A novel mutation that enhances the DNA cleavage activity of Acidaminococcus CPF1

Abstract

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

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a joint application under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 62 / 870,268, filed July 3, 2019, entitled "OPTIMIZED CAS12A (CPF1) PROTEINS FOR EFFICIENT GENOME EDITING IN EUKARYOTIC CELLS," U.S. Provisional Patent Application No. 62 / 749,607, filed October 23, 2018, entitled "DEEP-SCANNING MUTAGENESIS UNCOVERS 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 No. 62 / 749,607, filed August 8, 2018, entitled "NOVEL MUTATIONS THAT ENHANCE THE DNA CLEAVAGE ACTIVITY OF ACIDAMINOCOCCUS This application claims priority to U.S. Provisional Patent Application No. 62 / 716,138, entitled "SP. CPF1," the contents of each of which are incorporated herein by reference in their entirety.

[0002] (Technical field) The present invention relates to the ability to precisely cleave double-stranded DNA in living organisms using the CRISPR / Cas12a (Cpf1) nuclease system. In particular, a series of recombinant Cas12a proteins useful in eukaryotic contexts are described.

[0003] (Sequence Listing) The instant application contains a sequence listing delivered in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy made with The name is and the size is It's a part-time job. [Background technology]

[0004] Cas12a is an RNA-guided endonuclease found in bacterial species, including Acidaminococcus sp., and is part of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) adaptive immune system. Cas12a is guided to a 21- to 24-nt DNA target sequence, commonly referred to as a protospacer, by a target-site-specific 21- to 24-nt complementary RNA. The Cas12a-gRNA ribonucleoprotein (RNP) complex mediates double-stranded DNA breaks (DSBs), which are repaired by either nonhomologous end joining (NHEJ, which typically introduces a mutation or indel at the break site) or homology-directed repair (HDR) systems for precise editing in the presence of appropriate template nucleic acid.

[0005] Critical to Cas12a's recognition of the correct DNA target are both the standard "TTTV" protospacer adjacent motif (PAM), a 4-bp sequence immediately upstream of the crRNA and protospacer. Compared to the 2-bp NGG PAM of Streptococcus pyogenes-derived Cas9, Cas12a expands the range of loci that can be targeted for genome editing, particularly at AT-rich sites inaccessible to the Cas9 system. However, due to its relatively low enzymatic activity, the likelihood of achieving efficient genome editing at a given site is much lower than with the Cas9 system, limiting its broad application. As a result, the Cas12a system is often considered an alternative approach only when a genomic site is not targetable by Cas9.

[0006] Mutagenesis-based protein engineering can alter the PAM sequence preference of CRISPR systems. Previous studies identified two AsCpf1 variants compatible with the TYCV and TATV PAMs, respectively, through structure-guided mutagenesis screening of residues adjacent to the PAM sequence. Collectively, these variants expanded the targetable sites of the Cpf1 system threefold in the coding region of the human genome. However, the utility of individual variants remains limited due to mutually exclusive PAM sequence requirements (TYCV vs. TATV vs. TTTV). It is highly desirable to identify Cpf1 variants with short PAMs and highly flexible sequences without sacrificing activity at standard PAM sites.

[0007] Therefore, there is a need to enhance the utility of Cas12a. One aspect of the present application is to enhance the utility of Cpf1 by expanding its PAM compatibility. In this regard, certain novel AsCas12a variants with enhanced activity have been discovered. Another desirable goal is to maximize the delivery of bacterial proteins to the nucleus of eukaryotic cells while avoiding disruption of basic Cas12a function. Because Cas12a is a bacterial protein, certain molecular genetic obstacles must first be overcome before proteins can be successfully delivered into eukaryotic cells. The present invention provides a unique solution to achieve these goals. Summary of the Invention

[0008] In a first aspect, there is provided a CRISPR-associated protein comprising a polypeptide encoding a variant of AsCpf1, wherein the variant of AsCpf1 is selected from the group consisting of M537R (SEQ ID NO: 472), F870L (SEQ ID NO: 473), and M537R / F870L (SEQ ID NO: 465).

[0009] In a second aspect, a CRISPR ribonucleoprotein complex is provided. The CRISPR ribonucleoprotein complex includes a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein includes a polypeptide encoding a variant of AsCpf1. 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).

[0010] In a third aspect, TTT in cells having CRISPR ribonucleoprotein complexes. N A method for increasing the efficiency of gene editing at a PAM site is provided. The method includes contacting a cell with a CRISPR ribonucleoprotein complex. The CRISPR ribonucleoprotein complex includes a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein includes a polypeptide encoding a variant of AsCpf1. 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).

[0011] In a fourth aspect, a kit is provided comprising a guide RNA and a CRISPR-associated protein, the CRISPR-associated protein comprising a polypeptide encoding a variant of AsCpf1.

[0012] In a fifth aspect, a CRISPR-associated protein comprises a polypeptide encoding a variant of AsCas12a, wherein the variant AsCas12a 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 improved CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

[0013] 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 a variant of AsCas12a, wherein the variant AsCas12a 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 results in improved CRISPR / AsCas12a-associated nuclease activity at non-canonical TTTT PAM sites.

[0014] In a seventh aspect, a method for enhancing the efficiency of gene editing at a non-canonical TTTT PAM site in a cell harboring a CRISPR ribonucleoprotein complex is provided. The method comprises contacting the cell with a CRISPR ribonucleoprotein complex comprising a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding a variant of AsCas12a, wherein the variant AsCas12a 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 results in improved CRISPR / AsCas12a-associated nuclease activity at the non-canonical TTTT PAM site.

[0015] In an eighth aspect, a kit is provided that includes a guide RNA and a CRISPR-associated protein comprising a polypeptide encoding a variant of AsCas12a, wherein the variant AsCas12a 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 improved CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

[0016] In a ninth aspect, a nucleic acid encoding a CRISPR-associated protein is provided, comprising a polypeptide encoding a variant of AsCas12a, wherein the variant AsCas12a 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 improved CRISPR / AsCas12a-associated nuclease activity at a non-canonical TTTT PAM site.

[0017] In a tenth aspect, there is provided a polynucleotide sequence encoding a Cas12a polypeptide, the polynucleotide sequence comprising one element selected from the group consisting of SEQ ID NOs: 5-17.

[0018] In an eleventh aspect, there is provided an amino acid sequence encoding a Cas12a polypeptide, the amino acid sequence comprising one element selected from the group consisting of SEQ ID NOs: 18-30.

[0019] In a twelfth aspect, a CAS endonuclease system is provided comprising an expression cassette encoding a polynucleotide sequence encoding a Cas12a polypeptide, the polynucleotide sequence comprising one member selected from the group consisting of SEQ ID NOs: 5-17.

[0020] In a thirteenth aspect, there is provided a CAS endonuclease system comprising an amino acid sequence encoding a Cas12a polypeptide, the amino acid sequence comprising one element selected from the group consisting of SEQ ID NOs: 18-30.

[0021] In a fourteenth aspect, there is provided a method of performing genome editing in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a CAS endonuclease system comprising an expression cassette encoding a polynucleotide sequence encoding a Cas12a polypeptide comprising an element selected from the group consisting of SEQ ID NOs: 5-17.

[0022] In a fifteenth aspect, there is provided a method of genome editing in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a CAS endonuclease system comprising an amino acid sequence encoding a Cas12a polypeptide comprising one element selected from the group consisting of SEQ ID NOs: 18-30.

[0023] In a sixteenth aspect, there is provided a CRISPR-associated protein comprising a fusion polypeptide, the fusion polypeptide comprising an AsCas12a open reading frame, a nuclear localization signal, an optional amino acid linker, and an optional affinity tag.

