( Figure 3B ). In this assay, the double mutant (M537R / F870L) had similar activity to M537R. However, it is important to note that this is most likely due to the limited resolution of this particular assay, which was unable to resolve further differences between these highly active variants. In summary, these results suggest that the reported mutations increase Cpf1 activity by increasing intrinsic DNA cleavage. Therefore, it is expected that the benefits observed with these mutants will be broadly applicable and not dependent on the delivery method and / or cellular context of a particular experiment.

[0187] Example 4. Novel mutants broadly improve targeting efficiency of the TTTN PAM site in human cell lines.

[0188] The following examples demonstrate that when the Cpf1-crRNA complex is delivered into cells as RNP, the present invention increases TTT N The ability to enhance gene editing efficiency at the PAM site.

[0189] CRISPR / Cpf1 cell editing experiments were performed by first incubating cells with purified Cpf1 protein and Alt-R in Opti-MEM at 25°C. TM The crRNA formed a 4mM RNP complex for 5 minutes. Table 2 shows the targeted pro-spacer and PAM sequences in the CTNNB1 locus. The RNP complex was then transfected into HEK293 cells by Lonza nuclear transfection. The experiment was performed in triplicate in a biological manner. After 48 hours at 37°C and 5% CO2, the adherent cells were washed with 0.1 ml PBS and 0.05 ml QuickExtract TMThe DNA was extracted and dissolved in the solution. The cell lysate was incubated at 65°C for 15 minutes and then heat-inactivated at 98°C for 3 minutes. The crude DNA sample was then diluted 3 times with 0.1 ml ddH2O and used as a PCR template. The PCR primers are shown in Table 2. A 1 kb fragment of the CTNNB1 locus was amplified by PCR using KAPA HiFi DNA polymerase and the following cycle parameters: 95 5:00 , (98 0:20 , 64 0:15 , 72 0:30 ) Repeat 29 times, 72 2:00 The following cycling parameters were used to form heteroduplexes: 95 10:00 After 1min cooling to 85,85 1:00 After 1min cooling to 75,75 1:00 After 1min cooling to 65,65 1:00 After 1min cooling to 55,55 1:00 After 1min cooling to 45,45 1:00 After 1min cooling to 35,35 1:00 After 1min cooling to 25, 25 1:00 The heteroduplexes were cleaved by adding 2 U T7 Endonuclease I (New England Biolabs) for 1 hour at 37°C, and the cleavage products were analyzed by capillary electrophoresis (Fragment Analyzer, Advanced Analytical).

[0190] Table 2. Sequences of DNA target sites and primers used for PCR amplification (SEQ ID NOs.: 31-58).

[0191]

[0192] Reference Figure 4, T7EI assay reveals that M537R and F870L mutations significantly improve targeting efficiency. First, M537R, F870L or double mutants (M537R / F870L) can achieve effective cutting at all 15 sites with TTTT PAM, of which 11 of 15 have no detectable WT-Cpf1 cutting. For other sites with canonical TTTV PAM, these variants maintain or improve targeting efficiency. At those low-activity sites such as CTNNB1111-s (3-fold increase relative to WT), this benefit is particularly significant. Among these variants, the double mutant (M537R / F870) has the most consistent improvement in targeting efficiency at all test sites, with single mutants showing more site-dependent variations, such as F870L at 323-S (inactive, identical to WT). In general, the described invention exhibits much better on-target efficacy than WT-Cpf1.

[0193] Example 5. High-throughput measurement of DNA cleavage activity of AsCas12a variants at the TTTT PAM site in Escherichia coli

