CRISPR / Cpf1 systems and methods

Abstract

"TTTN" PAM Site Protospacer domain target binding site 5' 3° Human Genomic DNA ....... HPRT1 38595 Site 3' TTTTAAATCCTTTCTCTTAACAAAAGAGGAAGGTCGTGGA 5' suguagauggaaagagaauuguuuucuccuuo 3' 13 1111 As Cpf1 crRNA C 1111 ucaucuuuaau 5 / This invention pertains to recombinant AsCPf1 nucleic acids and polypeptides for use in CRISPR / Cpfl endonuclease sysems and mammalian cell lines encloding recombinant AsCpflor LbCpf1 polypeptides. The invention includes recombinant ribonucleoprotein compleses and CRISPR / Cpfl endonuclease systems having a sjuitable AsCpfl or crRNA is selected from a length-truncated AsCpfl crRNA, a chemically modified AsCPf1 crRNA, or an AsCpfl CrRNA comprising both length truncations and chemical modifications. Methods of performing gene editing using these system and reagents are also provided. 20 24 20 40 29 13 J un 2 02 4 A B S T R A C T 2 0 2 4 2 0 4 0 2 9 1 3 J u n 2 0 2 4 " T T T N " P A M S i t e 5 ' A A A A T T T A G G A A A G A G A A T T G T T T T C T U C T T C C A G C A C C T i n H u m a n G e n o m i c D N A = = = = = = = = H P R T 1 3 8 5 9 5 S i t e u g u a g a u g g a a a g a g a a u u g u u u u c u c c u u c 3 ' 1 1 1 1 1 1 1

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of Application No. 2017364084, entered on 18 April 2019, is related to PCT / US2017 / 063161, filed 22 November 2017 and claims benefit of priority under 35 U.S.C. 119 to U.S. Provisional Patent Application Serial Number 62 / 425,307, filed 22 November 2016 and entitled "CPF1 CRISPR SYSTEMS AND METHODS," and U.S. Provisional Patent Application Serial Number 62 / 482,896, filed 7 April 2017 and entitled "HEK293 CELL LINE WITH STABLE EXPRESSION OF ACIDAMINOCOCCUS SP. BV3L6 CPF1," the contents of which are herein incorporated by reference in their entirety. SEQUENCE LISTING

[0002] Preceding applications contained a sequence listing which was originally submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, is named IDT01-010-US_ST25.txt. The instant application contains a sequence listing which has been submitted electronically as an XML document in the ST.26 format and is hereby incorporated by reference in its entirety. Said XML copy, created on 19 September 2023, is named IDT01-010-PCT and is 558517 bytes in size. FIELD OF THE INVENTION

[0003] This invention pertains to Cpfl-based CRISPR genes, polypeptides encoded by the same, mammalian cell lines that stably express Cpfl, crRNAs and the use of these materials in compositions of CRISPR-Cpfl systems and methods. BACKGROUND OF THE INVENTION [0003a] Any reference to any prior art in this specification is not, and should not be taken as an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge. la 2024204029   13 Jun 2024

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

[0005] CRISPR-Cas systems are native to bacteria and Archaea and provide adaptive immunity against viruses and plasmids. Three classes of CRISPR-Cas systems could potentially be adapted for research and therapeutic reagents. Type-II CRISPR systems have a desirable characteristic in utilizing a single CRISPR associated (Cas) nuclease [Text continues on page 2.] 2024204029   13 Jun 2024 (specifically Cas9) in a complex with the appropriate guide RNAs (gRNAs). In bacteria or Archaea, Cas9 guide RNAs comprise 2 separate RNA species. A target-specific CRISPR-activating RNA (crRNA) directs the Cas9 / gRNA complex to bind and target a specific DNA sequence. The crRNA has 2 functional domains, a 5’-domain that is target specific and a 3’-domain that directs binding of the crRNA to the transactivating crRNA (tracrRNA). The tracrRNA is a longer, universal RNA that binds the crRNA and mediates binding of the gRNA complex to Cas9. Binding of the tracrRNA induces an alteration of Cas9 structure, shifting from an inactive to an active conformation. The gRNA function can also be provided as an artificial single guide RNA (sgRNA), where the crRNA and tracrRNA are fused into a single species (see Jinek, M., et al., Science 337 p816-21, 2012). The sgRNA format permits transcription of a functional gRNA from a single transcription unit that can be provided by a double-stranded DNA (dsDNA) cassette containing a transcription promoter and the sgRNA sequence. In mammalian systems, these RNAs have been introduced by transfection of DNA cassettes containing RNA Pol III promoters (such as U6 or Hl) driving RNA transcription, viral vectors, and single-stranded RNA following in vitro transcription (see Xu, T., et al., Appl Environ Microbiol, 2014. 80(5): p. 1544-52). In bacterial systems, these RNAs are expressed as part of a primitive immune system, or can be artificially expressed from a plasmid that is introduced by transformation (see Fonfara, I., et al., Nature, 2016. 532(7600): p. 517-21).

[0006] In the CRISPR-Cas system, using the system present in Streptococcus pyogenes as an example (S.py. or Spy), native crRNAs are about 42 bases long and contain a 5’-region of about 20 bases in length that is complementary to a target sequence (also referred to as a protospacer sequence or protospacer domain of the crRNA) and a 3’ region typically of about 22 bases in length that is complementary to a region of the tracrRNA sequence and mediates binding of the crRNA to the tracrRNA. A crRNA:tracrRNA complex comprises a functional gRNA capable of directing Cas9 cleavage of a complementary target DNA. The native tracrRNAs are about 85-90 bases long and have a 5’-region containing the region complementary to the crRNA. The remaining 3’ region of the tracrRNA includes secondary structure motifs (herein referred to as the “tracrRNA 3’-tail”) that mediate binding of the crRNA:tracrRNA complex to Cas9. 2024204029   13 Jun 2024

[0007] Jinek et al. extensively investigated the physical domains of the crRNA and tracrRNA that are required for proper functioning of the CRISPR-Cas system (Science, 2012. 337(6096): p. 816-21). They devised a truncated crRNA:tracrRNA fragment that could still function in CRISPR-Cas wherein the crRNA was the wild type 42 nucleotides and the tracrRNA was truncated to 75 nucleotides. They also developed an embodiment wherein the crRNA and tracrRNA are attached with a linker loop, forming a single guide RNA (sgRNA), which varies between 99-123 nucleotides in different embodiments.

[0008] At least three groups have elucidated the crystal structure of Streptococcus pyogenes Cas9 (SpyCas9). In Jinek, M., et al., the structure did not show the nuclease in complex with either a guide RNA or target DNA. They carried out molecular modeling experiments to reveal predictive interactions between the protein in complex with RNA and DNA (Science, 2014. 343, p. 1215, DOI: 10.1126 / science / 1247997).

[0009] In Nishimasu, H., et al., the crystal structure of Spy Cas9 is shown in complex with sgRNA and its target DNA at 2.5 angstrom resolution (Cell, 2014. 156(5): p. 935-49, incorporated herein in its entirety). The crystal structure identified two lobes to the Cas9 enzyme: a recognition lobe (REC) and a nuclease lobe (NUC). The sgRNA:target DNA heteroduplex (negatively charged) sits in the positively charged groove between the two lobes. The REC lobe, which shows no structural similarity with known proteins and therefore likely a Cas9-specific functional domain, interacts with the portions of the crRNA and tracrRNA that are complementary to each other.

[0010] Another group, Briner et al. (Mol Cell, 2014. 56(2): p. 333-9, incorporated herein in its entirety), identified and characterized the six conserved modules within native crRNA:tracrRNA duplexes and sgRNA. Anders et al. (Nature, 2014, 513(7519) p. 569-73) elucidated the structural basis for DNA sequence recognition of protospacer associate motif (PAM) sequences by Cas9 in association with an sgRNA guide.

[0011] The CRISPR-Cas endonuclease system is utilized in genomic engineering as follows: the gRNA complex (either a crRNA:tracrRNA complex or an sgRNA) binds to Cas9, inducing a conformational change that activates Cas9 and opens the DNA binding cleft, the protospacer domain of the crRNA (or sgRNA) aligns with the complementary target DNA and Cas9 binds the PAM sequence, initiating unwinding of the target DNA followed by annealing of the protospacer domain to the target, after which cleavage of 2024204029   13 Jun 2024 the target DNA occurs. The Cas9 contains two domains, homologous to endonucleases HNH and RuvC respectively, wherein the HNH domain cleaves the DNA strand complementary to the crRNA and the RuvC-like domain cleaves the non-complementary strand. This results in a double-stranded break in the genomic DNA. When repaired by non-homologous end joining (NHEJ) the break is typically repaired in an imprecise fashion, resulting in the DNA sequence being shifted by 1 or more bases, leading to disruption of the natural DNA sequence and, in many cases, leading to a frameshift mutation if the event occurs in a coding exon of a protein-encoding gene. The break may also be repaired by homology directed recombination (HDR), which permits insertion of new genetic material based upon exogenous DNA introduced into the cell with the Cas9 / gRNA complex, which is introduced into the cut site created by Cas9 cleavage.

[0012] While SpyCas9 is the protein being most widely used, it does hold some barriers to its effectiveness. SpyCas9 recognizes targeted sequences in the genome that are immediately followed by a GG dinucleotide sequence, and this system is therefore limited to GC-rich regions of the genome. AT-rich species or genomic regions are therefore often not targetable with the SpyCas9 system. Furthermore, the fact that the Cas9 system includes a gRNA having both a crRNA and a tracrRNA moiety that comprise over 100 bases means that more RNA must be optimized and synthesized for sequence-specific targeting. As such, a shorter simpler gRNA would be desirable.

[0013] A second class 2 CRISPR system, assigned to type V, has been identified. This type V CRISPR-associated system contains Cpfl, which is a -1300 amino acid protein - slightly smaller than Cas9 from S. pyogenes. The PAM recognition sequence of Cpfl from Acidaminococcus sp. BV3L6 or Lachnospiraceae bacterium ND2006 is TTTN, in contrast to the NGG PAM recognition domain of S.pyogenes Cas9 (FIG. 1). Having the ability to target AT- rich areas of the genome will be greatly beneficial to manipulate and study gene targets in regions that are lacking GG dinucleotide motifs. The Cpfl system is also remarkably simple in that it does not utilize a separate tracrRNA, and only requires a single short crRNA of 40-45 base length that both specifies target DNA sequence and directs binding of the RNA to the Cpfl nuclease.

[0014] In contrast to Cas9 which produces blunt-ended cleavage products, Cpfl facilitates double stranded breaks with 4-5 nucleotide overhangs. The advantage of this 2024204029   13 Jun 2024 is that it may ensure proper orientation as well as providing microhomology during non-homologous end joining (NHEJ). This could also be advantageous in non-dividing cell types that tend to be resistant to homology-directed repair (HDR). Furthermore, when Cpfl cleaves, it does so further away from PAM than Cas9, which is also further away from the target site. As a result, the protospacer, and especially the seed sequence of the protospacer, are less likely to be edited, thereby leaving open the potential for a second round of cleavage if the desired repair event doesn’t happen the first time.

[0015] The Cpfl protein forms a complex with a single stranded RNA oligonucleotide to mediate targeted DNA cleavage. The single strand guide RNA oligonucleotide consists of a constant region of 20 nt and a target region of 21-24 nt for an overall length of 41-44 nt. There are many known orthologs of Cpfl from a variety of different bacterial and Archaea sources that differ with respect to activity and target preference and may be candidates for use in genome editing applications. For the purposes of this invention, we primarily studied, as representative examples, the Cpfl nucleases from A.s. (Acidaminococcus sp. BV3L6) Cpfl and L.b. (Lachnospiraceae bacterium ND2006), both of which have already been shown to be active in mammalian cells as a tool for genome editing. Of note, the PAM recognition sequence is TTTN. The structure of the Cpfl crRNA and relationship of RNA binding to the PAM site in genomic DNA is shown in FIG. 1.

[0016] Since the discovery of Cpfl as another CRISPR pathway with potential utility for genome editing in mammalian cells, several publications have confirmed that the system works in mammals, can be used for embryo engineering, and the crystal structure and mechanism of PAM site recognition have been described. This system has also shown utility for screening purposes in genetically-tractable bacterial species such as E. coli. The system therefore has proven utility and developing optimized reagents to perform genome editing using Cpfl would be beneficial.

[0017] Previous work done on the SpyCas9 crRNA and tracrRNA demonstrated that significant shortening of the naturally occurring crRNA and tracrRNA species could be done for RNAs made by chemical synthesis and that such shortened RNAs were 1) higher quality, 2) less costly to manufacture, and 3) showed improved performance in mammalian genome editing compared with the wild-type (WT) RNAs. See Collingwood, 2024204029   13 Jun 2024 M.A., Jacobi, A.M, Rettig, G.R., Schubert, M.S., andBehlke, M.A., “CRISPR-BASED COMPOSITIONS AND METHOD OF USE,” U.S. Patent Application Serial No. 14 / 975,709, filed December 18, 2015, published now as U.S. Patent Application Publication No. US2016 / 0177304A1 on June 23, 2016 and issued as U.S. Patent. No. 9840702 on December 12, 2017.