[0024] In a seventeenth aspect, there is provided a method of performing genome editing in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a CAS endonuclease system, wherein the CAS endonuclease system comprises a CRISPR-associated protein according to the sixteenth aspect. [Brief explanation of the drawings]

[0025] [Figure 1]This figure shows how multiple recombinant forms of Cas12a result in varying editing efficiencies. A series of recombinant Cas12a proteins with varying compositions and arrangements of NLS sequences, purification tags, and linkers (A-M; corresponding to SEQ ID NOs: 18-30) were cloned and purified to homogeneity. The resulting purified Cas12a derivatives were electroporated into HEK293 cells complexed with RNA guides targeting the HPRT-38186 (SEQ ID NO: 1) and HPRT-38228 (SEQ ID NO: 2) loci. DNA was isolated from genome editing experiments 48 hours later, and editing efficiencies were measured by PCR amplification of the edited loci using HPRT-FWD (SEQ ID NO: 3) and HPRT-REV (SEQ ID NO: 4) primers with the Alt-R Genome Editing Detection Kit (Integrated DNA Technologies). The following abbreviations apply to the depicted constructs: V5 refers to the V5 epitope tag (SEQ ID NO: 485), SV40 refers to the simian virus SV40 large tumor antigen nuclear localization signal (SEQ ID NO: 475), Cas12a refers to the Cas12a coding sequence, HIS refers to a 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 bipartite nuclear localization signal (SEQ ID NO: 481), and BIP2 refers to the second bipartite nuclear localization signal (SEQ ID NO: 483). Arrows in the constructs indicate the transcription start sites of mRNA transcripts generated from the DNA. [Figure 2]Exemplary results of the M537R and F870L mutations, which improve the cleavage activity of Cpf1, are shown in a bacteria-based activity assay. The screened E. coli strain was transformed with a Cpf1 expression vector and crRNA targeting the HPRT-38346 site of a toxin expression plasmid. The apparent activity of Cpf1 against TTTT or TTTC PAMs can be estimated by the number of colonies that survived under arabinose selection when the same amount of plasmid was delivered. Both mutations clearly showed higher survival rates at TTTC and TTTT PAM sites, indicating improved cleavage activity compared to WT-Cpf1. [Figure 3A] An example of an in vitro cleavage assay followed by SDS-PAGE analysis of AsCpf1 variants used for genome editing in human cell lines is shown. The indicated amounts of protein were introduced and no differences were observed compared to WT-Cpf1. These results demonstrate that the M537R and F870L mutations can enhance the in vitro cleavage activity of Cpf1 at non-canonical TTTT PAM sites while maintaining high activity at canonical TTTV sites. [Figure 3B] Figure 1 shows exemplary DNA cleavage activity of Cpf1 variants with TTTC or TTTT PAM added at the HPRT-38346 site. Both variants exhibited a higher rate of DNA cleavage at TTTT PAM sites than WT-Cpf1. These results demonstrate that the M537R and F870L mutations can enhance the in vitro cleavage activity of Cpf1 at non-canonical TTTT PAM sites while maintaining high activity at the canonical TTTV site. [Figure 4]This paper summarizes exemplary results showing the significantly improved genome targeting efficiency of AsCpf1 at the TTTT PAM site in human cell lines with the M537R and F870L mutations. The genome targeting efficiency of the Cpf1 variants was tested in human cell line models using the T7 endonuclease I assay (T7EI). Twenty-four crRNAs targeting the CTNNB1 gene with a TTTN PAM were synthesized, assembled into RNP complexes with the Cpf1 variants, and delivered by nucleofection (Lonza). Genomic DNA was collected 48 hours after delivery, and indel formation was assessed by T7EI. Not only were the variants able to cleave DNA at all TTTT PAM sites, many of which were not targeted by WT-Cpf1, but the reported variants, particularly the double mutant (M537R / F870L), significantly improved the targeting efficiency of Cpf1 at 22 of the 24 sites tested, regardless of the PAM sequence. [Figure 5A] An exemplary correlation is shown between the relative viability of M537R / F870L-AsCas12a in condition 1 (X-axis) and the relative viability of M537R / F870L-AsCas12a in condition 2 (Y-axis). Consistent phenotypic measurements were obtained (ρ approximately 0.7). [Figure 5B] An exemplary correlation is shown between the relative viability of M537R / F870L-AsCas12a in condition 1 (X-axis) and the relative viability of WT-AsCas12a in condition 3 (Y-axis). Consistent phenotypic measurements were obtained (ρ approximately 0.7). [Figure 5C] An exemplary correlation is shown between the relative viability of M537R / F870L-AsCas12a (X-axis) in condition 2 and the relative viability of WT-AsCas12a (Y-axis) in condition 2. Consistent phenotypic measurements were obtained (ρ approximately 0.7). [Figure 5D] 1 shows exemplary variant scores for phenotypes (relative survival) of a selected number of AsCas12a single point mutations at positions 537 and 870 in condition 1 and condition 2. [Figure 5E]Exemplary variant scores for the phenotype (relative survival) of a selected number of AsCas12a single point mutations at positions 537 and 870 in condition 1 and condition 2 are shown. Figure 5D and Figure 5E show that M537R and F870L are the optimal substitutions at the respective positions, which is consistent with our previous screening and mutant characterization results. [Figure 5F] 1 shows exemplary variant scores for phenotypes (relative survival) of a select number of AsCas12a single point mutations at positions 505, 510, 569, and 599 in conditions 1, 2, and 3. [Figure 6A] Figure 1 shows E. coli survival due to the DNA cleavage activity of WT-AsCas12a at the TTTT PAM. The cleavage activity of AsCas12a variants at the TTTT PAM site was measured using a bacteria-based activity assay. The initial dish containing bacteria is shown on the left. The dish with surviving bacterial colonies is shown on the right. E. coli survival under selection depends on successful cleavage of the toxin-expressing plasmid containing the TTTT PAM site. [Figure 6B] Figure 6 shows the survival rate of E. coli due to the DNA cleavage activity of the L505K-AsCas12a variant at TTTT PAM. The petri dish presentation and experimental details are as shown in Figure 6A. [Figure 6C] Figure 6 shows the survival rate of E. coli due to the DNA cleavage activity of the S510L-AsCas12a variant at TTTT PAM. The petri dish presentation and experimental details are as shown in Figure 6A. [Figure 6D] Figure 6 shows the survival rate of E. coli due to the DNA cleavage activity of the M537R-AsCas12a variant at TTTT PAM. The petri dish presentation and experimental details are as shown in Figure 6A. [Figure 6E] Figure 6 shows the survival rate of E. coli due to the DNA cleavage activity of the P569D-AsCas12a variant in the TTTT PAM. The petri dish presentation and experimental details are as shown in Figure 6A. [Figure 6F]Figure 6 shows the survival rate of E. coli due to the DNA cleavage activity of the P599G-AsCas12a variant in the TTTT PAM. The petri dish presentation and experimental details are as shown in Figure 6A. [Figure 7A] Figure 1 shows E. coli survival due to the DNA cleavage activity of M537R / F870L-AsCas12a at the TTTT PAM. The cleavage activity of the M537R / F870L-AsCas12a variant at the TTTT PAM site was measured using a bacteria-based activity assay. The initial dish containing bacteria is shown on the left. The dish with surviving bacterial colonies is shown on the right. E. coli survival under selection depends on successful cleavage of the toxin-expressing plasmid containing the TTTT PAM site. [Figure 7B] Figure 7 shows the survival rate of E. coli due to the DNA cleavage activity of the L505K / M537R / F870L-AsCas12a variant in TTTT PAM. The petri dish presentation and experimental details are as shown in Figure 7A. [Figure 7C] Figure 7 shows the survival rate of E. coli due to the DNA cleavage activity of the M537R / F870L-AsCas12a variant in TTTT PAM. The petri dish presentation and experimental details are as shown in Figure 7A. [Figure 7D] Figure 7 shows the survival rate of E. coli due to the DNA cleavage activity of the P569D / M537R / F870L-AsCas12a variant in TTTT PAM. The petri dish presentation and experimental details are as shown in Figure 7A. [Figure 7E] Figure 7 shows the survival rate of E. coli due to the DNA cleavage activity of the P599G / M537R / F870L-AsCas12a variant in TTTT PAM. The petri dish presentation and experimental details are as shown in Figure 7A. [Figure 7F] Figure 7 shows the survival rate of E. coli due to the DNA cleavage activity of the S510L / M537R / F870L-AsCas12a variant in TTTT PAM. The petri dish presentation and experimental details are as shown in Figure 7A. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention relates to compositions of Cas12a variants for expression in eukaryotic cells and methods for enhancing the utility of Cas12a and its variants.