[0194] The following examples demonstrate the robustness of a novel high-throughput screening strategy that can directly measure the cleavage activity of thousands of AsCas12a variants at the TTTT PAM site in a bacteria-based activity assay ( FIG. 5 ). Figure 5A -F shows an exemplary high-throughput phenotypic measurement of AsCas12a point mutations by deep scanning mutagenesis. In the context of WT-AsCas12a or M537R / F870L-AsCas12a, a library covering every possible single point mutation of AsCas12a in the targeted region (499-640 and 840-913) was generated. The relative survival rate of each variant relative to the reference protein in the E. coli-based activity assay was determined by deep sequencing. In the context of the third condition (condition 3) in M537R / F870L (conditions 1 and 2) and WT-AsCas12a background, the phenotype of the single point mutation was measured under multiple selection stringency. As Figure 5A -C, the phenotypic scores of the variants (i.e., the natural logarithm of the relative survival rate) are positively correlated under different conditions (ρ ~ 0.7), demonstrating the consistency and reproducibility of this method. As positive controls, only M537R and F870L / I, but not any other substitutions at these positions, survived longer than WT-AsCas12a ( Figure 5D ). In contrast, mutations R537 or L870 on M537R / F870L-AsCas12a generally reduced survival ( Figure 5E ). These results indicate that the phenotypic scores measured by the bacterial screen reflect the DNA cleavage activity of previously characterized AsCas12a variants at the TTTT PAM.

[0195] To further validate the results of the bacterial screening, 444 samples were screened under all three conditions (L505K, S510L, P569D, and P599G, Fig. 5F ) studied four point mutations of AsCas12a, all of which had survival rates higher than the reference value. It should be noted that P599G has been shown to enhance the cleavage activity of AsCas12a at TTCC PAM. So far, the other three point mutations have not been characterized in any published studies. Therefore, the survival rate of E. coli cells transformed with plasmids expressing single AsCas12a variants under selection was measured. Compared with WT-AsCas12a, all selected point mutations increased survival when targeting the TTTT-PAM site (Figure 6). Unexpectedly, the benefits of these point mutations still hold true even when the survival rate of M537R / F870L-AsCas12a is further improved (Figure 7). Overall, these results show that high-throughput screening can accurately predict the phenotype of uncharacterized AsCas12a variants.

[0196] Table 3 lists the phenotypic scores of 3194 AsCas12a variants with single point mutations covered by screening with sufficient sequencing counts. Overall, ~60% showed some benefit (ie, phenotypic score>0) in one of the three conditions.

[0197] Table 3. Summary of selected variants under different background conditions. 1

[0198]

[0199]

[0200]

[0201]

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[0215]

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[0217]

[0218]

[0219]

[0220]

[0221]

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[0278]

[0279] 1Key to title abbreviations: Mutants are defined by a single-letter amino acid (wild type) at a given position in the wild-type AsCas12a polypeptide (e.g., amino acid position 500) followed by the subsequent variant amino acid changes; "Enrichment score (1)" refers to the enrichment score of the variant in the context of condition 1; "Standard error (1)" refers to the standard error of experiments performed on a given variant in the context of condition 1; "Enrichment score (2)" refers to the enrichment score of the variant in the context of condition 2; "Standard error (2)" refers to the standard error of experiments performed on a given variant in the context of condition 2; "Enrichment score (3)" refers to the enrichment score of the variant in the context of condition 3; "Standard error (3)" refers to the standard error of experiments performed on a given variant in the context of condition 3; "Any positive enrichment?" refers to the phenomenon that a given variant undergoes positive enrichment in at least one experimental condition; "Consistent positive enrichment?" refers to the phenomenon that a given variant undergoes positive enrichment in the context of all tested experimental conditions.

[0280] One hundred and eighty-seven (187) variants (~6% of the total) consistently improved survival across all conditions (Table 4). These variants, including four individually validated variants, can be stacked on WT- or M537R / F870L-AsCas12a to enhance its DNA cleavage activity at TTTT-PAM.

[0281] Table 4. Point mutations are consistently enriched over the reference protein under all conditions. 1

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291] 1A phenotypic score (i.e., natural logarithm of relative enrichment) is provided for each point mutation. Error bars estimate the precision of the measurement, which depends on the sequencing counts of each variant in the library. Only variants with counts greater than 50 in all libraries were included in the analysis.

[0292] Table 5 shows the sequences of primers used to generate the AsCas12a saturation mutagenesis library. Standard recombinant methods and techniques were used. The screening library was constructed using the method described by Wrenbeck et al. (2016).

[0293] Table 5. Primer sequences for generating AsCas12a saturation mutagenesis library.

[0294]

[0295]

[0296]

[0297]

[0298] a For "NNK", N refers to A, C, T or G; K refers to G or T.