[0018] Prior work demonstrated that reducing the length of the FnCpfl crRNA from 22 to 18 base length with deletions from the 3’-end supported cleavage of target DNA but that lengths of 17 or shorter showed reduced activity. Deletions or mutations that disrupted base-pairing in the universal loop domain disrupted activity. See Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., Essletzbichler, P., Volz, S.E., Joung, J., van der Oost, J., Regev, A., Koonin, E.V., and Zhang, F. (2015) Cpfl is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:113. The FnCpfl nuclease, however, does not work in mammalian cells to perform genome editing. It is unknown if the same length rules apply to the AsCpfl crRNA as were observed for the FnCpfl crRNA. We establish herein the shortest version of AsCpfl crRNAs having full activity in mammalian genome editing applications. We also establish chemical modification patterns that maintain or improve functioning of synthetic Cpfl crRNAs when used in mammalian or prokaryotic cells. BRIEF SUMMARY OF THE INVENTION

[0019] This invention pertains to Cpfl-based CRISPR genes, polypeptides encoded by the same, mammalian cell lines that stably express Cpfl, and chemically synthesized Cpfl crRNAs and their use in compositions of CRISPR-Cpfl systems and methods. Examples are shown employing the Cpfl systems from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006, however this is not intended to limit scope, which extends to Cpfl homologs or orthologs isolated from other species.

[0020] In a first aspect, an isolated nucleic acid is provided. The isolated nucleic acid encodes an As Cpfl polypeptide codon optimized for expression in H. sapiens as seen in SEQ ID NO:8, SEQ ID NO: 15 and SEQ ID NO:22 which includes the use of nuclear localization signals as well as an epitope tag. The isolated nucleic acid also encodes as As Cpfl polypeptide codon optimized for expression in E. coli which comprises SEQ ID NO:5 and may be fused or linked to a nuclear localization signal, multiple nuclear 2024204029   13 Jun 2024 localization signals, or sequences encoding an epitope tag enabling detection by antibodies or other methods, and / or an affinity tag that enables simple purification of recombinants proteins expressed from the nucleic acid, such as a His-Tag as seen in SEQ ID NO: 12 and SEQ ID NO: 19.

[0021] In a second aspect, an isolated polypeptide encoding a wild-type As Cpfl protein is provided. In a first respect, the isolated polypeptide comprises SEQ ID NO:2. The protein may be fused or linked to a nuclear localization signal, multiple nuclear localization signals, or sequences encoding an epitope tag enabling detection by antibodies or other methods, and / or an affinity tag that enables simple purification of recombinants proteins expressed from the nucleic acid, such as a His-Tag as seen in SEQ ID NO: 12, SEQ ID NO: 16 and SEQ ID NO: 19.

[0022] In a third aspect, an isolated nucleic acid is provided. The isolated nucleic acid encodes an Lb Cpfl polypeptide codon optimized for expression in H. sapiens as seen in SEQ ID NO:9 and SEQ ID NO: 17, which includes the use of nuclear localization signals as well as an epitope tag. The isolated nucleic acid also encodes as Lb Cpfl polypeptide codon optimized for expression inE. coli which comprises SEQ ID NO:6 and may be fused or linked to a nuclear localization signal, multiple nuclear localization signals, or sequences encoding an epitope tag enabling detection by antibodies or other methods, and / or an affinity tag that enables simple purification of recombinants proteins expressed from the nucleic acid, such as a His-Tag as seen in SEQ ID NO: 13.

[0023] In a fourth aspect, an isolated polypeptide encoding a wild-type Lb Cpfl protein is provided. In a first respect, the isolated polypeptide comprises SEQ ID NO:7 and SEQ ID NO: 10. The protein may be fused or linked to a nuclear localization signal, multiple nuclear localization signals, or sequences encoding an epitope tag enabling detection by antibodies or other methods, and / or an affinity tag that enables simple purification of recombinants proteins expressed from the nucleic acid, such as a His-Tag as seen in SEQ ID NO: 14.

[0024] In a fifth aspect, an isolated expression vector encoding SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 is provided. The isolated expression vectors include a transcriptional initiator element, such as a promoter and enhancer, operably-linked to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 to 2024204029   13 Jun 2024 permit expression of the polypeptide encoded by SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO:16.

[0025] In a sixth aspect, a host cell including an isolated expression vector encoding SEQIDNO:11, SEQIDNO:13, SEQIDNO:15 and SEQ ID NO: 17 is provided. The isolated expression vector encoding SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17 is operably linked to a suitable promoter and other genetic elements (as necessary) to permit expression of a polypeptide comprising SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.

[0026] In a seventh aspect, an isolated CRISPR / Cpfl endonuclease system is provided. The system includes an AsCpfl polypeptide and a suitable AsCpfl crRNA.

[0027] In an eighth aspect, an isolated CRISPR / Cpfl endonuclease system is provided. The system includes a human cell line expressing a AsCpfl polypeptide and a suitable AsCpfl crRNA.

[0028] In a ninth aspect, an isolated AsCpfl crRNA is provided. The isolated AsCpfl crRNA is active in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated protein endonuclease system. Different variants of the crRNA are provided including species optimized for performance in mammalian cells and species optimized for performance in bacteria.

[0029] In a tenth aspect, a method of performing gene editing is provided. The method includes the step of contacting a candidate editing target site locus with an active CRISPR / Cpfl endonuclease system having a wild-type AsCpfl polypeptide and a suitable AsCpfl crRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is a graphical representation of Cpfl PAM recognition sites and alignment of guide crRNA to target DNA. Genomic DNA sequence of the human HPRTI gene is shown at site ‘38595’. The “TTTN” PAM site that identifies As Cpfl sites is highlighted and the sequence of the guide-binding site is underlined. DNA is shown in uppercase and RNA is shown in lowercase. In the Cpfl crRNA, the protospacer target-specific domain is underlined and comprises the 3’-domain. The universal hairpin RNA sequence that mediates binding to Cpfl protein comprises the 5’-domain. 2024204029   13 Jun 2024

[0031] FIG. 2 depicts the map of a plasmid vector designed to express recombinant, synthetic, codon-optimized AsCpfl.

[0032] FIG. 3 depicts a schematic showing the final plasmid construct used to generate AsCpfl stable cell lines.

[0033] FIG. 4 depicts an exemplary Western blot showing expression of V5-tagged proteins. Cell extract from a monoclonal HEK cell line that stably expresses Cas9 with a V5 tag was run in Lane 2. Cell extract from the new polyclonal HEK cell culture that expresses a V5-tagged AsCpfl was run in Lane 3. Beta-actin is indicated and represents a mass loading control. Lane 1 was run with mass standard markers.

[0034] FIG. 5 depicts exemplary expression profiles of AsCpfl mRNA normalized to internal control HPRT1 mRNA in 10 clonal transgenic cell lines. RT-qPCR assay locations vary in position along the AsCpfl mRNA. Negative control non-transgenic HEKI cells are shown on the far right.

[0035] FIG. 6 depicts exemplary Western blot showing relative expression levels of AsCpfl protein in 10 monoclonal transgenic cell lines based on detection of the V5 epitope. Beta-actin loading control is seen below the AsCpfl bands.

[0036] FIG. 7 depicts a modification tolerance map of AsCpfl crRNAs at 2 sequence target sites, HPRT1-38351 (panel (z)) and HPRT1-38595 (panel (zz)), wherein the sequence of the universal 5’-loop domain is shown (5’-3’ orientation) for both the 24-nt protospacer domains (panels (i.a) and (H.a)) and the 21-nt protospacer domains (panels (i.h) and (ii-b}). The sequence of the variable 3’-target specific protospacer domain is indicated as “N” bases, as this sequence varies for every target. Positions that did not suffer loss of activity when modified as a 2’0Me RNA residue in the single base walk are indicated in upper case whereas positions that showed loss of activity with modification are indicated in lower case. Above the lower case residues an arrow is shown that indicates the relative magnitude of the loss of activity, wherein a large arrow represents a large loss of activity, a mid-sized arrow represents a medium loss of activity, and a small arrow represents a minor loss of activity when the respective RNA residues are changed to 2’0Me RNA. 2024204029   13 Jun 2024

[0037] FIG. 8 depicts exemplary modified variants AsCpfl crRNAs that are active in genome editing applications in mammalian cells at multiple target sites and therefore are not site-specific. The sequence of the universal 5’-loop domain is shown (5’-3’ orientation) and indicated with underline. The sequence of the variable 3’-target specific protospacer domain is indicated as “N” bases, as this sequence varies for every target. 2’0Me RNA modifications are indicated in uppercase and RNA residues are indicated in lowercase. “X” indicates a terminal non-base modifier, such as a C3 spacer (propanediol) or ZEN (napthyl-azo) group. indicates a phosphorothioate (PS) internucleotide linkage.

[0038] FIG. 9 depicts exemplary results that compare the target editing activity of LbCpfl with that of AsCpfl and SpyCas9 for 12 regions of the HPRT gene with low GC content via T7EI mismatch endonuclease assay. In this study, all enzymes and crRNA were delivered as RNP complexes (5 pM), into HEK293 cells by nucleofection using the Amaxa system from Lonza, and DNA was extracted after 48 hr. Percent editing was determined by T7E1 mismatch endonuclease assay. Error bars represent standard errors of the means. Of note, the crRNA’s for LbCpfl were tested at the native 23mer nucleotide length as well as the previously optimized AsCpfl length of 21 bases. DETAILED DESCRIPTION OF THE INVENTION

[0039] The methods and compositions of the invention described herein provide wild-type AsCpfl nucleic acids and polypeptides for use in a CRISPR / Cpfl system. The present invention describes an HEK293 cell line that has stable, low levels of expression of AsCpfl in HEK293 and can be used as a platform for investigation and optimization of the nucleic acid components of the system. AsCpfl provides a useful complement to SpyCas9 by expanding the range of PAM sequences that can be targeted from GC-rich areas (Cas9) to AT-rich areas of the genome (Cpfl), thereby expanding the range of sequences that can be modified using CRISPR genome engineering methods. In addition to having a T-rich PAM site, another advantage of the AsCpfl system compared with Cas9 is the use of a single, short RNA molecule. However, unlike Cas9 that shows activity at most sites in the human genome, AsCpfl shows little to no activity at half of TTTN PAM sites. Thus, exploiting the full potential of the AsCpfl CRISPR system will be enhanced by the availability of suitable predictive software that enriches for high 2024204029   13 Jun 2024 activity sites based on sequence context. The use of a stable constitutive Cpfl-expressing cell line makes the development of an algorithm easier to develop with reduced effort and cost as compared to using alternative methods, such as electroporation of ribonucleoprotein protein (RNP) complexes. HEK293 cells are an immortalized cell line that are easily cultured, passaged and cryogenically preserved. We established clonal cell lines that constitutively express SpyCas9 and AsCpfl as suitable test vehicles for algorithm development or rapid testing / optimization of the chemical structure of guide RNAs. The present invention describes length and chemical modification of length-optimized variants of the AsCpfl and LbCpfl crRNAs that improve function in genome editing. AsCpfl-encoded genes, polypeptides, expression vectors and host cells

[0040] The term “wild-type AsCpfl protein” (“WT-AsCpfl” or “WT-AsCpfl protein”) encompasses a protein having the identical amino acid sequence of the naturally-occurring Acidaminococcus sp. BV3L6 Cpfl (e.g., SEQ ID NO:2) and that has biochemical and biological activity when combined with a suitable crRNA to form an active CRISPR / Cpfl endonuclease system.

[0041] The term “wild-type LbCpfl protein” (“WT-LbCpfl” or “WT-LbCpfl protein”) encompasses a protein having the identical amino acid sequence of the naturally-occurring Lachnospiraceae bacterium ND2006 Cpfl (e.g., SEQ ID NO:4) and that has biochemical and biological activity when combined with a suitable crRNA to form an active CRISPR / Cpfl endonuclease system.

[0042] The term “wild-type CRISPR / Cpfl endonuclease system” refers to a CRISPR / Cpfl endonuclease system that includes wild-type AsCpfl protein and a suitable AsCpfl crRNA as a guide RNA.

[0043] The term “polypeptide” refers to any linear or branched peptide comprising more than one amino acid. Polypeptide includes protein or fragment thereof or fusion thereof, provided such protein, fragment or fusion retains a useful biochemical or biological activity.