[0027] (definition) When introducing elements of aspects of the disclosure or specific embodiments, the articles "a," "an," "the," and "said" are intended to mean 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 other than the listed elements. The term "or" means any one element of a particular list, and unless otherwise specified, also includes any combination of elements of that list.

[0028] As intended herein, the terms "substantially," "approximately," and "about" and similar terms are intended to have broad meanings consistent with common and accepted usage in the art to which the subject matter of this disclosure pertains. It will be understood by those skilled in the art who review this disclosure that these terms are intended to enable description of the particular features being described and claimed without limiting the scope of those features to the precise numerical ranges provided. Accordingly, these terms should be interpreted as indicating that insubstantial or insignificant modifications or variations of the subject matter described and claimed are considered to be within the scope of the invention as set forth in the appended claims.

[0029] Definitions associated with certain terms and phrases applicable herein may be found in relevant U.S. patents and publications, such as U.S. Provisional Patent Applications Nos. 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.

[0030] The term "substantially purified" as applied to a composition refers to a composition having a purity of at least 90% or greater, including 90% purity, 95% purity, 99% purity, and greater than 99% purity.

[0031] 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. Further, with respect to an isolated ribonucleoprotein complex, preferably all components are substantially purified.

[0032] The terms "nucleic acid" and "polynucleotide" are interchangeable and have the same meaning.

[0033] The terms "amino acid sequence," "polypeptide," and "protein" are interchangeable and have the same meaning.

[0034] The term "affinity tag" refers to a ligand that allows for detection and / or selection of an oligonucleotide sequence to which the ligand is bound. For purposes of this disclosure, a bait may comprise an affinity tag. In particular, affinity tags are typically placed at either or both the N'-terminus and / or C'-terminus of a polypeptide through the use of conventional chemical conjugation techniques or recombinant DNA technology. Exemplary affinity tags include biotin, digoxigenin, streptavidin, polyhistine (e.g., (His6)), glutathione-S-transferase (GST), HaloTag®, Avi-tag, calmodulin tag, polyglutamic acid tag, FLAG tag, HA tag, Myc tag, S-tag, SBP tag, Softag 3, V5 tag, Xpress tag, haptens, and others.

[0035] The term "eukaryotic cell," as discussed herein, includes cells of or derived from specific organisms such as plants or mammals, including, but not limited to, humans, or non-human eukaryotic organisms or animals or mammals, such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates. In some embodiments, processes for altering the genetic identity of a human germline and / or processes for altering the genetic identity of an animal that may cause suffering to the human or animal and the animals resulting from such processes without substantial medical benefit may be excluded. Preferred human cells include somatic cells and cells derived from germline cells. Exemplary somatic cells include cells from all major organ and tissue systems, including the immune and hematopoietic systems.

[0036] As described herein, conditions 1, 2, and 3 refer to various combinations of background strains and the amount of gRNA introduced into the background strain before variant selection. Condition 1 is an M537R / F870L background containing the amount of gRNA introduced at which the variant was selected (100 pmol per 10 microliters of transformation / plating experiment). Condition 2 is an M537R / F870L background containing the amount of gRNA introduced at which the variant was selected (50 pmol per 10 microliters of transformation / plating experiment). Condition 3 is a wild-type AsCpf1 background containing the amount of gRNA introduced at which the variant was selected (200 pmol per 10 microliters of transformation / plating experiment).

[0037] Cas12a polypeptide with a eukaryotic nuclear localization signal Because Cas12a is a bacterial protein, it does not have a native targeting mechanism to reach the eukaryotic nucleus where the target DNA resides.

[0038] To more efficiently target proteins to the eukaryotic nucleus, short protein sequences called nuclear localization signals (NLSs) are typically added to the amino or carboxy terminus of a given open reading frame. NLSs are recognized by transduced proteins on the eukaryotic nuclear envelope, first binding to the nuclear membrane and then enabling pore transport into the nucleus through an energy-dependent process. While recombinant protein tags such as NLSs can significantly improve localization, the addition of exogenous amino acid sequences presents a reasonable possibility of perturbing protein function. Therefore, discovering recombinant Cas12a protein sequences that promote very large amounts of nuclear transport without adversely affecting its activity would ultimately be crucial and highly desirable, making it the most powerful Cas12a genome editing solution.

[0039] To improve nuclear delivery of Cas12a without disrupting its function, several different recombinant versions of Cas12a were constructed, varying the identity, position, and number of protein tags (NLS, hexahistidine tag (exemplary affinity tag)). Hexahistidine and V5 tags were added to Cas12a constructs to aid in protein purification and / or detection, while the NLS tag was added to aid in delivery to the eukaryotic nucleus. The domain-disrupting linker sequence was also varied in composition and position to empirically determine the best placement and context for the tagged sequence. All constructs were first expressed in E. coli, and the recombinant Cas12a protein was purified by immobilized metal affinity chromatography (IMAC) followed by ion-exchange chromatography as previously described.

[0040] (AsCpf1 polypeptide variants with novel cleavage activity) Using a bacterial screening approach, we describe amino acid substitutions in AsCpf1 that enhance cleavage activity at both canonical (TTTV) and non-canonical (TTTT) PAM sites. This screen contains two components: (i) a toxin plasmid encoding an arabinose-inducible cell proliferation toxin and a CRISPR / Cpf1 on-target cleavage site (HPRT-38346) with TTTPAM, and (ii) a chloramphenicol resistance plasmid with random mutation regions within the AsCpf1 sequence (approximately 5 mutations per kb). The screen was performed as follows: E. coli BW25141(λDE3) was transformed with the toxin plasmid containing the HPRT-38346 target site in the absence of arabinose, which prevents toxin production and allows cell viability. Next, cells stably replicating the toxin plasmid were transformed with an AsCpf1 expression plasmid and a crRNA targeting HPRT-38346, and the cells were then plated on medium containing both chloramphenicol and arabinose. The grown bacteria were (i) successfully transformed with the AsCpf1 expression plasmid and (ii) expressed sufficient AsCpf1 variants to cleave the toxin plasmid at the HPRT-38346 site using the TTTT PAM. The AsCpf1 expression plasmid in surviving cells was recovered and used in subsequent rounds of selection. After multiple rounds of selection, the identities of enriched AsCpf1 variants were determined by DNA sequencing and carried forward for analysis in mammalian cells.