[0299] With respect to Table 4, the reference (i.e., wild-type) polypeptide sequence is SEQ ID NO.: 462, on which these mutants are based by comparison. The polynucleotides codon-optimized for expression in Escherichia coli and human cells encoding SEQ ID NO.: 462 are SEQ ID NOs.: 463 and 464, respectively. The same mutations are also introduced into the M537R / F870L-AsCas12a background. The corresponding reference polypeptide sequence of M537R / F870L-Cas12a is SEQ ID NO.: 465 (the amino acids that have been changed are underlined). The polynucleotides codon-optimized for expression in Escherichia coli and human cells encoding SEQ ID NO.: 465 are SEQ ID NOs.: 466 and 467, respectively (the codons that have been changed are underlined).

[0300] SEQ ID NO.:462

[0301]

[0302] SEQ ID NO.:463

[0303]

[0304]

[0305]

[0306] SEQ ID NO.:464

[0307]

[0308]

[0309] SEQ ID NO.:465

[0310]

[0311] SEQ ID NO.:466

[0312]

[0313]

[0314]

[0315] SEQ ID NO.:467

[0316]

[0317]

[0318] Additional polynucleotides and polypeptides associated with this embodiment include Cas12a variants with single amino acid substitutions at M537R and F870L, as shown below. The underlined codons or amino acids correspond to changes relative to the corresponding WT Cas12a sequence.

[0319] SEQ ID NO.: 468 Escherichia coli optimized DNA M537R

[0320]

[0321]

[0322] SEQ ID NO.: 469 Escherichia coli optimized DNA F870L Cas12a

[0323]

[0324]

[0325] SEQ ID NO.: 470 Human optimized DNA M537R Cas12a

[0326]

[0327]

[0328]

[0329] SEQ ID NO.: 471 Human optimized DNA F870L Cas12a

[0330]

[0331]

[0332] SEQ ID NO.: 472 M537RCas12a AA

[0333]

[0334] SEQ ID NO.: 473 F870L Cas12a AA

[0335]

[0336]

[0337] Example 6. Rational design of fusion Cas12a polypeptides

[0338] Fusion Cas12 / a polypeptides with additional motifs (collectively referred to as "NLS" or "NLS sequences") that enable nuclear localization into eukaryotic cells and / or purification and labeling detection motifs (collectively referred to as "affinity tags") fall within the scope of the present invention. Exemplary nuclear localization signals ("NLS" or "NLS sequences") are well known in the art and include those identified by the polynucleotide and amino acid sequences shown in Table 6.

[0339] Table 6. Exemplary NLS sequences

[0340]

[0341] Exemplary purification and / or labeling detection motifs include affinity tags also known in the art. Typically, additional amino acid linkers inserted before or after additional motifs can improve the expression and / or stability of the expressed fusion Cas12 / a polypeptides. Two examples of affinity tags are defined by the polynucleotide and amino acid sequences shown in Table 7 below.

[0342] Table 7. Exemplary affinity tags

[0343]

[0344] Fusion Cas12a polypeptides comprising nuclear localization signals, linker amino acids and / or affinity tags can be easily constructed using chemical polypeptide methods or expressed from engineered polynucleotides encoding in-frame polypeptides created using recombinant DNA technology. Such techniques are well known and within the capabilities of those skilled in the art. Working examples of such polynucleotides and polypeptides are shown by SEQ ID NO.: 5-30. Fusion Cas12a polypeptide variants encoding open reading frames of SEQ ID NO.: 59-245 with nuclear localization sequences and / or affinity tags and optionally optional amino acid linkers fall within the scope of the present disclosure. Exemplary Cas12a variants with nuclear localization signals are shown below.

[0345] Briefly, site-directed mutagenesis (SDM) was used to create expression constructs with AsCas12a coding sequences containing different nuclear localization signals (NLS's). Site-directed mutagenesis was performed by designing complementary primers containing the desired nucleotide base changes and flanking plasmid vector sequences, where the melting temperature (T m ) is at least 60°C. The polymerase chain reaction (PCR) operation is then performed using standard cycling conditions for a total of 16 cycles. The restriction enzyme DPN I is added to digest the starting plasmid vector material, so that only new products containing base changes are retained. After DPN I treatment, a small amount of PCR product is transformed into competent E. coli cells, recovered in SOC medium, and plated on kanamycin-resistant Luria broth (LB) agar plates. Colonies are screened using the Sanger sequencing method to verify the correct base changes in the selected clones.