[0044] Fusion proteins typically include extra amino acid information that is not native to the protein to which the extra amino acid information is covalently attached. 2024204029   13 Jun 2024 Such extra amino acid information may include tags that enable purification or identification of the fusion protein. Such extra amino acid information may include peptides that enable the fusion proteins to be transported into cells and / or transported to specific locations within cells. Examples of tags for these purposes include the following: AviTag, which is a peptide allowing biotinylation by the enzyme BirA so the protein can be isolated by streptavidin (GLNDIFEAQKIEWHE); Calmodulin-tag, which is a peptide bound by the protein calmodulin (KRRWKKNFIAVSAANRFKKISSSGAL); polyglutamate tag, which is a peptide binding efficiently to anion-exchange resin such as Mono-Q (EEEEEE); E-tag, which is a peptide recognized by an antibody (GAPVPYPDPLEPR); FLAG-tag, which is a peptide recognized by an antibody (DYKDDDDK); HA-tag, which is a peptide from hemagglutinin recognized by an antibody (YPYDVPDYA); His-tag, which is typically 5-10 histidines and can direct binding to a nickel or cobalt chelate (HHHHHH); Myc-tag, which is a peptide derived from c-myc recognized by an antibody (EQKLISEEDL); NE-tag, which is a novel 18-amino-acid synthetic peptide (TKENPRSNQEESYDDNES) recognized by a monoclonal IgGl antibody, which is useful in a wide spectrum of applications including Western blotting, ELISA, flow cytometry, immunocytochemistry, immunoprecipitation, and affinity purification of recombinant proteins; S-tag, which is a peptide derived from Ribonuclease A (KETAAAKFERQHMDS); SBP-tag, which is a peptide which binds to streptavidin; (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP); Softag 1, which is intended for mammalian expression (SLAELLNAGLGGS); Softag 3, which is intended for prokaryotic expression (TQDPSRVG); Strep-tag, which is a peptide which binds to streptavidin or the modified streptavidin called streptactin (Strep-tag II: WSHPQFEK); TC tag, which is a tetracysteine tag that is recognized by FlAsH and ReAsH biarsenical compounds (CCPGCC)V5 tag, which is a peptide recognized by an antibody (GKPIPNPLLGLDST); VSV-tag, a peptide recognized by an antibody (YTDIEMNRLGK); Xpress tag (DLYDDDDK); Isopeptag, which is a peptide which binds covalently topilin-C protein (TDKDMTITFTNKKDAE); SpyTag, which is a peptide which binds covalently to SpyCatcher protein (AHIVMVDAYKPTK); SnoopTag, a peptide which binds covalently to SnoopCatcher protein (KLGDIEFIKVNK); BCCP (Biotin Carboxyl Carrier Protein), which is a protein domain biotinylated by BirA to enable recognition by streptavidin; Glutathione-S-transferase- 2024204029   13 Jun 2024 tag, which is a protein that binds to immobilized glutathione; Green fluorescent proteintag, which is a protein which is spontaneously fluorescent and can be bound by antibodies; HaloTag, which is a mutated bacterial haloalkane dehalogenase that covalently attaches to a reactive haloalkane substrate to allow attachment to a wide variety of substrates; Maltose binding protein-tag, a protein which binds to amylose agarose; Nus-tag; Thioredoxin-tag; and Fc-tag, derived from immunoglobulin Fc domain, which allows dimerization and solubilization and can be used for purification on Protein-A Sepharose.

[0045] Nuclear localization signals (NLS), such as those obtained from SV40, allow for proteins to be transported to the nucleus immediately upon entering the cell. Given that the native AsCpfl protein is bacterial in origin and therefore does not naturally comprise a NLS motif, addition of one or more NLS motifs to the recombinant AsCpfl protein is expected to show improved genome editing activity when used in eukaryotic cells where the target genomic DNA substrate resides in the nucleus. Functional testing in HEK293 cells revealed that using a bipartite NLS (nucleoplasmin) increased editing in comparison to the current commercial design (3 SV40 NLS) and the use of single or dual OpT NLS that showed promise in the Cpfl protein. Additional combinations of NLS elements including the bipartite are envisioned. Of note, the nucleoplasmin functions best in mammalian cells while the SV40 NLS appears to function in almost any nucleated cell. The bipartite SV40 NLS is functional in both Cas9 and Cpfl. Having two different NLS domains may expand effectiveness across a broad spectrum of species.

[0046] One skilled in the art would appreciate these various fusion tag technologies, as well as how to make and use fusion proteins that include them.

[0047] The term “isolated nucleic acid” include DNA, RNA, cDNA, and vectors encoding the same, where the DNA, RNA, cDNA and vectors are free of other biological materials from which they may be derived or associated, such as cellular components. Typically, an isolated nucleic acid will be purified from other biological materials from which they may be derived or associated, such as cellular components.

[0048] The term “isolated wild-type AsCpfl nucleic acid” is an isolated nucleic acid that encodes a wild-type AsCpfl protein. Examples of an isolated wild-type AsCpfl nucleic acid include SEQ ID NO:1. 2024204029   13 Jun 2024

[0049] The term “isolated wild-type LbCpfl nucleic acid” is an isolated nucleic acid that encodes a wild-type LbCpfl protein. Examples of an isolated wild-type LbCpfl nucleic acid include SEQ ID NO:3.

[0050] In a first aspect, an isolated nucleic acid is provided. The isolated nucleic acid encodes an As Cpfl polypeptide codon optimized for expression in H. sapiens. In a first respect, the isolated nucleic acid comprises SEQ ID NO:8, SEQ ID NO: 15 and SEQ ID NO:22 which includes the use of nuclear localization signals as well as an epitope tag. The isolated nucleic acid also encodes as As Cpfl polypeptide codon optimized for expression in E. coli which comprises SEQ ID NO:5 and may be fused or linked to a nuclear localization signal, multiple nuclear localization signals, or sequences encoding an epitope tag enabling detection by antibodies or other methods, and / or an affinity tag that enables simple purification of recombinants proteins expressed from the nucleic acid, such as a His-Tag as seen in SEQ ID NO: 12 and SEQ ID NO: 19.

[0051] In a second aspect, an isolated polypeptide encoding a wild-type As Cpfl protein is provided. In a first respect, the isolated polypeptide comprises SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO: 16 or SEQ ID NO: 19.

[0052] In a third aspect, an isolated expression vector encoding SEQ ID NO: 15 is provided. The isolated expression vector includes transcriptional initiator elements, such as a promoter and enhancer, operably-linked to SEQ ID NO: 15 to permit expression of the polypeptide encoded by SEQ ID NO: 16. The isolated expression vector may additionally include transcriptional termination elements, posttranscriptional processing elements (for example, splicing donor and acceptor sequences and / or polyadenylation signaling sequences), mRNA stability elements and mRNA translational enhancer elements. Such genetic elements are understood and used by those having ordinary skill in the art.

[0053] In a fourth aspect, a host cell comprising an isolated expression vector encoding SEQ ID NO: 15 is provided. The isolated expression vector encoding SEQ ID NO: 15 is operably linked to a suitable promoter and other genetic elements (as necessary) to permit expression of a polypeptide comprising SEQ ID NO: 16. In a first respect, the host cell includes a human cell. In a second respect, the human cell comprises an immortalized cell line. In a third respect, the immortalized cell line is a 2024204029   13 Jun 2024 HEK293 cell line. As a further elaboration of this third respect, the immortalized cell line comprises an isolated AsCpfl crRNA capable of forming a ribonucleoprotein complex with the polypeptide comprising SEQ ID NO:2 to form a wild-type CRISPR / Cpfl endonuclease. Length- and chemical structure-optimized AsCpfl crRNAs

[0054] The term “length-modified,” as that term modifies RNA, refers to a shortened or truncated form of a reference RNA lacking nucleotide sequences or an elongated form of a reference RNA including additional nucleotide sequences.

[0055] The term “chemically-modified,” as that term modifies RNA, refers to a form of a reference RNA containing a chemically-modified nucleotide or a non-nucleotide chemical group covalently linked to the RNA. Chemically-modified RNA, as described herein, generally refers to synthetic RNA prepared using oligonucleotide synthesis procedures wherein modified nucleotides are incorporated during synthesis of an RNA oligonucleotide. However, chemically-modified RNA also includes synthetic RNA oligonucleotides modified with suitable modifying agents post-synthesis.

[0056] A competent CRISPR / Cpfl endonuclease system includes a ribonucleoprotein (RNP) complex formed with isolated AsCpfl protein and a guide RNA consisting of an isolated AsCpfl crRNA. In some embodiments, an isolated length-modified and / or chemically-modified form of AsCpfl crRNA is combined with purified AsCpfl protein, an isolated mRNA encoding AsCpfl protein or a gene encoding AsCpfl protein in an expression vector. In certain assays, an isolated length-modified and / or chemically-modified form of AsCpfl crRNA can be introduced into cell lines that stably express AsCpfl protein from an endogenous expression cassette encoding the AsCpfl gene.

[0057] It is desirable for synthesis of synthetic RNAs that sequences are shortened of unnecessary bases but not so shortened that loss of function results. The 5’-constant regions that mediates binding of the crRNA to the Cpfl nuclease shows loss of activity if truncated below 20 residues. The 3’-variable domain that comprises the protospacer guide region which confers target sequence specificity to the crRNA naturally occurs as long as 25 bases. This domain can be shortened to around 20-21 bases with no loss of 2024204029   13 Jun 2024 functional activity. The optimized length of the Cpfl crRNA is therefore 40-41 bases, comprising a 20 base 5’-constant domain and a 20-21 base 3’-variable domain.

[0058] The present invention provides suitable guide RNAs for triggering DNA nuclease activity of the AsCpfl nuclease. These optimized reagents, both in terms of length-modified and / or chemically-modified forms of crRNA’s, provide for improved genome editing in any application with AsCpfl. The applications of CRISPR-based tools include, but are not limited to: plant gene editing, yeast gene editing, rapid generation of knockout / knockin animal lines, generating an animal model of disease state, correcting a disease state, inserting reporter genes, and whole genome functional screening. The “tool-kit” could be further expanded by including nickase versions and a dead mutant of AsCpfl as a fusion protein with transcriptional activators CRISPRa) and repressors (CRISPRi).

[0059] RNA-guided DNA cleavage by AsCpfl is primarily useful for its ability to target AT-rich gene regions (as compared with the GC-rich targeting by SpyCas9). The newly-discovered AsCpfl crRNA truncation and modification variants will be suitable to promote AsCpfl-mediated staggered cutting and beneficial in gene silencing, homology directed repair or exon excision. The present invention defines the shortest AsCpfl guide RNA that has full potency to direct gene editing by the CRISPR / Cpfl endonuclease. This is useful for manufacturing to synthesize the shortest compound that fully functions, leading to higher quality, lower cost, while maximizing functionality.

[0060] Unlike S.py. Cas9 which requires a complex of 2 RNAs to recognize and cleave a target DNA sequence (comprising a hybridized crRNA:tracrRNA pair) or a long synthetic single-guide sgRNA, the Cpfl nuclease only requires a short, single crRNA species to direct target recognition. This RNA comprises 2 domains, a 5’-domain of 20 RNA residues that is universal and mediates binding of the RNA species to the Cpfl protein and a 3’domain of 21-24 RNA residues which is target specific and mediates binding of the RNP complex to a precise DNA sequence. A functional nuclease complex comprises a single crRNA (41-44 bases in length) and isolated Cpfl protein, which combine in a 1:1 molar ratio to form an active complex. The guide crRNA species can be expressed in mammalian cells from expression plasmids or viral vectors. The crRNA can also be made as an in vitro transcript (IVT) and isolated as a pure enzymatic RNA 2024204029   13 Jun 2024 species. More preferably, the crRNAs can be manufactured as a synthetic chemical RNA oligonucleotide. Chemical manufacturing enables use of modified residues, which have many advantages as will be outlined below.

[0061] Synthetic nucleic acids are attacked by cellular nucleases and rapidly degrade in mammalian cells or in serum. Chemical modification can confer relative nuclease resistance to the synthetic nucleic acids and prolong their half-lives, thereby dramatically improving functional performance and potency. As a further complication, synthetic nucleic acids are often recognized by the antiviral surveillance machinery in mammalian cells that are part of the innate immune system and lead to interferon response pathway activation, which can lead to cell death. Chemical modification can reduce or eliminate unwanted immune responses to synthetic RNAs. It is therefore useful to establish methods to chemically modify synthetic RNA oligonucleotides intended for use in live cells. Nucleic acid species that have specific interactions with protein factors, however, cannot be blindly modified as chemical modification will change tertiary structure of the nucleic acid and can block critical contact points between the nucleic acid and aminoacid residues. For example, the 2’-O-methyl RNA modification (2’0Me) will block the 2’-oxygen of RNA from interaction with amino-acid residues that in turn can disrupt functional interaction between a modified RNA and a protein. Likewise, a phosphorothioate modification can disrupt protein binding along the phosphate backbone of a nucleic acid through substitution of a non-bridging oxygen at the phosphate.

[0062] The 2’0Me modification is particularly useful in this setting as it has previously been shown to increase nuclease stability of antisense oligonucleotides (ASOs) and siRNAs and at the same kind can also reduce the risk that a chemically-synthesized RNA will trigger an innate immune response when introduced into mammalian cells. Specific modification patterns have been established that permit incorporation of this modified residue into an ASO or siRNA and retain function. Likewise, we have recently developed chemical modification patterns that improved the stability of the crRNA and tracrRNA that serve as guide RNA in the SpyCas9 system. Use of 2’OMe-modified residues in a CRISPR guide RNA improves RNA stability to nucleases and boosts the overall efficiency of editing in nuclease-rich environments while at the same time reduces cell death and toxicity associated with immunogenic triggers (such as is seen with long, unmodified RNAs). 2024204029   13 Jun 2024

[0063] The present invention relates to defining chemical modification patterns for the AsCpfl crRNA that retain function in forming an active RNP complex capable of use in genome editing in mammalian cells. Modification ‘walks’ were performed where a single 2’OMe residue was place sequentially at every position with the Cpfl crRNA. Sites that reduced or killed function of the RNP complex in genome editing were identified. Chemical modification patterns were defined that were compatible with high efficiency genome editing. The utility of 2’-fluoro (2’F) and locked nucleic acid (LNA) modifications at ‘modification competent’ position in the crRNA were also demonstrated. The use of phosphorothioate internucleotide linkages to modify select sites to reduce nuclease susceptibility was shown, as well as successful use of non-base modifiers as end blocks to reduce exonuclease attack on the synthetic RNAs. Taken together, these studies provide a ‘map’ of sites in the Cpfl crRNA amenable to chemical modification along with a suite of modification chemistries demonstrated to function in the intended application in mammalian cells.