[0041] This disclosure provides two novel point mutations and their combination in the AsCpf1 gene that enhance cleavage activity: M537R and F870L. The cleavage activity of each individual mutant was first tested in a bacterial-based activity assay. The purified protein was then further tested in vitro and in human cell lines. In summary, both substitutions significantly enhanced the DNA cleavage activity of Cpf1 at TTTT PAM sites in all assays. Furthermore, the combination of M537R and F870L significantly improved the targeting efficiency of AsCpf1 in human cell lines. Overall, this 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.

[0042] As explained in the Background section, the prior art consists of using wild-type Cpf1 protein or two variants that are compatible with the TYCV and TATV PAMs. As mentioned above, these variants have limited utility due to the complex and mutually exclusive requirements of the PAM sequence. Furthermore, the TTT T None of the variants showed improved cleavage activity at the PAM. Unfortunately, this occurs more frequently than other TTTV PAM sites (V = G / A / C) throughout the human genome. In contrast, the mutations reported in this study (M537R and F870L) not only enable efficient cleavage at the TTTT PAM, but also broadly enhanced the cleavage activity of Cpf1 at canonical TTTV sites tested in human cell lines. Together, the enhanced activity and expanded PAM flexibility (TTTN) of this study make it a superior CRISPR enzyme that can directly replace current wild-type Cpf1 in genome editing applications.

[0043] High-throughput generation of AsCas12a variants with cleavage activity against target sites containing TTTT PAM We describe the phenotypes of all point mutations in the following regions of AsCas12a: 499-640 and 840-913 in bacterial screens measuring DNA cleavage activity with the non-canonical TTTTPAM. Three sets of screens were performed to measure the phenotype of each point mutation in both WT-AsCas12a and M537R / F870L-AsCas12a backgrounds. Cross-comparison of the three datasets revealed consistent phenotypic measurements, enabling us to isolate novel AsCas12a variants with enhanced activity beyond M537R and F870L.

[0044] High-throughput characterization of Cas12a activity at the TTTT PAM site yielded the functional consequences of all possible single amino acid changes within the target region. Our unbiased approach allows us to identify a large collection of mutants to further enhance the cleavage activity of AsCa12a beyond our previous invention (M537R / F870L).

[0045] To improve the coverage and efficiency of screening, we generated AsCas12a deep-scanning mutagenesis libraries containing all possible point mutations at the protein level in the target regions (490-640 and 840-913), with most clones containing only a single mutation. This type of library allowed us to directly evaluate the phenotype of each point mutation by measuring its relative survival rate relative to the reference protein in bacterial screening. Briefly, the screening strain carrying the toxin plasmid was transformed with an AsCas12a library containing a target site containing a TTTT PAM on the toxin plasmid. After transformation, cells were plated on selective medium. The AsCas12a expression plasmid carried by surviving E. coli cells was extracted and purified. Both the input and selection plasmid libraries were PCR amplified and sequenced on an Illumina MiSeq with 1–2 million reads per library. The frequency of each AsCas12a variant in both libraries was determined using Enrich2 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 normalized frequencies between the selected and input libraries. The degree of cell survival indicates the DNA cleavage activity of each AsCas12a variant, so variants with higher survival rates than the reference protein have enhanced activity with TTTTPAM.

[0046] As provided herein, codon-optimized Cas12a polypeptides are provided, including codon-optimized Cas12a polypeptides for CRISPR ribonucleoprotein complexes. An example of a codon-optimized sequence is one optimized for expression in a eukaryote, such as a human (i.e., optimized for human expression), or for another eukaryote, animal, or mammal, as discussed herein. While this is preferred, other examples are possible, and it should be understood that codon optimization for host species other than humans, or for specific organs, is known. In some embodiments, the enzyme-coding sequence encoding the CRISPR Cas12a polypeptide is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells include cells of or derived from specific organisms, such as plants or mammals, including, but not limited to, humans, or non-human eukaryotes or animals or mammals, such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates. In some embodiments, processes for altering the germline genetic identity of humans and / or processes for altering the genetic identity of animals that may cause suffering to humans or animals and animals resulting from such processes without substantial medical benefit to them may be excluded.

[0047] Preferred human cells include cells derived from somatic and germline cells. Exemplary somatic cells include cells from all major organ and tissue systems, including the immune and hematopoietic systems.

[0048] Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by substituting at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with a codon more frequently or most frequently used in the host cell's genes while maintaining the native amino acid sequence. Different species exhibit specific biases toward certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which is thought to depend, among other things, on the characteristics of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of selected tRNAs within a cell generally reflects the codons most frequently used in peptide synthesis. Therefore, genes can be tailored for optimal gene expression in a given organism based on codon optimization. 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 are also available, such as Gene Forge (Aptagen; Jacobus, PA), to optimize codons for expression in a particular host cell. 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 a sequence encoding Cas12a correspond to the most frequently used codon for a particular amino acid.

[0049] Additionally provided are codon-optimized Cas12a polypeptides, including codon-optimized Cas12a polypeptides for CRISPR ribonucleoprotein complexes, wherein the Cas12a polypeptide sequence is optimized for expression in prokaryotes, such as bacteria (e.g., E. coli).

[0050] (Application) The present invention is useful for any CRISPR / Cas12a DNA cleavage and / or gene editing experiment or for either basic research or therapeutic applications. The superior activity of these recombinant variants can be applied to Cas12a from any species or potentially any CRISPR enzyme.

[0051] In a first aspect, a CRISPR-associated protein is provided, comprising a polypeptide encoding a variant of AsCpf1. 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 a first respect, the CRISPR-associated protein corresponds to the AsCpf1 variant M537R. In a second respect, the CRISPR-associated protein corresponds to the AsCpf1 variant F870L (SEQ ID NO: 473). In a third respect, the CRISPR-associated protein corresponds to the AsCpf1 variant M537R / F870L (SEQ ID NO: 465).

[0052] In a second aspect, a CRISPR ribonucleoprotein complex is provided. The CRISPR ribonucleoprotein complex includes a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein includes a polypeptide encoding a variant of AsCpf1. 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 a first aspect, the CRISPR ribonucleoprotein complex includes a variant of AsCpf1 that is M537R. In a second aspect, the CRISPR ribonucleoprotein complex includes a variant of AsCpf1 that is F870L (SEQ ID NO: 473). In a third aspect, the CRISPR ribonucleoprotein complex includes a variant of AsCpf1 that is M537R / F870L (SEQ ID NO: 465).

[0053] In a third aspect, TTT in cells having CRISPR ribonucleoprotein complexes. N A method for increasing the efficiency of gene editing at a PAM site is provided. The method includes contacting a cell with a CRISPR ribonucleoprotein complex. The CRISPR ribonucleoprotein complex includes a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein includes a polypeptide encoding a variant of AsCpf1. 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 a first aspect, TTT N The PAM site comprises one selected from the group of TTTA, TTTT, and TTTC PAM sites.

[0054] In a fourth aspect, a kit is provided that includes a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein includes a polypeptide encoding a variant of AsCpf1. The variant of AsCpf1 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 a first aspect, the variant of AsCpf1 is M537R. In a second aspect, the variant of AsCpf1 is F870L (SEQ ID NO: 473). In a third aspect, the variant of AsCpf1 is M537R / F870L (SEQ ID NO: 465).

[0055] In a fifth aspect, a CRISPR-associated protein comprises a polypeptide encoding a variant of AsCas12a, wherein the variant AsCas12a 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 improved CRISPR / AsCas12a-associated nuclease activity at non-canonical TTTT PAM sites. In a first aspect, the AsCas12a variant is selected from the group consisting of SEQ ID NOs: 59-245. In a second aspect, the AsCas12a variant further comprises the mutations M537R / F870L (SEQ ID NO: 465) as described in the first aspect or the preceding first aspect of the first aspect.