[0346] SEQ ID NO.: 488 E. coli optimized DNA WT Cas12a with NLS linker

[0347]

[0348]

[0349] The underlined sequence refers to the nucleotides encoding the amino acid linker sequence. The double underlined sequence refers to the nucleotides encoding the nuclear localization sequence (NLS linker). The italicized sequence refers to the nucleotides encoding the amino acid affinity tag sequence ((HIS)6).

[0350] SEQ ID NO.489 E. coli optimized DNA M537R F870L Cas12a with NLS linker

[0351]

[0352] The bold and underlined sequences refer to the mutant codons introduced into the Cas12a open reading frame. The underlined sequences refer to the nucleotides encoding the amino acid linker sequence. The double underlined sequences refer to the nucleotides encoding the nuclear localization sequence (NLS linker). The italic sequences refer to the nucleotides encoding the amino acid affinity tag sequence ((HIS)6).

[0353] SEQ ID NO.: 490 Human optimized DNA WT Cas12a with NLS linker

[0354]

[0355]

[0356] The underlined sequence refers to the nucleotides encoding the amino acid linker sequence. The double underlined sequence refers to the nucleotides encoding the nuclear localization sequence (NLS linker). The italicized sequence refers to the nucleotides encoding the amino acid affinity tag sequence ((HIS)6).

[0357] SEQ ID NO.: 491 Human optimized DNA M537R F870L Cas12a with NLS linker

[0358]

[0359]

[0360] The bold and underlined sequences refer to the mutant codons introduced into the Cas12a open reading frame. The underlined sequences refer to the nucleotides encoding the amino acid linker sequence. The double underlined sequences refer to the nucleotides encoding the nuclear localization sequence (NLS linker). The italic sequences refer to the nucleotides encoding the amino acid affinity tag sequence ((HIS)6).

[0361] SEQ ID NO.:492WT Cas12aAA with NLS linker

[0362]

[0363] The underlined sequence refers to the amino acid sequence encoding the amino acid linker sequence. The double underlined sequence refers to the amino acid sequence encoding the nuclear localization sequence (NLS linker). The italicized sequence refers to the amino acid sequence encoding the amino acid affinity tag sequence ((HIS)6).

[0364] SEQ ID NO.:493M537R F870L Cas12aAA with NLS linker

[0365]

[0366]

[0367] The bold and underlined sequences refer to the mutant amino acids introduced into the Cas12a polypeptide variants. The underlined sequences refer to the amino acid sequences encoding the amino acid linker sequences. The double underlined sequences refer to the amino acid sequences encoding the nuclear localization sequences (NLS linkers). The italic sequences refer to the amino acid sequences encoding the amino acid affinity tag sequences ((HIS)6).

[0368] References:

[0369] Chen, JS, et al., Enhanced proofreading governs CRISPR-Cs9 targeting accuracy. Nature, 2017.550(7676): p.407-410.

[0370] Gao, L., et al., Engineered Cpf1 variants with altered PAM specificitiesincrease genome targeting range. Nat Biotechnol. 2017;35(8):789-792.

[0371] Jinek M, et al. A programmable dual-RNA-guided DNA eudonuclease inadaptive bacterial immunity. Science. 2012; 337: 816-821. doi: 10.1126 / science.1225829.

[0372] Kleinsliver, BP, et al., High-fiddelity CRISPR-Cas9 nucleases with nodetectable genome-wide off-target effects. Nature, 2016.529(7587): p.490-5.

[0373] Slaymaker, IM, et al., Rationally engineered Cas9 nucleases witimproved specificity. Science, 2016.351(6268): p.84-8.

[0374] Suun, Y., et al., Factors influencing the nuclear targeting ability ofnuclear localization signals. J Drug Target, 2016.24(10): p.927-933.