[0064] Specific examples of modification patterns are shown in the examples below. The 20-base 5’-constant domain could be heavily modified and retain function. In particular, using a 20-base 5’-constant region and counting from the 5’-end, RNA residues at position 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, and 19 can all be substituted with 2’0Me RNA residues with no loss of activity. Such substitutions can be made single, multiply, or all 14 residues modified, such that 14 / 20 residues have been changed in this domain from RNA to 2’0Me RNA. Maximum modification patterns that are tolerated in the 21-base 3’-variable domain vary with sequence of the domain. Within this domain, residues 21, 22, 23, 28, 29, 30, 32, 34, 35, 39, 40, and 41 (counting from the first base of the 5’-constant region) can be substituted with 2’0Me residues with no loss of activity.

[0065] Only select positions within the 21-24-base 3’-target specific domain can be modified without compromising activity. Based on the crystal structure of Cpfl, there are many protein contact points within the constant region as well as the target region. For constant region modification, there is no obvious correlation that emerges when comparing the Cpfl crystal structure contact points with the identified functional positions that can be modified - meaning that a good modification pattern cannot be predicted from the crystal structure. Likewise, empirical testing was needed to 2024204029   13 Jun 2024 determine target region modification patterns. Based on the early 2’OMe modification testing, selected areas within the Cpfl crRNA were modified using 2’OMe as an attempt to narrow down an area that will tolerate modification. The position of single residues within the Cpfl crRNA that are sensitive to 2’OMe modification are shown in FIG. 7. Higher-level modification patterns that are potent triggers of Cpfl-mediated genome editing are shown in FIG. 8. 2’F modifications can be positioned at any residue that is tolerant to 2’OMe modification. Further, the 3’-variable domain is more tolerate of large blocks of 2’F modification than large blocks of 2’OMe modification. Hence a highly modified version of the Cpfl crRNA comprises 2’OMe modification in the 3’-domain and 2’F modification in the 5’-domain. For medium or light modification patterns, either 2’OMe or 2’F (or both) modifications can be used in both domains. Also, LNA residues can be incorporated into the crRNA without compromising function, as defined in the examples below.

[0066] As an alternative to extensive use of 2’OMe or other modified sugar approaches, blocking exonuclease attack with non-base modifiers at the 3’-end and 5’-end are compatible with crRNA function and improve function in cells. Small C3 spacer (propanediol) or large ZEN groups work equally well for this approach. Further, phosphorothioate intemucleotide linkages can be placed at select sites, such as between the terminal 2-3 bases on each end of the crRNA, but complete PS modification of the crRNA or complete modification of either the loop domain or the protospacer domain show reduced activity.

[0067] Guide RNAs are required in RNA-directed dsDNA cleavage by AsCpfl, which initiate the subsequent repair events that are involved in most CRISPR applications in mammalian cells. The use of modified synthetic AsCpfl crRNAs as guides for AsCpfl genome editing is provided. The utility of 2’OMe-modified AsCpfl crRNAs, 2’F-modified AsCpfl crRNAs, LNA modified AsCpfl crRNAs, and end-blocked AsCpfl crRNAs for CRISPR / Cpfl applications in mammalian cells is demonstrated. Those with skill in the art will recognize and appreciate additional chemical modifications are possible based upon this disclosure. It is expected that many of these base modifying groups will likewise function according to the patterns taught in the present invention. Heretofore, all crRNAs used with Cpfl for genome editing were 2024204029   13 Jun 2024 unmodified RNA. In the present invention, functional modification patterns that improve properties of the AsCpfl crRNA and lower risk of toxicity are provided.

[0068] AsCpfl crRNAs can be made in cells from RNA transcription vectors, as in vitro transcripts (IVTs), or by chemical synthesis. Synthetic RNA oligonucleotides offer a distinct advantage because they alone allow for precise insertion of modified bases at specific sites in the molecule. The present invention provides a map of positions amenable to chemical modification that can be used to improve AsCpfl crRNA performance in cells. For some applications, “minimal modification” approaches will be sufficient. In higher nuclease environments or for use in cells with particularly high innate immune reactivity, “high modification” approaches may work better. The present invention provides methods for low, medium, or high modification needs.

[0069] The applications of AsCpfl-based tools are many and varied. They include, but are not limited to: bacterial gene editing, plant gene editing, yeast gene editing, mammalian gene editing, editing of cells in the organs of live animals, editing of embryos, rapid generation of knockout / knock-in animal lines, generating an animal model of disease state, correcting a disease state, inserting a reporter gene, and whole genome functional screening.

[0070] In a fifth aspect, an isolated CRISPR / Cpfl endonuclease system is provided. The system includes an AsCpfl polypeptide and a suitable AsCpfl crRNA. In a first respect, the AsCpfl polypeptide comprises SEQ ID NO:2. In a second respect, the suitable AsCpfl crRNA is selected from a length-truncated AsCpfl crRNA or a chemically-modified AsCpfl crRNA, or an AsCpfl crRNA containing both length truncations and chemical modifications.

[0071] In a sixth aspect, an isolated CRISPR / Cpfl endonuclease system is provided. The system includes a human cell line expressing an AsCpfl polypeptide and a suitable AsCpfl crRNA. In a first respect, the AsCpfl polypeptide comprises at least one member selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 12, SEQ ID NO: 16 and SEQ ID NO: 19. In a second respect, the suitable AsCpfl crRNA is selected from a length-truncated AsCpfl crRNA or a chemically-modified AsCpfl crRNA, or an AsCpfl crRNA containing both length truncations and chemical modifications. 2024204029   13 Jun 2024

[0072] In a seventh aspect, an isolated AsCpfl crRNA is provided. The isolated AsCpfl crRNA is active in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated protein endonuclease system. In a first respect, the isolated AsCpfl crRNA is selected from length-truncated AsCpfl crRNA, a chemically-modified AsCpfl crRNA, or an AsCpfl crRNA containing both length truncations and chemical modifications.

[0073] In an eighth aspect, a method of performing gene editing is provided. The method includes the step of contacting a candidate editing target site locus with an active CRISPR / Cpfl endonuclease system having a wild-type AsCpfl polypeptide and a suitable AsCpfl crRNA. In a first respect, the wild-type AsCpfl polypeptide comprises at least one member selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 12, SEQ ID NO: 16 and SEQ ID NO: 19. In a second respect, the suitable AsCpfl crRNA is selected from a length-truncated AsCpfl crRNA, a chemically-modified AsCpfl crRNA, or an AsCpfl crRNA containing both length truncations and chemical modifications.

[0074] In another aspect, an isolated nucleic acid encoding an Lb Cpfl polypeptide codon optimized for expression in H. sapiens is provided. In a first respect the isolated nucleic acid comprises SEQ ID NO: 17 or SEQ ID NO:396.

[0075] In another aspect, an isolated polypeptide encoding a wild-type Lp Cpfl protein is provided. In a first respect, the isolated polypeptide comprises SEQ ID NO: 14 or SEQ ID NO:24.

[0076] In another aspect, an isolated expression vector encoding SEQ ID NO: 17 or SEQ ID NO:396 is provided.

[0077] In another aspect, a host cell including an isolated expression vector encoding SEQ ID NO: 17 or SEQ ID NO:396 is provided. The isolated expression vector encoding SEQ ID NO: 17 or SEQ ID NO:396 is operably linked to a suitable promoter to permit expression of a polypeptide comprising SEQ ID NO: 14 or SEQ ID NO:24, respectively. In a first respect, the host cell comprises a human cell. In a second respect, the human cell comprises an immortalized cell line. In a third respect, the immortalized cell line is a HEK293 cell line. In a further elaboration of this respect, the host cell includes an isolated Lb Cpfl crRNA capable of forming a ribonucleoprotein complex with the polypeptide selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 14, SEQ ID NO:20 and SEQ ID NO:24 to form a wild-type CRISPR / Cpfl endonuclease. 2024204029   13 Jun 2024

[0078] In another aspect, an isolated CRISPR / Cpfl endonuclease system having an Lb Cpfl polypeptide and a suitable Cpfl crRNA is provided. In a first respect, the CRISPR / Cpfl endonuclease system includes a Lb Cpfl polypeptide in the form of SEQ ID NO: 14. In a second respect, the isolated CRISPR / Cpfl endonuclease system includes a suitable Cpfl crRNA selected from a length-truncated Cpfl crRNA or a chemically-modified Cpfl crRNA, or a Cpfl crRNA comprising both length truncations and chemical modifications.

[0079] In another aspect, an isolated CRISPR / Cpfl endonuclease system having a human cell line expressing an Lb Cpfl polypeptide and a suitable Cpfl crRNA is provided. In a first respect, the Lb Cpfl polypeptide is SEQ ID NO: 14 or SEQ ID NO:24. In a second respect, the suitable Cpfl crRNA is selected from a length-truncated Cpfl crRNA or a chemically-modified Cpfl crRNA, or a Cpfl crRNA comprising both length truncations and chemical modifications.

[0080] In another respect, a method of performing gene editing is provided. The method includes the steps of contacting a candidate editing target site locus with an active CRISPR / Cpfl endonuclease system having a wild-type Lb Cpfl polypeptide and a suitable Cpfl crRNA. In a first respect, the method includes a wild-type Lb Cpfl polypeptide selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 14, SEQ ID NO:20 and SEQ ID NO:24. In a second respect, the suitable Cpfl crRNA is selected from a length-truncated Cpfl crRNA, a chemically-modified Cpfl crRNA, or a Cpfl crRNA comprising both length truncations and chemical modifications.

[0081] In another respect, a CRISPR endonuclease system having a recombinant Cpfl fusion protein and a suitable crRNA is provided. In a first respect, the recombinant Cpfl fusion protein is an isolated, purified protein. In a second respect, the recombinant Cpfl fusion protein includes an N-terminal NLS, a C-terminal NLS and a plurality of affinity tags located at either the N-terminal or C-terminal ends. In one preferred embodiment, the recombinant Cpfl fusion protein includes an N-terminal NLS, a C-terminal NLS and 3 N-terminal FLAG tags and a C-terminal 6xHis tag. In a third respect, the recombinant Cpfl fusion protein and a suitable crRNA is provided in a 1:1 stoichiometric ratio (that is, in equimolar amounts). 2024204029   13 Jun 2024 EXAMPLE 1

[0082] DNA and amino acid sequences of wild type As Cpfl polypeptide, as encoded in isolated nucleic acid vectors