[0056] 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 a variant of AsCas12a, wherein the variant AsCas12a 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 improved CRISPR / AsCas12a-associated nuclease activity at non-canonical TTTT PAM sites. In a first aspect, the AsCas12a variant is selected from the group consisting of SEQ ID NOs: 59-245. In a second aspect, the AsCas12a variant further comprises the mutations M537R / F870L (SEQ ID NO: 465) as described in the second aspect or the foregoing first aspect of the second aspect.

[0057] In a seventh aspect, a method for enhancing the efficiency of gene editing at non-canonical TTTT PAM sites in a cell harboring a CRISPR ribonucleoprotein complex is provided. The method includes contacting the cell with a CRISPR ribonucleoprotein complex comprising a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein includes a polypeptide encoding a variant of AsCas12a, wherein the variant AsCas12a provides improved CRISPR / AsCas12a-associated nuclease activity at the non-canonical TTTT PAM site, and 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. In a first aspect, the AsCas12a variant is selected from the group consisting of SEQ ID NOs: 59-245. In a second respect, the variant of AsCas12a as described in the third aspect or the above first respect of the third aspect further comprises the mutations M537R / F870L (SEQ ID NO: 465).

[0058] In an eighth aspect, a kit is provided that includes a CRISPR-associated protein comprising a guide RNA and a polypeptide encoding a variant of AsCas12a. 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 improved CRISPR / AsCas12a-associated nuclease activity at non-canonical TTTT PAM sites. In a first aspect, the AsCas12a variant is selected from the group consisting of SEQ ID NOs: 59-245. In a second aspect, the AsCas12a variant further comprises the mutations M537R / F870L (SEQ ID NO: 465) as described in the fourth aspect or the aforementioned first aspect of the fourth aspect.

[0059] In a ninth aspect, a nucleic acid encoding a CRISPR-associated protein is provided, comprising a polypeptide encoding a variant of AsCas12a. 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 improved CRISPR / AsCas12a-associated nuclease activity at non-canonical TTTT PAM sites. Highly preferred nucleic acids encoding CRISPR-associated proteins include isolated nucleic acids encoding CRISPR-associated proteins. In a first aspect, the AsCas12a variant is selected from the group consisting of SEQ ID NOs: 59-245. In a second aspect, the AsCas12a variant further comprises the mutation M537R / F870L (SEQ ID NO: 465). In a third aspect, the nucleic acid is operably linked to appropriate transcriptional elements for expressing the nucleic acid. In a fourth aspect, the nucleic acid is DNA or RNA.

[0060] In a tenth aspect, there is provided a polynucleotide sequence encoding a Cas12a polypeptide, the polynucleotide sequence comprising one element selected from the group consisting of SEQ ID NOs: 5-17.

[0061] In an eleventh aspect, there is provided an amino acid sequence encoding a Cas12a polypeptide, the amino acid sequence comprising one element selected from the group consisting of SEQ ID NOs: 18-30.

[0062] In a twelfth aspect, a CAS endonuclease system is provided comprising an expression cassette encoding a polynucleotide sequence encoding a Cas12a polypeptide, the polynucleotide sequence comprising one member selected from the group consisting of SEQ ID NOs: 5-17.

[0063] In a thirteenth aspect, there is provided a CAS endonuclease system comprising an amino acid sequence encoding a Cas12a polypeptide, the amino acid sequence comprising one element selected from the group consisting of SEQ ID NOs: 18-30.

[0064] In a fourteenth aspect, there is provided a method of performing genome editing in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a CAS endonuclease system comprising an expression cassette encoding a polynucleotide sequence encoding a Cas12a polypeptide comprising an element selected from the group consisting of SEQ ID NOs: 5-17.

[0065] In a fifteenth aspect, there is provided a method of performing genome editing in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a CAS endonuclease system comprising an amino acid sequence encoding a Cas12a polypeptide comprising one element selected from the group consisting of SEQ ID NOs: 18-30.

[0066] In a 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 a first aspect, the AsCas12a open reading frame is selected from the group consisting of SEQ ID NOs: 59-245. In a second aspect, the nuclear localization signal is selected from SEQ ID NOs: 475, 477, 479, 481, and 483. In a third aspect, the CRISPR-associated protein is encoded by SEQ ID NOs: 488-491. In a fourth aspect, the CRISPR-associated protein is selected from SEQ ID NOs: 492 and 493.

[0067] In a seventeenth aspect, there is provided a method of performing genome editing in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a CAS endonuclease system, the CAS endonuclease system comprising a CRISPR-associated protein according to aspect sixteen. Highly preferred CRISPR-associated proteins include isolated CRISPR-associated proteins. [Example]

[0068] Example 1. Recombinant Cas12a proteins with different tag / linker sequences result in a range of editing efficiencies. The following examples demonstrate that recombinant Cas12a proteins with only minor changes in the tag sequences at the amino and carboxy termini result in proteins that exhibit a wide range of editing efficiencies in human cells (Figure 1).

[0069] Briefly, the method of site-directed mutagenesis (SDM) was used to generate expression constructs carrying the AsCas12a coding sequence with various nuclear localization signals (NLS). Site-directed mutagenesis was performed by designing complementary primers encompassing the desired nucleotide base changes along with flanking plasmid vector sequences, where each flanking region had a melting temperature (T) of at least 60°C. m Next, polymerase chain reaction (PCR) was performed for a total of 16 cycles using standard cycling conditions. The restriction enzyme DPN I was added to digest the starting plasmid vector material, leaving only the new product containing the base change. After DPN I treatment, a small amount of the PCR product was transformed into competent E. coli cells, recovered in SOC medium, and plated onto kanamycin-resistant Luria Broth (LB) agar plates. To confirm the correct base change in selected clones, colonies were screened using Sanger sequencing.

[0070] This result indicates that the ideal sequence and placement of the NLS sequence on Cas12a is unclear and indicates that highly efficient Cas12a genome editing solutions must be empirically determined, as was done in this study. The protein was tested using guides targeting the HPRT-38186 (SEQ ID NO: 1) and HPRT-38228 (SEQ ID NO: 2) loci in human cells. SEQ ID NOs: 1-4 are shown in Table 1.

[0071] [Table 1]

[0072] The following nucleotide sequences encoding preferred Cas12a polypeptides of this example are provided below:

[0073] SEQ ID NO.: 5

[0074]

[0075] SEQ ID NO.:6

[0076]

[0077] SEQ ID NO.: 7

[0078]

[0079] SEQ ID NO.: 8 (SEQ ID NO: 8)

[0080]

[0081] SEQ ID NO.:9

[0082]

[0083] SEQ ID NO.: 10 (SEQ ID NO: 10)

[0084]

[0085] SEQ ID NO.: 11 (SEQ ID NO: 11)

[0086]

[0087] SEQ ID NO.: 12

[0088]

[0089] SEQ ID NO.: 13

[0090]

[0091] SEQ ID NO.: 14 (SEQ ID NO: 14)

[0092]

[0093] SEQ ID NO.: 15 (SEQ ID NO: 15)

[0094]

[0095] SEQ ID NO.: 16 (SEQ ID NO: 16)

[0096]

[0097] SEQ ID NO.: 17

[0098]

[0099] The amino acid sequences of the following preferred Cas12a polypeptides are shown below:

[0100] SEQ ID NO.: 18 (SEQ ID NO: 18)

[0101]

[0102] SEQ ID NO.: 19 (SEQ ID NO: 19)

[0103]