[0375] Wrenbeck EE, Klesmith JR, Stapleton JA, Adeniran A, Tyo KE, Whitehead TA. Plasmid-based one-pot saturation mutagenesis. Nat Methods. 2016; 13(11): 928-930. doi: 10.1038 / nneth.4029

[0376] Zetsche, B., et al., Cpfl Is a Single RNA-Guided Endonuclease of a Class2CRISPR-Cas System. Cell.2015;163:759-771.doi:10.1016 / j.cell.2015.09.038.

[0377] Incorporated by Reference

[0378] All patents, patent applications, patent application publications, and other publications cited herein are incorporated by reference as if fully set forth.

[0379] Preferred embodiments

[0380] The present invention has been described in conjunction with what are currently considered to be the most practical and preferred embodiments. However, the present invention has been presented by way of illustration, and the present invention is not intended to be limited to the disclosed embodiments. Therefore, those skilled in the art will recognize that the present invention is intended to cover all modifications and alternatives within the spirit and scope of the present invention as set forth in the appended claims.

Claims

1. A CRISPR-associated protein comprising a polypeptide encoding an AsCpf1 variant, wherein the AsCpf1 variant is selected from the group consisting of: M537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R / F870L (SEQ ID NO.: 465).

2. The CRISPR-associated protein according to claim 1, wherein the AsCpf1 variant is M537R (SEQ ID NO.: 472).

3. The CRISPR-associated protein according to claim 1, wherein the AsCpf1 variant is F870L (SEQ ID NO.: 473).

4. The CRISPR-associated protein according to claim 1, wherein the AsCpf1 variant is M537R / F870L (SEQ ID NO.: 465).

5. A CRISPR ribonucleoprotein complex comprising: guide RNA; and A CRISPR-associated protein comprising a polypeptide encoding an AsCpf1 variant, wherein the AsCpf1 variant is selected from the group consisting of: M537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R / F870L (SEQ ID NO.: 465).

6. The CRISPR ribonucleoprotein complex of claim 5, wherein the AsCpf1 variant is M537R (SEQ ID NO.: 472).

7. The CRISPR ribonucleoprotein complex of claim 5, wherein the AsCpf1 variant is F870L (SEQ ID NO.: 473).

8. The CRISPR ribonucleoprotein complex of claim 5, wherein the AsCpf1 variant is M537R / F870L (SEQ ID NO.: 465).

9. Using CRISPR ribonucleoprotein complexes to increase TTT in cells N A method for gene editing efficiency at a PAM site, the method comprising: Contacting a cell with the CRISPR ribonucleoprotein complex according to any one of claims 5-8.

10. The method according to claim 9, wherein the TTT N The PAM site consists of one selected from the group consisting of TTTA, TTTT and TTTCPAM sites.

11. A kit comprising a guide RNA and a CRISPR-associated protein, wherein the CRISPR-associated protein comprises a polypeptide encoding an AsCpf1 variant, wherein the AsCpf1 variant is selected from the group consisting of: M537R (SEQ ID NO.: 472), F870L (SEQ ID NO.: 473) and M537R / F870L (SEQ ID NO.: 465).

12. The kit according to claim 11, wherein the AsCpf1 variant is M537R (SEQ ID NO.: 472).

13. The kit according to claim 11, wherein the AsCpf1 variant is F870L (SEQ ID NO.: 473). The kit according to claim 11 , wherein the AsCpf1 variant is M537R / F870L (SEQ ID NO.: 465).

15. A CRISPR-associated protein comprising a polypeptide encoding an AsCas12a variant, wherein the AsCas12a variant is selected from the group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

16. The CRISPR-associated protein of claim 15, wherein the AsCas12a variant is selected from the group consisting of SEQ ID NO.: 59-245.

17. The CRISPR-associated protein of claim 15 or 16, wherein the AsCas12a variant further comprises a mutation of M537R / F870L (SEQ ID NO.: 465).

18. A CRISPR ribonucleoprotein complex comprising: guide RNA; and A CRISPR-associated protein comprising a polypeptide encoding an AsCas12a variant, wherein the AsCas12a variant is selected from the group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, with the proviso that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

19. The CRISPR ribonucleoprotein complex of claim 18, wherein the AsCas12a variant is selected from the group consisting of SEQ ID NO.: 59-245.