[0083] The list below shows wild type (WT) As Cpfl nucleases expressed as a polypeptide fusion protein described in the present invention. It will be appreciated by one with skill in the art that many different DNA sequences can encode / express the same amino acid (AA) sequence since in many cases more than one codon can encode for the same amino acid. The DNA sequences shown below only serve as example and other DNA sequences that encode the same protein (e.g., same amino acid sequence) are contemplated. It is further appreciated that additional features, elements or tags may be added to said sequences, such as NLS domains and the like. Examples are shown for WT AsCpfl showing amino acid and DNA sequences for those proteins as Cpfl alone and Cpfl fused to both C-terminal and N-terminal SV40 NLS domains and a HIS-tag. Amino acid sequences that represent NLS sequences, domain linkers, or purification tags are indicated in bold font. SEQ ID NO:1 AsCpfl Native Nucleotide Sequence ATGACCCAATTTGAAGGTTTTACCAATTTATACCAAGTTTCGAAGACCCTTCGTTTTGAACTGAT TCCCCAAGGAAAAACACTCAAACATATCCAGGAGCAAGGGTTCATTGAGGAGGATAAAGCTCGCA ATGACCATTACAAAGAGTTAAAACCAATCATTGACCGCATCTATAAGACTTATGCTGATCAATGT CTCCAACTGGTACAGCTTGACTGGGAGAATCTATCTGCAGCCATAGACTCCTATCGTAAGGAAAA AACCGAAGAAACACGAAATGCGCTGATTGAGGAGCAAGCAACATATAGAAATGCGATTCATGACT ACTTTATAGGTCGGACGGATAATCTGACAGATGCCATAAATAAGCGCCATGCTGAAATCTATAAA GGACTTTTTAAAGCTGAACTTTTCAATGGAAAAGTTTTAAAGCAATTAGGGACCGTAACCACGAC AGAACATGAAAATGCTCTACTCCGTTCGTTTGACAAATTTACGACCTATTTTTCCGGCTTTTATG AAAACCGAAAAAATGTCTTTAGCGCTGAAGATATCAGCACGGCAATTCCCCATCGAATCGTCCAG GACAATTTCCCTAAATTTAAGGAAAACTGCCATATTTTTACAAGATTGATAACCGCAGTTCCTTC TTTGCGGGAGCATTTTGAAAATGTCAAAAAGGCCATTGGAATCTTTGTTAGTACGTCTATTGAAG AAGTCTTTTCCTTTCCCTTTTATAATCAACTTCTAACCCAAACGCAAATTGATCTTTATAATCAA CTTCTCGGCGGCATATCTAGGGAAGCAGGCACAGAAAAAATCAAGGGACTTAATGAAGTTCTCAA TCTGGCTATCCAAAAAAATGATGAAACAGCCCATATAATCGCGTCCCTGCCGCATCGTTTTATTC CTCTTTTTAAACAAATTCTTTCCGATCGAAATACGTTATCCTTTATTTTGGAAGAATTCAAAAGC GATGAGGAAGTCATCCAATCCTTCTGCAAATATAAAACCCTCTTGAGAAACGAAAATGTACTGGA GACTGCAGAAGCCCTTTTCAATGAATTAAATTCCATTGATTTGACTCATATCTTTATTTCCCATA AAAAGTTAGAAACCATCTCTTCAGCGCTTTGTGACCATTGGGATACCTTGCGCAATGCACTTTAC GAAAGACGGATTTCTGAACTCACTGGCAAAATAACAAAAAGTGCCAAAGAAAAAGTTCAAAGGTC ATTAAAACATGAGGATATAAATCTCCAAGAAATTATTTCTGCTGCAGGAAAAGAACTATCAGAAG CATTCAAACAAAAAACAAGTGAAATTCTTTCCCATGCCCATGCTGCACTTGACCAGCCTCTTCCC 2024204029   13 Jun 2024 ACAACATTAAAAAAACAGGAAGAAAAAGAAATCCTCAAATCACAGCTCGATTCGCTTTTAGGCCT TTATCATCTTCTTGATTGGTTTGCTGTCGATGAAAGCAATGAAGTCGACCCAGAATTCTCAGCAC GGCTGACAGGCATTAAACTAGAAATGGAACCAAGCCTTTCGTTTTATAATAAAGCAAGAAATTAT GCGACAAAAAAGCCCTATTCGGTGGAAAAATTTAAATTGAATTTTCAAATGCCAACCCTTGCCTC TGGTTGGGATGTCAATAAAGAAAAAAATAATGGAGCTATTTTATTCGTAAAAAATGGTCTCTATT ACCTTGGTATCATGCCTAAACAGAAGGGGCGCTATAAAGCCCTGTCTTTTGAGCCGACAGAAAAA ACATCAGAAGGATTCGATAAGATGTACTATGACTACTTCCCAGATGCCGCAAAAATGATTCCTAA GTGTTCCACTCAGCTAAAGGCTGTAACCGCTCATTTTCAAACTCATACCACCCCCATTCTTCTCT CAAATAATTTCATTGAACCTCTTGAAATCACAAAAGAAATTTATGAGCTGAACAATCCTGAAAAG GAGCCTAAAAAGTTTCAAACGGCTTATGCAAAGAAGACAGGCGATCAAAAAGGCTATAGAGAAGC GCTTTGCAAATGGATTGACTTTACGCGGGATTTTCTCTCTAAATATACGAAAACAACTTCAATCG ATTTATCTTCACTCCGCCCTTCTTCGCAATATAAAGATTTAGGGGAATATTACGCCGAACTGAAT CCGCTTCTCTATCATATCTCCTTCCAACGAATTGCTGAAAAGGAAATCATGGATGCTGTAGAAAC GGGAAAATTGTATCTGTTCCAAATCTACAATAAGGATTTTGCGAAGGGCCATCACGGGAAACCAA ATCTCCACACCCTGTATTGGACAGGTCTCTTCAGTCCTGAAAACCTTGCGAAAACCAGCATCAAA CTTAATGGTCAAGCAGAATTGTTCTATCGACCTAAAAGCCGCATGAAGCGGATGGCCCATCGTCT TGGGGAAAAAATGCTGAACAAAAAACTAAAGGACCAGAAGACACCGATTCCAGATACCCTCTACC AAGAACTGTACGATTATGTCAACCACCGGCTAAGCCATGATCTTTCCGATGAAGCAAGGGCCCTG CTTCCAAATGTTATCACCAAAGAAGTCTCCCATGAAATTATAAAGGATCGGCGGTTTACTTCCGA TAAATTTTTCTTCCATGTTCCCATTACACTGAATTATCAAGCAGCCAATAGTCCCAGTAAATTCA ACCAGCGTGTCAATGCCTACCTTAAGGAGCATCCGGAAACGCCCATCATTGGTATCGATCGTGGA GAACGCAATCTAATCTATATTACCGTCATTGACAGTACTGGGAAAATTTTGGAGCAGCGTTCCCT GAATACCATCCAGCAATTTGACTACCAAAAAAAATTGGACAACAGGGAAAAAGAGCGTGTTGCCG CCCGTCAAGCCTGGTCCGTCGTCGGAACGATCAAAGACCTTAAACAAGGCTACTTGTCACAGGTC ATCCATGAAATTGTAGACCTGATGATTCATTACCAAGCTGTTGTCGTCCTTGAAAACCTCAACTT CGGATTTAAATCAAAACGGACAGGCATTGCCGAAAAAGCAGTCTACCAACAATTTGAAAAGATGC TAATAGATAAACTCAACTGTTTGGTTCTCAAAGATTATCCTGCTGAGAAAGTGGGAGGCGTCTTA AACCCGTATCAACTTACAGATCAGTTCACGAGCTTTGCAAAAATGGGCACGCAAAGCGGCTTCCT TTTCTATGTACCGGCCCCTTATACCTCAAAGATTGATCCCCTGACTGGTTTTGTCGATCCCTTTG TATGGAAGACCATTAAAAATCATGAAAGTCGGAAGCATTTCCTAGAAGGATTTGATTTCCTGCAT TATGATGTCAAAACAGGTGATTTTATCCTCCATTTTAAAATGAATCGGAATCTCTCTTTCCAGAG AGGGCTTCCTGGCTTCATGCCAGCTTGGGATATTGTTTTCGAAAAGAATGAAACCCAATTTGATG CAAAAGGGACGCCCTTCATTGCAGGAAAACGAATTGTTCCTGTAATCGAAAATCATCGTTTTACG GGTCGTTACAGAGACCTCTATCCCGCTAATGAACTCATTGCCCTTCTGGAAGAAAAAGGCATTGT CTTTAGAGACGGAAGTAATATATTACCCAAACTTTTAGAAAATGATGATTCTCATGCAATTGATA CGATGGTCGCCTTGATTCGCAGTGTACTCCAAATGAGAAACAGCAATGCCGCAACGGGGGAAGAC TACATCAACTCTCCCGTTAGGGATCTGAACGGGGTGTGTTTCGACAGTCGATTCCAAAATCCAGA ATGGCCAATGGATGCGGATGCCAACGGAGCTTATCATATTGCCTTAAAAGGGCAGCTTCTTCTGA ACCACCTCAAAGAAAGCAAAGATCTGAAATTACAAAACGGCATCAGCAACCAAGATTGGCTGGCC TACATTCAGGAACTGAGAAACTGA SEQ ID NO:2 AsCpfl Native Protein Sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQC LQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYK GLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQ DNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQ 2024204029   13 Jun 2024 LLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKS DEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALY ERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLP TTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNY ATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEK TSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEK EPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELN PLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK LNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARAL LPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRG ERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQV IHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVL NPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLH YDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFT GRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGED YINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA YIQELRN SEQ ID NO:5 E.coli optimized AsCpfl DNA ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGAT TCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTA AC GAT CAC TACAAAGAAC T GAAACC GAT TAT C GAC C GCAT C TATAAAAC C TAT GCAGAT CAGT GT CTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAA AACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATT ATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAA GGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCAC CGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATG AGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAG GATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAG CCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAG AAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAA CTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAA TCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTC CGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCC GAT GAAGAGGT GAT T CAGAGC T T T T GCAAATACAAAAC GC T GC T GC GCAAT GAAAAT GT T C T GGA AACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACA AAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTAT GAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAG TCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAG CATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCG ACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCT GTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCAC GTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTAT GCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAG CGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATT 2024204029   13 Jun 2024 ATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAA ACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAA ATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGA GCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAA GAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGC GCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCG ATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAAT CCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAAC CGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGA ATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAA CTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCT GGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATC AAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTG CTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGA CAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTA ACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGT GAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCT GAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAG CACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTT ATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTT TGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGC TGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTG AATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCT GTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTG TTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCAT TACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCG TGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATG CAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACC GGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGT TTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATA CCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGAT TACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGA ATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGA ACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCA TATATCCAAGAACTGCGTAACTGA SEQ ID NO:8 AsCpfl Human Codon Optimized Nucleotide Sequence ATGACCCAGTTCGAGGGCTTCACCAACCTGTACCAGGTGTCCAAGACCCTGAGATTCGAGCTGAT CCCCCAGGGCAAGACACTGAAGCACATCCAGGAACAGGGCTTCATCGAAGAGGACAAGGCCCGGA AC GAC CAC TACAAAGAGC T GAAGCC CAT CAT C GAC C GGAT C TACAAGAC C TAC GC C GAC CAGT GC CTGCAGCTGGTGCAGCTGGACTGGGAGAATCTGAGCGCCGCCATCGACAGCTACCGGAAAGAGAA AACCGAGGAAACCCGGAACGCCCTGATCGAGGAACAGGCCACCTACAGAAACGCCATCCACGACT ACTTCATCGGCCGGACCGACAACCTGACCGACGCCATCAACAAGCGGCACGCCGAGATCTATAAG GGCCTGTTCAAGGCCGAGCTGTTCAACGGCAAGGTGCTGAAGCAGCTGGGCACCGTGACCACCAC CGAGCACGAAAACGCCCTGCTGCGGAGCTTCGACAAGTTCACCACCTACTTCAGCGGCTTCTACG AGAACCGGAAGAACGTGTTCAGCGCCGAGGACATCAGCACCGCCATCCCCCACAGAATCGTGCAG GACAACTTCCCCAAGTTCAAAGAGAACTGCCACATCTTCACCCGGCTGATCACCGCCGTGCCCAG 2024204029   13 Jun 2024 CCTGAGAGAACACTTCGAGAACGTGAAGAAGGCCATCGGCATCTTCGTGTCCACCAGCATCGAGG AAGTGTTCAGCTTCCCATTCTACAACCAGCTGCTGACCCAGACCCAGATCGACCTGTATAATCAG C T GCT GGGC GGCATCAGCAGAGAGGCC GGCACC GAGAAGAT CAAGGGCC T GAACGAAGT GC T GAA CCTGGCCATCCAGAAGAACGACGAGACAGCCCACATCATTGCCAGCCTGCCCCACCGGTTCATCC CTCTGTTCAAGCAGATCCTGAGCGACAGAAACACCCTGAGCTTCATCCTGGAAGAGTTCAAGTCC GATGAGGAAGTGATCCAGAGCTTCTGCAAGTATAAGACCCTGCTGAGGAACGAGAATGTGCTGGA AACCGCCGAGGCCCTGTTCAATGAGCTGAACAGCATCGACCTGACCCACATCTTTATCAGCCACA AGAAGCTGGAAACAATCAGCAGCGCCCTGTGCGACCACTGGGACACACTGCGGAATGCCCTGTAC GAGCGGCGGATCTCTGAGCTGACCGGCAAGATCACCAAGAGCGCCAAAGAAAAGGTGCAGCGGAG CCTGAAGCACGAGGATATCAACCTGCAGGAAATCATCAGCGCCGCTGGCAAAGAACTGAGCGAGG CCTTTAAGCAGAAAACCAGCGAGATCCTGTCCCACGCCCACGCCGCACTGGATCAGCCTCTGCCT ACCACCCTGAAGAAGCAGGAAGAGAAAGAGATCCTGAAGTCCCAGCTGGACAGCCTGCTGGGCCT GTACCATCTGCTGGATTGGTTCGCCGTGGACGAGAGCAACGAGGTGGACCCCGAGTTCTCCGCCA GACTGACAGGCATCAAACTGGAAATGGAACCCAGCCTGTCCTTCTACAACAAGGCCAGAAACTAC GCCACCAAGAAACCCTACAGCGTGGAAAAGTTTAAGCTGAACTTCCAGATGCCCACCCTGGCCAG CGGCTGGGACGTGAACAAAGAGAAGAACAACGGCGCCATCCTGTTCGTGAAGAACGGACTGTACT ACCTGGGCATCATGCCTAAGCAGAAGGGCAGATACAAGGCCCTGTCCTTTGAGCCCACCGAAAAG ACCAGCGAGGGCTTTGACAAGATGTACTACGATTACTTCCCCGACGCCGCCAAGATGATCCCCAA GTGCAGCACCCAGCTGAAGGCCGTGACCGCCCACTTTCAGACCCACACCACCCCCATCCTGCTGA GCAACAACTTCATCGAGCCCCTGGAAATCACCAAAGAGATCTACGACCTGAACAACCCCGAGAAA GAGCCCAAGAAGTTCCAGACCGCCTACGCCAAGAAAACCGGCGACCAGAAGGGCTACCGCGAGGC TCTGTGCAAGTGGATCGACTTTACCCGGGACTTCCTGAGCAAGTACACCAAGACCACCTCCATCG ATCTGAGCAGCCTGCGGCCCAGCTCCCAGTACAAGGATCTGGGCGAGTACTACGCCGAGCTGAAC CCTCTGCTGTACCACATCAGCTTCCAGCGGATCGCCGAAAAAGAAATCATGGACGCCGTGGAAAC CGGCAAGCTGTACCTGTTCCAGATCTATAACAAGGACTTCGCCAAGGGCCACCACGGCAAGCCCA ATCTGCACACCCTGTACTGGACCGGCCTGTTTAGCCCCGAGAATCTGGCCAAGACCAGCATCAAG CTGAACGGCCAGGCCGAACTGTTTTACCGGCCCAAGAGCCGGATGAAGCGGATGGCCCATAGACT GGGCGAGAAGATGCTGAACAAGAAACTGAAGGACCAGAAAACCCCTATCCCCGACACACTGTATC AGGAACTGTACGACTACGTGAACCACCGGCTGAGCCACGACCTGTCCGACGAAGCTAGAGCACTG CTGCCCAACGTGATCACAAAAGAGGTGTCCCACGAGATCATCAAGGACCGGCGGTTTACCTCCGA TAAGTTCTTCTTCCACGTGCCCATCACCCTGAACTACCAGGCCGCCAACAGCCCCAGCAAGTTCA ACCAGAGAGTGAACGCCTACCTGAAAGAGCACCCCGAGACACCCATCATTGGCATCGACAGAGGC GAGC GGAAC C T GAT C TACAT CAC C GT GAT C GACAGCACAGGCAAAAT CC T GGAACAGAGAAGC C T GAACACCATCCAGCAGTTCGACTACCAGAAGAAACTGGACAACCGGGAAAAAGAACGGGTGGCCG CCAGACAGGCTTGGAGCGTCGTGGGCACCATTAAGGACCTGAAGCAGGGCTACCTGAGCCAAGTG ATTCACGAGATCGTGGACCTGATGATCCACTATCAGGCTGTGGTGGTGCTGGAAAACCTGAACTT CGGCTTCAAGAGCAAGCGGACCGGAATCGCCGAGAAAGCCGTGTACCAGCAGTTTGAGAAAATGC TGATCGACAAGCTGAATTGCCTGGTGCTGAAAGACTACCCCGCTGAGAAAGTGGGAGGCGTGCTG AATCCCTACCAGCTGACCGACCAGTTCACCTCCTTTGCCAAGATGGGAACCCAGAGCGGCTTCCT GTTCTACGTGCCAGCCCCCTACACCAGCAAGATCGACCCTCTGACCGGCTTCGTGGACCCCTTCG TGTGGAAAACCATCAAGAACCACGAGTCCCGGAAGCACTTCCTGGAAGGCTTTGACTTCCTGCAC TACGACGTGAAAACAGGCGATTTCATCCTGCACTTCAAGATGAATCGGAATCTGTCCTTCCAGAG GGGCCTGCCCGGCTTCATGCCTGCCTGGGATATCGTGTTCGAGAAGAATGAGACACAGTTCGACG CCAAGGGAACCCCCTTTATCGCCGGCAAGAGGATCGTGCCTGTGATCGAGAACCACAGATTCACC GGCAGATACCGGGACCTGTACCCCGCCAACGAGCTGATTGCCCTGCTGGAAGAGAAGGGCATCGT GTTCCGGGACGGCAGCAACATCCTGCCCAAGCTGCTGGAAAATGACGACAGCCACGCCATCGATA CCATGGTGGCACTGATCCGCAGCGTGCTGCAGATGCGGAACAGCAATGCCGCCACCGGCGAGGAC TACATCAATAGCCCAGTGCGGGACCTGAACGGCGTGTGCTTCGACAGCAGATTCCAGAACCCCGA GTGGCCCATGGATGCCGACGCCAATGGCGCCTACCACATTGCCCTGAAGGGACAGCTGCTGCTGA ACCATCTGAAAGAGAGCAAAGACCTGAAACTGCAGAACGGCATCTCCAACCAGGACTGGCTGGCC TATATCCAGGAACTGCGGAACTGA 2024204029   13 Jun 2024 SEQ ID NO:11 E.coli optimized As Cpfl with flanking NLS's, V5 tag and 6x His - DNA ATGGGTCGGGATCCAGGTAAACCGATTCCGAATCCGCTGCTGGGTCTGGATAGCACCGCACCGAA AAAAAAACGTAAAGTTGGTATTCATGGTGTTCCGGCAGCAACCCAGTTTGAAGGTTTCACCAATC TGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGATTCCGCAGGGTAAAACCCTGAAACATATT CAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTAACGATCACTACAAAGAACTGAAACCGAT TATCGACCGCATCTATAAAACCTATGCAGATCAGTGTCTGCAGCTGGTTCAGCTGGATTGGGAAA ATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAAAACCGAAGAAACCCGTAATGCACTGATT GAAGAACAGGCAACCTATCGTAATGCCATCCATGATTATTTCATTGGTCGTACCGATAATCTGAC CGATGCAATTAACAAACGTCACGCCGAAATCTATAAAGGCCTGTTTAAAGCCGAACTGTTTAATG GCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCACCGAACATGAAAATGCACTGCTGCGTAGC TTTGATAAATTCACCACCTATTTCAGCGGCTTTTATGAGAATCGCAAAAACGTGTTTAGCGCAGA AGATATTAGCACCGCAATTCCGCATCGTATTGTGCAGGATAATTTCCCGAAATTCAAAGAGAACT GCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAGCCTGCGTGAACATTTTGAAAACGTTAAA AAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAGAAGTTTTTAGCTTCCCGTTTTACAATCA GCTGCTGACCCAGACCCAGATTGATCTGTATAACCAACTGCTGGGTGGTATTAGCCGTGAAGCAG GCACC GAAAAAAT CAAAGGT C T GAAT GAAGT GC T GAAT C T GGC CAT T CAGAAAAAT GAT GAAACC GCACATATTATTGCAAGCCTGCCGCATCGTTTTATTCCGCTGTTCAAACAAATTCTGAGCGATCG TAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCCGATGAAGAGGTGATTCAGAGCTTTTGCA AATACAAAACGCTGCTGCGCAATGAAAATGTTCTGGAAACTGCCGAAGCACTGTTTAACGAACTG AATAGCATTGATCTGACCCACATCTTTATCAGCCACAAAAAACTGGAAACCATTTCAAGCGCACT GTGTGATCATTGGGATACCCTGCGTAATGCCCTGTATGAACGTCGTATTAGCGAACTGACCGGTA AAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAGTCTGAAACATGAGGATATTAATCTGCAA GAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAGCATTTAAACAGAAAACCAGCGAAATTCT GTCACATGCACATGCAGCACTGGATCAGCCGCTGCCGACCACCCTGAAAAAACAAGAAGAAAAAG AAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCTGTATCATCTGCTGGACTGGTTTGCAGTT GATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCACGTCTGACCGGCATTAAACTGGAAATGGA ACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTATGCCACCAAAAAACCGTATAGCGTCGAAA AATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAGCGGTTGGGATGTTAATAAAGAAAAAAAC AACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATTATCTGGGTATTATGCCGAAACAGAAAGG TCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAAACCAGTGAAGGTTTTGATAAAATGTACT ACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAAATGTAGCACCCAGCTGAAAGCAGTTACC GCACATTTTCAGACCCATACCACCCCGATTCTGCTGAGCAATAACTTTATTGAACCGCTGGAAAT CACCAAAGAGATCTACGATCTGAATAACCCGGAAAAAGAGCCGAAAAAATTCCAGACCGCATATG CAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGCGCTGTGTAAATGGATTGATTTCACCCGT GATTTTCTGAGCAAATACACCAAAACCACCAGTATCGATCTGAGCAGCCTGCGTCCGAGCAGCCA GTATAAAGATCTGGGCGAATATTATGCAGAACTGAATCCGCTGCTGTATCATATTAGCTTTCAGC GTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAACCGGTAAACTGTACCTGTTCCAGATCTAC AATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGAATCTGCATACCCTGTATTGGACCGGTCT GTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAACTGAATGGTCAGGCGGAACTGTTTTATC GTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCTGGGTGAAAAAATGCTGAACAAAAAACTG AAAGACCAGAAAACCCCGATCCCGGATACACTGTATCAAGAACTGTATGATTATGTGAACCATCG TCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTGCTGCCGAATGTTATTACCAAAGAAGTTA GCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGACAAATTCTTTTTTCATGTGCCGATTACC C T GAAT TAT CAGGCAGCAAATAGCC C GAGCAAAT T TAAC CAGC GT GT TAAT GCATAT C T GAAAGA ACATCCAGAAACGCCGATTATTGGTATTGATCGTGGTGAACGTAACCTGATTTATATCACCGTTA 2024204029   13 Jun 2024 TTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCTGAATACCATTCAGCAGTTTGATTACCAG AAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAGCACGTCAGGCATGGTCAGTTGTTGGTAC AATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTTATTCATGAAATTGTGGATCTGATGATTC ACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTTTGGCTTTAAAAGCAAACGTACCGGCATT GCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGCTGATTGACAAACTGAATTGCCTGGTGCT GAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTGAATCCGTATCAGCTGACCGATCAGTTTA CCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCTGTTTTATGTTCCGGCACCGTATACGAGC AAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTGTTTGGAAAACCATCAAAAACCATGAAAG CCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCATTACGACGTTAAAACGGGTGATTTCATCC TGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCGTGGCCTGCCTGGTTTTATGCCTGCATGG GATATTGTGTTTGAGAAAAACGAAACACAGTTCGATGCAAAAGGCACCCCGTTTATTGCAGGTAA ACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACCGGTCGTTATCGCGATCTGTATCCGGCAA ATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGTTTTTCGTGATGGCTCAAACATTCTGCCG AAACTGCTGGAAAATGATGATAGCCATGCAATTGATACCATGGTTGCACTGATTCGTAGCGTTCT GCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGATTACATTAATAGTCCGGTTCGTGATCTGA ATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGAATGGCCGATGGATGCAGATGCAAATGGT GCATATCATATTGCACTGAAAGGACAGCTGCTGCTGAACCACCTGAAAGAAAGCAAAGATCTGAA ACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCATATATCCAAGAACTGCGTAACCCTAAAA AAAAACGCAAAGTGAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGA SEQ ID NO:12 E.coli optimized As Cpfl with 5'- and 3'-flanking NLS's, 5'-V5 tag and 3'-6x His MGRDPGKPIPNPLLGLDSTAPKKKRKVGIHGVPAATQFEGFTNLYQVSKTLRFELIPQGKTLKHI QEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALI EEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRS FDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDET AHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNEL NSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQ EIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAV DESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKN NGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVT AHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTR DFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIY NKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKL KDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPIT LNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQ KKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGI AEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTS KIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAW DIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILP KLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANG AYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNPKKKRKVKLAAALEHHHHHH SEQ ID NO:15 Hs optimized As Cpfl with flanking NLS's, V5 tag and 6x His - DNA 2024204029   13 Jun 2024 ATGGGCAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACAGCACAGCCCCTAAGAAAAAGCGGAA AGTGGGCATCCATGGCGTGCCAGCCGCCACACAGTTTGAGGGCTTCACCAACCTGTACCAGGTGT CCAAGACACTGCGCTTCGAGCTGATCCCTCAGGGCAAGACCCTGAAGCACATCCAAGAGCAGGGC TTCATCGAAGAGGACAAGGCCCGGAACGACCACTACAAAGAGCTGAAGCCCATCATCGACCGGAT CTACAAGACCTACGCCGACCAGTGTCTGCAGCTGGTGCAGCTCGATTGGGAGAATCTGAGCGCCG CCATCGACAGCTACCGGAAAGAGAAAACCGAGGAAACCCGGAACGCCCTGATCGAGGAACAGGCC ACCTACAGAAACGCCATCCACGACTACTTCATCGGCCGGACCGACAACCTGACCGACGCCATCAA CAAGAGACACGCCGAGATCTATAAGGGCCTGTTCAAGGCCGAGCTGTTCAACGGCAAGGTGCTGA AGCAGCTGGGCACCGTGACAACCACCGAGCACGAAAATGCCCTGCTGCGGAGCTTCGACAAGTTC ACCACCTACTTCAGCGGCTTCTACGAGAACCGGAAGAACGTGTTCAGCGCCGAGGACATCAGCAC CGCCATTCCTCACAGAATCGTGCAGGACAACTTCCCCAAGTTCAAAGAGAACTGCCACATCTTCA CCCGGCTGATCACAGCCGTGCCTAGCCTGAGAGAACACTTCGAGAACGTGAAGAAGGCCATCGGC ATCTTCGTGTCCACCAGCATCGAGGAAGTGTTCAGCTTCCCATTCTACAACCAGCTGCTGACCCA GACACAGATCGACCTGTATAATCAGCTGCTCGGCGGCATCAGCAGAGAGGCCGGAACAGAGAAGA TCAAGGGCCTGAACGAAGTGCTGAACCTGGCCATCCAGAAGAACGACGAGACAGCCCACATCATT GCCAGCCTGCCTCACCGGTTCATCCCTCTGTTCAAGCAGATCCTGAGCGACAGAAACACCCTGAG CTTCATCCTGGAAGAGTTCAAGTCCGATGAGGAAGTGATCCAGAGCTTCTGCAAGTATAAGACCC TGCTGAGGAACGAGAATGTGCTGGAAACCGCCGAGGCTCTGTTTAACGAGCTGAACAGCATCGAT CTGACCCACATCTTTATCAGCCACAAGAAGCTCGAGACAATCAGCAGCGCCCTGTGCGACCACTG GGATACCCTGAGAAACGCCCTGTACGAGCGGAGAATCAGCGAGCTGACCGGCAAGATCACCAAGA GC GCCAAAGAAAAGGT GCAGC GGAGCC T GAAACAC GAGGATAT CAAC C T GCAAGAGAT CAT CAGC GCCGCTGGCAAAGAACTGAGCGAGGCCTTTAAGCAGAAAACCAGCGAGATCCTGTCTCACGCCCA CGCTGCTCTT GAT CAGC C T C T GC C TAC CACAC T GAAGAAGCAAGAGGAAAAAGAGAT CC T GAAGT CCCAGCTGGACAGCCTGCTGGGACTGTACCATCTGCTGGATTGGTTCGCCGTGGACGAGAGCAAT GAGGTGGACCCTGAGTTCTCCGCCAGACTGACAGGCATCAAGCTGGAAATGGAACCCAGCCTGTC CTTCTACAACAAGGCCAGAAACTACGCCACCAAGAAGCCCTACAGCGTCGAGAAGTTCAAGCTCA ACTTCCAGATGCCTACACTGGCCAGCGGCTGGGACGTGAACAAAGAGAAGAACAACGGCGCCATC CTGTTCGTGAAGAACGGACTGTACTACCTGGGCATCATGCCAAAGCAGAAGGGCAGATACAAGGC CCTGTCCTTTGAGCCCACCGAAAAGACCAGCGAGGGCTTCGATAAGATGTACTACGATTACTTCC CCGACGCCGCCAAGATGATCCCCAAGTGTAGCACACAGCTGAAGGCCGTGACCGCTCACTTTCAG ACCCACACCACACCTATCCTGCTGAGCAACAACTTCATCGAGCCCCTGGAAATCACCAAAGAGAT CTACGACCTGAACAACCCCGAGAAAGAGCCCAAGAAGTTCCAGACCGCCTACGCCAAGAAAACCG GCGACCAGAAGGGCTACAGAGAAGCCCTGTGCAAGTGGATCGACTTTACCCGGGACTTCCTGAGC AAGTACACCAAGACCACCTCCATCGACCTGAGCAGCCTGAGGCCTAGCAGCCAGTATAAGGACCT GGGCGAGTACTACGCCGAGCTGAATCCACTGCTGTACCACATCAGCTTCCAGCGGATCGCCGAAA AAGAAATCATGGACGCCGTGGAAACCGGCAAGCTGTACCTGTTCCAGATATACAACAAAGACTTC GCCAAGGGCCACCACGGCAAGCCTAATCTGCACACCCTGTACTGGACCGGCCTGTTTAGCCCTGA GAATCTGGCCAAGACCTCTATCAAGCTGAACGGCCAGGCCGAACTGTTTTACAGACCCAAGAGCC GGAT GAAGC GGAT GGCC CACAGAC T GGGAGAGAAGAT GC T GAACAAGAAAC T GAAGGAC CAGAAA ACGCCCATTCCGGACACACTGTACCAAGAGCTGTACGACTACGTGAACCACCGGCTGAGCCACGA TCTGAGCGACGAAGCTAGAGCACTGCTGCCCAACGTGATCACAAAAGAGGTGTCCCACGAGATCA TTAAGGACCGGCGGTTTACCTCCGATAAGTTCTTCTTCCACGTGCCGATCACACTGAACTACCAG GCCGCCAACTCTCCCAGCAAGTTCAACCAGAGAGTGAACGCCTACCTGAAAGAGCACCCCGAGAC ACCCATCATTGGCATCGACAGAGGCGAGCGGAACCTGATCTACATCACCGTGATCGACTCCACAG GCAAGATCCTGGAACAGCGGTCCCTGAACACCATCCAGCAGTTCGACTACCAGAAGAAGCTGGAC AACCGAGAGAAAGAAAGAGTGGCCGCCAGACAGGCTTGGAGCGTTGTGGGCACAATCAAGGATCT 2024204029   13 Jun 2024 GAAGCAGGGCTACCTGAGCCAAGTGATTCACGAGATCGTGGACCTGATGATCCACTATCAGGCTG TGGTGGTGCTCGAGAACCTGAACTTCGGCTTCAAGAGCAAGCGGACCGGAATCGCCGAGAAAGCC GTGTACCAGCAGTTTGAGAAAATGCTGATCGACAAGCTGAATTGCCTGGTCCTGAAGGACTACCC CGCTGAGAAAGTTGGCGGAGTGCTGAATCCCTACCAGCTGACCGATCAGTTCACCAGCTTTGCCA AGATGGGAACCCAGAGCGGCTTCCTGTTCTACGTGCCAGCTCCTTACACCTCCAAGATCGACCCT CTGACCGGCTTCGTGGACCCCTTCGTGTGGAAAACCATCAAGAACCACGAGTCCCGGAAGCACTT CCTGGAAGGCTTTGACTTCCTGCACTACGACGTGAAAACAGGCGATTTCATCCTGCACTTCAAGA TGAATCGGAATCTGTCCTTCCAGAGGGGCCTGCCTGGCTTCATGCCTGCTTGGGATATCGTGTTC GAGAAGAATGAGACTCAGTTCGACGCCAAGGGGACCCCTTTTATCGCCGGCAAGAGAATTGTGCC TGTGATCGAGAACCACAGGTTCACCGGCAGATACCGGGATCTGTACCCCGCCAATGAGCTGATCG CCCTGCTGGAAGAGAAGGGCATCGTGTTTAGAGATGGCAGCAACATCCTGCCTAAGCTGCTGGAA AACGACGACAGCCACGCCATCGATACCATGGTGGCACTGATCAGATCCGTGCTGCAGATGCGGAA CAGCAATGCCGCTACCGGCGAGGACTACATCAATAGCCCCGTGCGGGATCTGAACGGCGTGTGCT TCGACAGCAGATTTCAGAACCCCGAGTGGCCTATGGATGCCGACGCCAATGGCGCCTATCACATT GCCCTGAAAGGACAGCTGCTGCTGAACCATCTGAAAGAGAGCAAGGACCTGAAACTGCAGAACGG CATCTCCAACCAGGACTGGCTGGCCTACATTCAAGAGCTGCGGAATCCCAAAAAGAAACGGAAAG T GAAGC T GGCCGCTGCTCT GGAACACCAC CACCAT CACCAT SEQ ID NO:16 Hs optimized As Cpfl with 5'- and 3'-flanking NLS's, 5'-V5 tag and 3'-6x His - AA MGKPIPNPLLGLDSTAPKKKRKVGIHGVPAATQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQG FIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQA TYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKF TTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIG IFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHII ASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSID LTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIIS AAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESN EVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAI LFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLS KYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDF AKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQK TPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQ AANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLD NREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKA VYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDP LTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVF EKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLE NDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHI ALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNPKKKRKVKLAAALEHHHHHH SEQ ID NO:18 DNA E.coli optimized As Cpfl with OpT NLS and 6x His - 2024204029   13 Jun 2024 ATGACCCAGTTTGAAGGTTTCACCAATCTGTATCAGGTTAGCAAAACCCTGCGTTTTGAACTGAT TCCGCAGGGTAAAACCCTGAAACATATTCAAGAACAGGGCTTCATCGAAGAGGATAAAGCACGTA AC GAT CAC TACAAAGAAC T GAAACC GAT TAT C GAC C GCAT C TATAAAAC C TAT GCAGAT CAGT GT CTGCAGCTGGTTCAGCTGGATTGGGAAAATCTGAGCGCAGCAATTGATAGTTATCGCAAAGAAAA AACCGAAGAAACCCGTAATGCACTGATTGAAGAACAGGCAACCTATCGTAATGCCATCCATGATT ATTTCATTGGTCGTACCGATAATCTGACCGATGCAATTAACAAACGTCACGCCGAAATCTATAAA GGCCTGTTTAAAGCCGAACTGTTTAATGGCAAAGTTCTGAAACAGCTGGGCACCGTTACCACCAC CGAACATGAAAATGCACTGCTGCGTAGCTTTGATAAATTCACCACCTATTTCAGCGGCTTTTATG AGAATCGCAAAAACGTGTTTAGCGCAGAAGATATTAGCACCGCAATTCCGCATCGTATTGTGCAG GATAATTTCCCGAAATTCAAAGAGAACTGCCACATTTTTACCCGTCTGATTACCGCAGTTCCGAG CCTGCGTGAACATTTTGAAAACGTTAAAAAAGCCATCGGCATCTTTGTTAGCACCAGCATTGAAG AAGTTTTTAGCTTCCCGTTTTACAATCAGCTGCTGACCCAGACCCAGATTGATCTGTATAACCAA CTGCTGGGTGGTATTAGCCGTGAAGCAGGCACCGAAAAAATCAAAGGTCTGAATGAAGTGCTGAA TCTGGCCATTCAGAAAAATGATGAAACCGCACATATTATTGCAAGCCTGCCGCATCGTTTTATTC CGCTGTTCAAACAAATTCTGAGCGATCGTAATACCCTGAGCTTTATTCTGGAAGAATTCAAATCC GAT GAAGAGGT GAT T CAGAGC T T T T GCAAATACAAAAC GC T GC T GC GCAAT GAAAAT GT T C T GGA AACTGCCGAAGCACTGTTTAACGAACTGAATAGCATTGATCTGACCCACATCTTTATCAGCCACA AAAAACTGGAAACCATTTCAAGCGCACTGTGTGATCATTGGGATACCCTGCGTAATGCCCTGTAT GAACGTCGTATTAGCGAACTGACCGGTAAAATTACCAAAAGCGCGAAAGAAAAAGTTCAGCGCAG TCTGAAACATGAGGATATTAATCTGCAAGAGATTATTAGCGCAGCCGGTAAAGAACTGTCAGAAG CATTTAAACAGAAAACCAGCGAAATTCTGTCACATGCACATGCAGCACTGGATCAGCCGCTGCCG ACCACCCTGAAAAAACAAGAAGAAAAAGAAATCCTGAAAAGCCAGCTGGATAGCCTGCTGGGTCT GTATCATCTGCTGGACTGGTTTGCAGTTGATGAAAGCAATGAAGTTGATCCGGAATTTAGCGCAC GTCTGACCGGCATTAAACTGGAAATGGAACCGAGCCTGAGCTTTTATAACAAAGCCCGTAATTAT GCCACCAAAAAACCGTATAGCGTCGAAAAATTCAAACTGAACTTTCAGATGCCGACCCTGGCAAG CGGTTGGGATGTTAATAAAGAAAAAAACAACGGTGCCATCCTGTTCGTGAAAAATGGCCTGTATT