[0104] SEQ ID NO.: 20 (SEQ ID NO: 20)

[0105]

[0106] SEQ ID NO.: 21 (SEQ ID NO: 21)

[0107]

[0108] SEQ ID NO.: 22

[0109]

[0110] SEQ ID NO.: 23 (SEQ ID NO: 23)

[0111]

[0112] SEQ ID NO.: 24

[0113]

[0114] SEQ ID NO.: 25

[0115]

[0116] SEQ ID NO.: 26 (SEQ ID NO: 26)

[0117]

[0118] SEQ ID NO.: 27

[0119]

[0120] SEQ ID NO.: 28 (SEQ ID NO: 28)

[0121]

[0122] SEQ ID NO.: 29

[0123]

[0124] SEQ ID NO.: 30 (SEQ ID NO: 30)

[0125]

[0126] Example 2. Novel Cpf1 mutants enhance DNA cleavage activity at TTTT PAM sites in a bacterial-based activity assay. The following example demonstrates the enhanced activity of the present invention at both TTTT and TTTC PAM sites in a bacterial-based activity assay (Figure 2). Screening strains carrying the toxin plasmid were transformed with WT, M537R, or F870L AsCpf1 expression plasmids. After recovery and IPTG induction, cells were plated on LB-chloramphenicol medium with or without arabinose. The degree of cell survival under arabinose selection compared to the transformation input control (no arabinose) indicates the cleavage activity of the Cpf1 variant at the HPRT-38346 protospacer on the toxin plasmid in the context of either the TTTT or TTTC PAM.

[0127] In the case of WT-Cpf1, the survival rate of transformed E. coli with TTTC PAM was significantly higher than that with TTTT PAM, which is consistent with the prior knowledge that TTTT is a less active PAM site. [6] This is in good agreement with the results. In contrast, both M537R and F870L increased survival at the TTTT PAM, indicating that these mutants broadened the PAM compatibility of AsCpf1 at this alternative PAM site. More importantly, survival of both mutants at the canonical TTTC PAM was even higher than that of WT-Cpf1, suggesting that these mutants generally enhanced the performance of the AsCpf1 protein at other TTTC PAM sites. Given these positive results, we expressed and purified individual AsCpf1 variants and the double mutant (M537R / F870L) to determine their intrinsic cleavage activity in vitro.

[0128] Example 3. A novel Cpf1 mutant enhances endogenous DNA cleavage activity at TTTT PAM sites in vitro. 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. Briefly, the Cpf1-crRNA ribonucleoprotein (RNP) complex was first assembled by incubating purified protein (Figure 3A) with HPRT-38346 crRNA in 1X cleavage buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 5 mM MgCl2, 10% glycerol, and 1 mM DTT) at 37 °C for 15 min. The cleavage reaction was initiated by titrating the RNP complex (8–500 nM) in 10 nM dsDNA substrate containing the HPRT-38346 protospacer in the context of TTTC or TTTT PAM. Cleavage reactions at various time points were sampled and quenched with 50 mM EDTA. After removing AsCpf1 protein by proteinase K treatment (56°C, 30 min), the reaction was resolved using capillary electrophoresis (Fragment Analyzer, AATI). The relative concentrations of cleavage products and uncleaved dsDNA were quantified to calculate the percentage of DNA cleavage.

[0129] The intrinsic DNA cleavage activity of WT and Cpf1 variants at the TTTT and TTTC PAM sites is compared in Figure 3B. For simplicity, only a single RNP concentration (31 nM) at the 20 s time point is shown. As expected, the TTT C From TTT TA single nucleotide change in the PAM sequence to α-Cpf1 reduced the cleavage activity of WT-Cpf1 from approximately 95% to approximately 40%. Consistent with observations in bacterial-based activity assays, both mutants significantly increased DNA cleavage at the TTTT PAM while maintaining high activity at the TTTC PAM (Figure 3B). In this assay, the double mutant (M537R / F870L) exhibits similar activity to M537R. However, it should be noted that this is likely due to the limited resolution of this particular assay to resolve further differences between these highly active variants. Overall, these results demonstrated that the reported mutations improve the activity of Cpf1 by enhancing endogenous DNA cleavage. Therefore, we envision that the observed benefits of these mutants are broadly applicable and not dependent on the delivery method and / or cellular context of a particular experiment.

[0130] Example 4. Novel mutants significantly enhance targeting efficiency of TTTN PAM sites in human cell lines. The following example demonstrates that when the Cpf1-crRNA complex is delivered to cells as an RNP, TTT N 1 demonstrates the ability of the present invention to increase the efficiency of gene editing at PAM sites.

[0131] CRISPR / Cpf1 cell editing experiments were first performed using purified Cpf1 protein and Alt-R TM This was carried out by forming a 4 mM RNP complex with crRNA in Opti-MEM at 25°C for 5 minutes. The target protospacer and PAM sequences at the CTNNB1 locus are shown in Table 2. The RNP complex was then transfected into HEK293 cells by Lonza nucleofection. Experiments were performed in biological triplicates. After 48 hours at 37°C and 5% CO2, adherent cells were washed with 0.1 ml of PBS and transfected with 0.05 ml of QuickExtract. TMThe cells were lysed in DNA extraction solution. The cell lysate was incubated at 65°C for 15 minutes, followed by heat inactivation at 98°C for 3 minutes. The unpurified DNA sample was then diluted 3-fold with 0.1 ml of ddH2O and used as a PCR template. PCR primers are listed in Table 2. PCR was used to amplify a 1 kb fragment of the CTNNB1 locus using KAPA HiFi DNA polymerase. The following cycle parameters were used: 95 cycles, 95 min, 96 min, 10 min, 15 min, 10 ... 5:00 , (98 0:20 , 64 0:15 , 72 0:30 ) 29 times, 72 2:00 The heteroduplex was amplified using the following cycle parameters: 95 cycles for 1 minute; 10:00 Cools to 85°C in 1 minute 1:00 Cools to 75°C in 1 minute 1:00 Cools to 65°C in 1 minute 1:00 Cooling from 55 to 55 in 1 minute 1:00 Cools to 45°C in 1 minute 1:00 Cools to 35°C in 1 minute 1:00 Cooling from 25 to 25 1:00 The heteroduplex was cleaved by adding 2 U T7 Endonuclease I (New England Biolabs) for 1 h at 37°C, and the cleaved products were analyzed by capillary electrophoresis (Fragment Analyzer, Advanced Analytical).

[0132] [Table 2]

[0133] Referring to Figure 4, T7EI assays revealed significant improvements in targeting efficiency with the M537R and F870L mutations. First, the M537R, F870L, or double mutant (M537R / F870L) enabled efficient cleavage at all 15 sites with a TTTT PAM, where 11 of the 15 had no detectable cleavage by WT-Cpf1. At other sites using the standard TTTV PAM, targeting efficiency was maintained or improved by these variants. This advantage was particularly significant at low-activity sites, such as CTNNB1 111-s (3-fold improvement over WT). Among these variants, the double mutant (M537R / F870) showed the most consistent improvement in targeting efficiency at all sites tested, where single mutants showed more site-dependent variability, such as F870L at 323-s (no activity, similar to WT). Overall, the described invention exhibits much better on-target potency than WT-Cpf1.