20. The CRISPR ribonucleoprotein complex of claim 18 or 19, wherein the AsCas12a variant further comprises a mutation of M537R / F870L (SEQ ID NO.: 465).

21. A method for improving gene editing efficiency at a non-canonical TTTN PAM site in a cell using a CRISPR ribonucleoprotein complex, the method comprising: Contacting a cell with the CRISPR ribonucleoprotein complex of any one of claims 18-20.

22. A kit comprising a guide RNA and a CRISPR-associated protein, wherein the CRISPR-associated protein comprises a polypeptide encoding an AsCas12a variant, wherein the AsCas12a variant is selected from the group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

23. A kit according to claim 22, wherein the AsCas12a variant is selected from the group consisting of SEQ ID NO.: 59-245.

24. A kit according to claim 22 or 23, wherein the AsCas12a variant further comprises a mutation of M537R / F870L (SEQ ID NO.: 465).

25. A nucleic acid encoding a CRISPR-associated protein comprising a polypeptide encoding an AsCas12a variant, wherein the AsCas12a variant is selected from the group consisting of at least one variant amino acid selected from amino acid positions 499-640 and 840-913, provided that the variant AsCas12a provides an improvement in CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

26. A nucleic acid according to claim 25, wherein the AsCas12a variant is selected from the group consisting of SEQ ID NO.: 59-245.

27. A nucleic acid according to claim 25 or 26, wherein the AsCas12a variant further comprises a mutation of M537R / F870L (SEQ ID NO.: 465).

28. The nucleic acid of claims 25-27, wherein the nucleic acid is operably linked to a suitable transcription element for expressing the nucleic acid.

29. The nucleic acid of claims 25-28, wherein the nucleic acid is DNA or RNA.

30. A polynucleotide sequence encoding a Cas12a polypeptide, comprising a member selected from the group consisting of SEQ ID NO.: 5-17.

31. An amino acid sequence encoding a Cas12a polypeptide, comprising a member selected from the group consisting of SEQ ID NO.: 18-30.

32. A CAS endonuclease system comprising an expression cassette encoding a polynucleotide sequence encoding a Cas12a polypeptide, wherein the polynucleotide sequence comprises a member selected from the group consisting of SEQ ID NO.: 5-17.

33. A CAS endonuclease system, comprising an amino acid sequence encoding a Cas12a polypeptide, wherein the amino acid sequence comprises a member selected from the group consisting of SEQ ID NO.: 18-30.

34. A method for genome editing in a eukaryotic cell, the method comprising: A CAS endonuclease system is introduced into the eukaryotic cell, wherein the CAS endonuclease system comprises an expression cassette encoding a polynucleotide sequence, wherein the polynucleotide sequence encodes a Cas12a polypeptide, and the polynucleotide sequence comprises a member selected from the group consisting of SEQ ID NO.: 5-17.

35. A method for genome editing in a eukaryotic cell, the method comprising: A CAS endonuclease system is introduced into the eukaryotic cell, wherein the CAS endonuclease system comprises an amino acid sequence encoding a Cas12a polypeptide, wherein the amino acid sequence comprises a member selected from the group consisting of SEQ ID NO.: 18-30.

36. A CRISPR-associated protein comprising a fusion polypeptide encoding an AsCas12a open reading frame, a nuclear localization signal, an optional amino acid linker, and an optional affinity tag.

37. The CRISPR-associated protein of claim 36, wherein the AsCas12a open reading frame is selected from the group consisting of SEQ ID NO.: 59-245.

38. The CRISPR-associated protein of claim 36, wherein the nuclear localization signal is selected from SEQ ID NO.: 475, 477, 479, 481 and 483.

39. The CRISPR-associated protein of claim 36, wherein the CRISPR-associated protein is encoded by SEQ ID NOs.: 488-491.

40. The CRISPR-associated protein of claim 36, wherein the CRISPR-associated protein is selected from SEQ ID NO.: 492 and 493.

41. A method for genome editing in a eukaryotic cell, the method comprising: A CAS endonuclease system is introduced into the eukaryotic cell, wherein the CAS endonuclease system comprises the CRISPR-associated protein of claims 36-40.

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