[0134] Example 5. High-throughput measurement of DNA cleavage activity of AsCas12a variants at TTTT PAM sites in E. coli The following example demonstrates the robustness of our novel high-throughput screening strategy, which directly measures the cleavage activity at the TTTT PAM site of thousands of AsCas12a variants in a bacterial-based activity assay (Figure 5). Figures 5A-F show exemplary high-throughput phenotypic measurements of AsCas12a point mutations by deep-scanning mutagenesis. Comprehensive libraries encompassing all possible single point mutations in AsCas12a in the target regions (499-640 and 840-913) were generated in the context of WT-AsCas12a or M537R / F870L-AsCas12a. The relative viability of each variant relative to the reference protein in an E. coli-based activity assay was determined by deep sequencing. The phenotypes of individual point mutations were measured under multiple selection stringencies in the context of M537R / F870L (conditions 1 and 2) and a third condition in the WT-AsCas12a background (condition 3). As shown in Figure 5A-C, the phenotypic scores of the variants (i.e., the natural logarithm of relative viability) were positively correlated across different conditions (ρ approximately 0.7), demonstrating the consistency and reproducibility of this approach. As positive controls, only M537R and F870L / I, but not other substitutions at these positions, outlived WT-AsCas12a (Figure 5D). Conversely, mutating R537 or L870 in M537R / F870L-AsCas12a ubiquitously reduced viability (Figure 5E). These results demonstrated that the phenotypic scores measured by bacterial screening reflect the DNA cleavage activity of AsCas12a variants previously characterized with the TTTT PAM.

[0135] To further validate the results of our bacterial screening, we investigated four point mutations in AsCas12a that conferred higher survival rates than the reference in all three conditions (L505K, S510L, P569D, and P599G, Figure 5F). Notably, P599G enhances the cleavage activity of AsCas12a at the TTCC PAM. The other three point mutations have not been characterized in any published studies to date. Therefore, we measured the viability of E. coli cells transformed with plasmids expressing the individual AsCas12a variants under selection. Compared to WT-AsCas12a, all selected point mutations increased survival when targeting the TTTT-PAM site (Figure 6). Unexpectedly, the benefit of these point mutations also held true in the context of M537R / F870L-AsCas12a, where survival was further increased (Figure 7). Collectively, these results demonstrate that high-throughput screening can accurately predict the phenotype of uncharacterized AsCas12a variants.

[0136] The phenotypic scores of 3,194 AsCas12a variants with single point mutations covered by screening with sufficient sequencing counts are shown in Table 3. Overall, approximately 60% showed some benefit (i.e., phenotypic score >0) in one of the three conditions.

[0137] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19 Table 3-20 Table 3-21 Table 3-22 Table 3-23 Table 3-24 Table 3-25 Table 3-26 Table 3-27 Table 3-28 Table 3-29 Table 3-30 Table 3-31 Table 3-32 Table 3-33 Table 3-34 Table 3-35 Table 3-36 Table 3-37 Table 3-38 Table 3-39 Table 3-40 Table 3-41 Table 3-42 Table 3-43 Table 3-44 Table 3-45 Table 3-46 Table 3-47 Table 3-48 Table 3-49 Table 3-50 Table 3-51 Table 3-52 Table 3-53 Table 3-54 Table 3-55 Table 3-56 Table 3-57 Table 3-58 Table 3-59 Table 3-60 Table 3-61 Table 3-62 Table 3-63 Table 3-64 Table 3-65 Table 3-66 Table 3-67 Table 3-68 Table 3-69 Table 3-70 Table 3-71 Table 3-72 Table 3-73 Table 3-74 [Table 3-75] [Table 3-76] [Table 3-77]

[0138] 1 Header abbreviation key: Mutants are defined by the single-letter amino acid (wild-type) at a given position (e.g., amino acid position 500) of the wild-type AsCas12a polypeptide, followed by a variant amino acid change. "Enrichment score (1) ("Enrich. Score(1)")" refers to the enrichment score of a variant in a background of condition 1. "Standard error (1) ("Stand. Error(1)")" refers to the standard error of an experiment performed on a given variant in a background of condition 1. "Enrichment score (2) ("Enrich. Score(2)")" refers to the enrichment score of a variant in a background of condition 2. "Standard error (2) ("Stand. Error(2)")" refers to the standard error of an experiment performed on a given variant in a background of condition 2. "Enrichment score (3) ("Enrich. Score(3)")" refers to the enrichment score of a variant in a background of condition 3. "Stand. Error(3)" refers to the standard error of an experiment performed for a given variant in the background of condition 3. "Any Positive Enrichment?" refers to the occurrence of positive enrichment for a given variant in the background of at least one experimental condition. "Consistent positive enrichment?" refers to the occurrence of positive enrichment for a given variant in the background of all experimental conditions tested.

[0139] 187 variants (approximately 6% of the total) consistently improved viability under all conditions (Table 4). These variants, including four individually validated variants, can be stacked with WT- or M537R / F870L-AsCas12a to enhance DNA cleavage activity at TTTT-PAM.

[0140] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0141] 1 A phenotypic score (i.e., the natural logarithm of relative enrichment) is provided for each point mutation. Error bars estimate the precision of the measurement, which depends on the sequence count of each variant in the library. Only variants with counts >50 across all libraries were included in the analysis.

[0142] Table 5 shows the sequences used as primers to generate the AsCas12a saturation mutagenesis library. Standard recombination methods and techniques were used. The screening library was constructed using the method described by Wrenbeck et al. (2016).

[0143] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0144] For Table 4, the reference (i.e., wild-type) polypeptide sequence is SEQ ID NO:462, to which these variants are based by comparison. Codon-optimized polynucleotides encoding SEQ ID NO:462 for expression in E. coli and human cells are SEQ ID NOs:463 and 464, respectively. The same mutations were also introduced into the M537R / F870L-AsCas12a background. The reference polypeptide sequence corresponding to M537R / F870L-Cas12a is SEQ ID NO:465 (the altered amino acids are underlined). Codon-optimized polynucleotides encoding SEQ ID NO:465 for expression in E. coli and human cells are SEQ ID NOs:466 and 467, respectively (the altered codons are underlined).

[0145] SEQ ID NO.: 462

[0146]

[0147] SEQ ID NO.: 463

[0148]

[0149] SEQ ID NO.: 464

[0150]

[0151] SEQ ID NO.: 465

[0152] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQ R PTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKF LFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSNLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTI KNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEGGIVFRDGSNILKPLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN

[0153] SEQ ID NO.: 466(SEQ ID NO:466)

[0154] CGTCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTC CTG

[0155] SEQ ID NO.: 467

[0156] CGTCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTC CTG

[0157] Additional polynucleotides and polypeptides related to this example include Cas12a variants with single amino acid substitutions at M537R and F870L, as shown below, where the underlined codons or amino acids correspond to changes relative to the corresponding WT Cas12a sequence.

[0158] SEQ ID NO.: 468 (SEQ ID NO: 468) E. coli optimized DNA M537R

[0159] CGT

[0160] SEQ ID NO.: 469 (SEQ ID NO: 469) E. coli optimized DNA F870L Cas12a

[0161] CTG

[0162] SEQ ID NO.: 470 (SEQ ID NO: 470) Human optimized DNA M537R Cas12a

[0163] CGT

[0164] SEQ ID NO.:471 (SEQ ID NO:471) Human optimized DNA F870L Cas12a

[0165] CTG

[0166] SEQ ID NO.:472(SEQ ID NO:472)M537R Cas12a AA

[0167] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQ RPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDS TKGILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN

[0168] SEQ ID NO.:473(SEQ ID NO:473)F870L Cas12a AA

[0169] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPS LREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHGKPNHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKF LFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEI VDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTI KNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGS NILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN

[0170] Example 6. Rational design of fusion Cas12a polypeptides Fusion Cas12 / a polypeptides with additional motifs (collectively "NLS" or "NLS sequences") that allow for nuclear localization in eukaryotic cells and / or purification and labeling detection motifs (collectively "affinity tags") are 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 and listed by the polynucleotide and amino acid sequences set forth in Table 6.

[0171] [Table 6]

[0172] Exemplary purification and / or labeling detection motifs include affinity tags, which are well known in the art. In many cases, an additional amino acid linker inserted before or after the additional motif can improve the expression and / or stability of the expressed fusion Cas12 / a polypeptide. Two examples of affinity tags are defined by the polynucleotide and amino acid sequences shown in Table 7 below.

[0173] [Table 7]

[0174] Fusion Cas12a polypeptides containing a nuclear localization signal, linker amino acids, and / or affinity tag can be readily constructed using chemical polypeptide methods and expressed from engineered polynucleotides encoding in-frame polypeptides created by recombinant DNA technology. Such techniques are well known and within the skill of the art. Examples of such polynucleotides and polypeptides are shown in SEQ ID NOS: 5-30. Fusion Cas12a polypeptide variants encoding the open reading frames of SEQ ID NOS: 59-245 with a nuclear localization sequence and / or affinity tag and, optionally, an amino acid linker, are within the scope of this disclosure. Exemplary Cas12a variants with a nuclear localization signal are shown below.

[0175] Briefly, the method of site-directed mutagenesis (SDM) was used to generate expression constructs carrying the AsCas12a coding sequence with various nuclear localization signals (NLS). Site-directed mutagenesis was performed by designing complementary primers encompassing the desired nucleotide base changes along with flanking plasmid vector sequences, and each flanking region was engineered to have a melting temperature (T) of at least 60°C. mNext, polymerase chain reaction (PCR) was performed for a total of 16 cycles using standard cycling conditions. The restriction enzyme DPN I was added to digest the starting plasmid vector material, leaving only the new product containing the base change. After DPN I treatment, a small amount of the PCR product was transformed into competent E. coli cells, recovered in SOC medium, and plated onto kanamycin-resistant Luria Broth (LB) agar plates. To confirm the correct base change in selected clones, colonies were screened using Sanger sequencing.

[0176] SEQ ID NO.:488 (SEQ ID NO:488) E. coli optimized DNA WT Cas12a with NLS linker

[0177] [ka] [ka]

[0178] The underlined sequence indicates nucleotides encoding the amino acid linker sequence, the double underlined sequence indicates nucleotides encoding the nuclear localization sequence (NLS linker), and the italicized sequence indicates nucleotides encoding the amino acid affinity tag sequence ((HIS)6).

[0179] SEQ ID NO. 489 (SEQ ID NO: 489) E. coli optimized DNA M537R F870L Cas12a

[0180] [ka] [ka]

[0181] The bold and underlined sequences indicate mutant codons introduced into the Cas12a open reading frame. The underlined sequences indicate nucleotides encoding the amino acid linker sequence. The double underlined sequences indicate nucleotides encoding the nuclear localization sequence (NLS linker). The italicized sequences indicate nucleotides encoding the amino acid affinity tag sequence ((HIS)6).

[0182] SEQ ID NO.:490 (SEQ ID NO:490) Human optimized DNA WT Cas12a with NLS linker

[0183] [ka] [ka]

[0184] The underlined sequence indicates nucleotides encoding the amino acid linker sequence, the double underlined sequence indicates nucleotides encoding the nuclear localization sequence (NLS linker), and the italicized sequence indicates nucleotides encoding the amino acid affinity tag sequence ((HIS)6).

[0185] SEQ ID NO.: 491 (SEQ ID NO: 491) Human optimized DNA M537R F870L Cas12a with NLS linker

[0186] [ka] [ka] [ka]

[0187] The bold and underlined sequences indicate mutant codons introduced into the Cas12a open reading frame. The underlined sequences indicate nucleotides encoding the amino acid linker sequence. The double underlined sequences indicate nucleotides encoding the nuclear localization sequence (NLS linker). The italicized sequences indicate nucleotides encoding the amino acid affinity tag sequence ((HIS)6).

[0188] SEQ ID NO.:492 (SEQ ID NO:492) WT Cas12a with NLS linker

[0189] [ka]

[0190] The underlined sequences indicate amino acid sequences encoding the amino acid linker sequence, the double underlined sequences indicate amino acid sequences encoding the nuclear localization sequence (NLS linker), and the italicized sequences indicate amino acid sequences encoding the amino acid affinity tag sequence ((HIS)6).

[0191] SEQ ID NO.: 493 (SEQ ID NO: 493) M537R F870L Cas12a AA with NLS linker

[0192] [ka] [ka]

[0193] The bold and underlined sequences indicate mutant amino acids introduced into the Cas12a polypeptide variant. The underlined sequences indicate amino acid sequences encoding the amino acid linker sequence. The double underlined sequences indicate amino acid sequences encoding the nuclear localization sequence (NLS linker). The italicized sequences indicate amino acid sequences encoding the amino acid affinity tag sequence ((HIS)6). [Primary Technology Documents]

Non-licensed literature

[0194]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

[0195] (Incorporated by reference)

[0196] All patents, patent applications, patent application publications, and other publications cited herein are hereby incorporated by reference as if set forth in their entirety.

[0197] (Preferred embodiment)

[0198] The present invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the present invention has been presented by way of example and is not intended to be limited to the disclosed embodiments. Accordingly, those skilled in the art will recognize that the present invention is intended to cover all modifications and alternative arrangements that are within the spirit and scope of the present invention as set forth in the appended claims.

Claims

1. A CRISPR-associated protein comprising a variant of Cas12a, wherein the variant of Cas12a comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 472, 473, and 465.

2. 2. The CRISPR-associated protein of claim 1, wherein the variant of Cas12a consists of SEQ ID NO:

472.

3. 2. The CRISPR-associated protein of claim 1, wherein the variant of Cas12a consists of SEQ ID NO:

473.

4. 2. The CRISPR-associated protein of claim 1, wherein the variant of Cas12a consists of SEQ ID NO:

465.

5. A guide RNA, A CRISPR-associated protein comprising a variant of AsCasl2a, wherein the variant of Casl2a comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 472, 473, and 465; A CRISPR ribonucleoprotein complex comprising:

6. 6. The CRISPR ribonucleoprotein complex of claim 5, wherein the variant of Cas12a consists of SEQ ID NO:

472.

7. 6. The CRISPR ribonucleoprotein complex of claim 5, wherein the variant of Cas12a consists of SEQ ID NO:

473.

8. 6. The CRISPR ribonucleoprotein complex of claim 5, wherein the variant of Cas12a consists of SEQ ID NO:

465.

9. 1. A method for gene editing at a TTTN PAM site in a cell in vitro with a CRISPR ribonucleoprotein complex, comprising:

9. A method comprising contacting the CRISPR ribonucleoprotein complex of any one of claims 5 to 8 with a cell in vitro.

10. 10. The method of claim 9, wherein the TTTN PAM site consists of one selected from the group of TTTA, TTTT, and TTTC PAM sites.

11. 1. A kit comprising a guide RNA and a CRISPR-associated protein comprising a variant of Cas12a, wherein the variant of Cas12a is selected from the group consisting of SEQ ID NOs: 472, 473, and 465.

12. The kit of claim 11, wherein the variant of Cas12a is SEQ ID NO:

472.

13. 12. The kit of claim 11, wherein the variant of Cas12a is SEQ ID NO:

473.

14. 12. The kit of claim 11, wherein the variant of Cas12a is SEQ ID NO:

465.

15. 1. An isolated nucleic acid encoding a variant of Cas12a, wherein said variant of Cas12a is selected from the group consisting of SEQ ID NOs: 472, 473, and 465.

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