ATCTGGGTATTATGCCGAAACAGAAAGGTCGTTATAAAGCGCTGAGCTTTGAACCGACGGAAAAA ACCAGTGAAGGTTTTGATAAAATGTACTACGACTATTTTCCGGATGCAGCCAAAATGATTCCGAA ATGTAGCACCCAGCTGAAAGCAGTTACCGCACATTTTCAGACCCATACCACCCCGATTCTGCTGA GCAATAACTTTATTGAACCGCTGGAAATCACCAAAGAGATCTACGATCTGAATAACCCGGAAAAA GAGCCGAAAAAATTCCAGACCGCATATGCAAAAAAAACCGGTGATCAGAAAGGTTATCGTGAAGC GCTGTGTAAATGGATTGATTTCACCCGTGATTTTCTGAGCAAATACACCAAAACCACCAGTATCG ATCTGAGCAGCCTGCGTCCGAGCAGCCAGTATAAAGATCTGGGCGAATATTATGCAGAACTGAAT CCGCTGCTGTATCATATTAGCTTTCAGCGTATTGCCGAGAAAGAAATCATGGACGCAGTTGAAAC CGGTAAACTGTACCTGTTCCAGATCTACAATAAAGATTTTGCCAAAGGCCATCATGGCAAACCGA ATCTGCATACCCTGTATTGGACCGGTCTGTTTAGCCCTGAAAATCTGGCAAAAACCTCGATTAAA CTGAATGGTCAGGCGGAACTGTTTTATCGTCCGAAAAGCCGTATGAAACGTATGGCACATCGTCT GGGTGAAAAAATGCTGAACAAAAAACTGAAAGACCAGAAAACCCCGATCCCGGATACACTGTATC AAGAACTGTATGATTATGTGAACCATCGTCTGAGCCATGATCTGAGTGATGAAGCACGTGCCCTG CTGCCGAATGTTATTACCAAAGAAGTTAGCCACGAGATCATTAAAGATCGTCGTTTTACCAGCGA CAAATTCTTTTTTCATGTGCCGATTACCCTGAATTATCAGGCAGCAAATAGCCCGAGCAAATTTA ACCAGCGTGTTAATGCATATCTGAAAGAACATCCAGAAACGCCGATTATTGGTATTGATCGTGGT GAACGTAACCTGATTTATATCACCGTTATTGATAGCACCGGCAAAATCCTGGAACAGCGTAGCCT GAATACCATTCAGCAGTTTGATTACCAGAAAAAACTGGATAATCGCGAGAAAGAACGTGTTGCAG CACGTCAGGCATGGTCAGTTGTTGGTACAATTAAAGACCTGAAACAGGGTTATCTGAGCCAGGTT ATTCATGAAATTGTGGATCTGATGATTCACTATCAGGCCGTTGTTGTGCTGGAAAACCTGAATTT TGGCTTTAAAAGCAAACGTACCGGCATTGCAGAAAAAGCAGTTTATCAGCAGTTCGAGAAAATGC 2024204029   13 Jun 2024 TGATTGACAAACTGAATTGCCTGGTGCTGAAAGATTATCCGGCTGAAAAAGTTGGTGGTGTTCTG AATCCGTATCAGCTGACCGATCAGTTTACCAGCTTTGCAAAAATGGGCACCCAGAGCGGATTTCT GTTTTATGTTCCGGCACCGTATACGAGCAAAATTGATCCGCTGACCGGTTTTGTTGATCCGTTTG TTTGGAAAACCATCAAAAACCATGAAAGCCGCAAACATTTTCTGGAAGGTTTCGATTTTCTGCAT TACGACGTTAAAACGGGTGATTTCATCCTGCACTTTAAAATGAATCGCAATCTGAGTTTTCAGCG TGGCCTGCCTGGTTTTATGCCTGCATGGGATATTGTGTTTGAGAAAAACGAAACACAGTTCGATG CAAAAGGCACCCCGTTTATTGCAGGTAAACGTATTGTTCCGGTGATTGAAAATCATCGTTTCACC GGTCGTTATCGCGATCTGTATCCGGCAAATGAACTGATCGCACTGCTGGAAGAGAAAGGTATTGT TTTTCGTGATGGCTCAAACATTCTGCCGAAACTGCTGGAAAATGATGATAGCCATGCAATTGATA CCATGGTTGCACTGATTCGTAGCGTTCTGCAGATGCGTAATAGCAATGCAGCAACCGGTGAAGAT TACATTAATAGTCCGGTTCGTGATCTGAATGGTGTTTGTTTTGATAGCCGTTTTCAGAATCCGGA ATGGCCGATGGATGCAGATGCAAATGGTGCATATCATATTGCACTGAAAGGACAGCTGCTGCTGA ACCACCTGAAAGAAAGCAAAGATCTGAAACTGCAAAACGGCATTAGCAATCAGGATTGGCTGGCA TATATCCAAGAACTGCGTAACGGTCGTAGCAGTGATGATGAAGCAACCGCAGATAGCCAGCATGC AGCACCGCCTAAAAAGAAACGTAAAGTTGGTGGTAGCGGTGGTTCAGGTGGTAGTGGCGGTAGTG GTGGCTCAGGGGGTTCTGGTGGCTCTGGTGGTAGCCTCGAGCACCACCACCACCACCACTGA SEQ ID NO:19 Amino acid sequence for AsCpfl fusion with OpT NLS and 6x His used for gene editing in both E. coll and human cells MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQC LQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYK GLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQ DNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQ LLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKS DEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALY ERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLP TTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNY ATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEK TSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEK EPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELN PLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK LNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARAL LPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRG ERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQV IHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVL NPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLH YDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFT GRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGED YINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA YIQELRNGRSSDDEATADSQHAAPPKKKRKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH SEQ ID NO:21 Hs optimized As Cpfl with OpT NLS and 6x His - DNA ATGGGCGACCCTCTGAAGAACGTGGGCATCGACAGACTGGACGTGGAAAAGGGCAGAAAGAACAT GAGCAAGCTCGAGAAGTTCACCAACTGCTACAGCCTGAGCAAGACCCTGCGGTTCAAGGCCATTC CTGTGGGCAAGACCCAAGAGAACATCGACAACAAGCGGCTGCTGGTGGAAGATGAGAAGAGAGCC 2024204029   13 Jun 2024 GAGGACTACAAGGGCGTGAAGAAGCTGCTGGACCGGTACTACCTGAGCTTCATCAACGACGTGCT GCACAGCATCAAGCTGAAGAACCTGAACAACTACATCAGCCTGTTCCGGAAGAAAACCCGGACCG AGAAAGAGAACAAAGAGCTGGAAAACCTCGAGATCAACCTGCGGAAAGAGATCGCCAAGGCCTTC AAGGGCAACGAGGGCTACAAGAGCCTGTTCAAGAAGGACATCATCGAGACAATCCTGCCTGAGTT CCTGGACGACAAGGACGAGATCGCCCTGGTCAACAGCTTCAACGGCTTCACAACCGCCTTCACCG GCTTTTTCGACAACCGCGAGAATATGTTCAGCGAGGAAGCCAAGAGCACCTCTATCGCCTTCCGG TGCATCAACGAGAATCTGACCCGGTACATCAGCAACATGGATATCTTCGAGAAGGTGGACGCCAT C T T C GACAAGCAC GAGGT GCAAGAGAT CAAAGAAAAGAT CC T GAACAGC GAC TAC GAC GT C GAGG ACTTCTTCGAGGGCGAGTTCTTCAACTTCGTGCTGACACAAGAGGGCATCGATGTGTACAACGCC ATCATCGGCGGCTTCGTGACAGAGAGCGGCGAGAAGATCAAGGGCCTGAACGAGTACATCAACCT CTACAACCAGAAAACGAAGCAGAAGCTGCCCAAGTTCAAGCCCCTGTACAAACAGGTGCTGAGCG ACAGAGAGAGCCTGTCCTTTTACGGCGAGGGCTATACCAGCGACGAAGAGGTGCTGGAAGTGTTC AGAAACACCCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAGCTCGAAAAGCTGTTTAA GAACTTCGACGAGTACAGCAGCGCCGGCATCTTCGTGAAGAATGGCCCTGCCATCAGCACCATCT CCAAGGACATCTTCGGCGAGTGGAACGTGATCCGGGACAAGTGGAACGCCGAGTACGACGACATC CACCTGAAGAAAAAGGCCGTGGTCACCGAGAAGTACGAGGACGACAGAAGAAAGAGCTTCAAGAA GATCGGCAGCTTCAGCCTGGAACAGCTGCAAGAGTACGCCGACGCCGATCTGAGCGTGGTGGAAA AGCTGAAAGAGATTATCATCCAGAAGGTCGACGAGATCTACAAGGTGTACGGCAGCAGCGAGAAG CTGTTCGACGCCGACTTTGTGCTGGAAAAGAGCCTCAAAAAGAACGACGCCGTGGTGGCCATCAT GAAGGACCTGCTGGATAGCGTGAAGTCCTTCGAGAACTATATTAAGGCCTTCTTTGGCGAGGGCA AAGAGACAAACCGGGACGAGAGCTTCTACGGCGATTTCGTGCTGGCCTACGACATCCTGCTGAAA GTGGACCACATCTACGACGCCATCCGGAACTACGTGACCCAGAAGCCTTACAGCAAGGACAAGTT TAAGCTGTACTTCCAGAATCCGCAGTTCATGGGCGGCTGGGACAAAGACAAAGAAACCGACTACC GGGCCACCATCCTGAGATACGGCTCCAAGTACTATCTGGCCATTATGGACAAGAAATACGCCAAG TGCCTGCAGAAGATCGATAAGGACGACGTGAACGGCAACTACGAGAAGATTAACTACAAGCTGCT GCCCGGACCTAACAAGATGCTGCCTAAGGTGTTCTTTAGCAAGAAATGGATGGCCTACTACAACC CCAGCGAGGATATCCAGAAAATCTACAAGAACGGCACCTTCAAGAAAGGCGACATGTTCAACCTG AACGACTGCCACAAGCTGATCGATTTCTTCAAGGACAGCATCAGCAGATACCCCAAGTGGTCCAA CGCCTACGACTTCAATTTCAGCGAGACAGAGAAGTATAAGGATATCGCCGGGTTCTACCGCGAGG TGGAAGAACAGGGCTATAAGGTGTCCTTTGAGAGCGCCAGCAAGAAAGAGGTGGACAAGCTGGTC GAAGAGGGCAAGCTGTACATGTTCCAGATCTATAACAAGGACTTCTCCGACAAGAGCCACGGCAC CCCTAACCTGCACACCATGTACTTTAAGCTGCTGTTCGATGAGAACAACCACGGCCAGATCAGAC TGTCTGGCGGAGCCGAGCTGTTTATGAGAAGGGCCAGCCTGAAAAAAGAGGAACTGGTCGTTCAC CCCGCCAACTCTCCAATCGCCAACAAGAACCCCGACAATCCCAAGAAAACCACCACACTGAGCTA CGACGTGTACAAGGATAAGCGGTTCTCCGAGGACCAGTACGAGCTGCACATCCCTATCGCCATCA ACAAGTGCCCCAAGAATATCTTCAAGATCAACACCGAAGTGCGGGTGCTGCTGAAGCACGACGAC AACCCTTACGTGATCGGCATCGATCGGGGCGAGAGAAACCTGCTGTATATCGTGGTGGTGGACGG CAAGGGCAATATCGTGGAACAGTACTCCCTGAATGAGATCATCAACAACTTCAATGGCATCCGGA T CAAGAC GGAC TACCACAGCC T GC T GGACAAAAAAGAGAAAGAAC GC T T C GAGGC CC GGCAGAAC TGGACCAGCATCGAGAACATCAAAGAACTGAAGGCCGGCTACATCTCCCAGGTGGTGCACAAGAT CTGCGAGCTGGTTGAGAAGTATGACGCCGTGATTGCCCTGGAAGATCTGAATAGCGGCTTTAAGA ACAGCCGCGTGAAGGTCGAGAAACAGGTGTACCAGAAATTCGAGAAGATGCTGATCGACAAGCTG AACTACATGGTCGACAAGAAGTCTAACCCCTGCGCCACAGGCGGAGCCCTGAAGGGATATCAGAT CACCAACAAGTTCGAGTCCTTCAAGAGCATGAGCACCCAGAATGGCTTCATCTTCTACATCCCCG CCTGGCTGACCAGCAAGATCGATCCTAGCACCGGATTCGTGAACCTGCTCAAGACCAAGTACACC AGCATTGCCGACAGCAAGAAGTTCATCTCCAGCTTCGACCGGATTATGTACGTGCCCGAAGAGGA CCTGTTCGAATTCGCCCTGGATTACAAGAACTTCAGCCGGACCGATGCCGACTATATCAAGAAGT 2024204029   13 Jun 2024 GGAAGCTGTATAGCTACGGCAACCGCATCCGCATCTTCAGAAACCCGAAGAAAAACAACGTGTTC GACTGGGAAGAAGTGTGCCTGACCAGCGCCTACAAAGAACTCTTCAACAAATACGGCATCAACTA CCAGCAGGGCGACATCAGAGCCCTGCTGTGCGAGCAGAGCGACAAGGCCTTTTACAGCTCCTTCA TGGCCCTGATGAGCCTGATGCTGCAGATGCGGAATAGCATCACCGGCAGGACCGACGTGGACTTC CTGATCAGCCCTGTGAAGAATTCCGACGGGATCTTCTACGACAGCAGAAACTACGAGGCTCAAGA GAACGCCATCCTGCCTAAGAACGCCGATGCCAACGGCGCCTATAATATCGCCAGAAAGGTGCTGT GGGCCATCGGCCAGTTTAAGAAGGCCGAGGACGAGAAACTGGACAAAGTGAAGATCGCCATCTCT AACAAAGAGTGGCTGGAATACGCCCAGACCAGCGTGAAGCACGGCAGATCTAGTGACGATGAGGC CACCGCCGATAGCCAGCATGCAGCCCCTCCAAAGAAAAAGCGGAAAGTGCTGGAACACCACCACC ATCACCAC SEQ ID NO:22 Hs optimized As Cpfl with OpT NLS and 6x His - AA MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQC LQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYK GLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQ DNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQ LLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKS DEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALY ERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLP TTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNY ATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEK TSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEK EPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELN PLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIK LNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARAL LPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRG ERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQV IHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVL NPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLH YDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFT GRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGED YINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA YIQELRNGRSSDDEATADSQHAAPPKKKRKVGGSGGSGGSGGSGGSGGSGGSGGSLEHHHHHH EXAMPLE 2

[0084] Preparation of isolated vectors expressing nucleic acid encoding human codon-optimized AsCpfl polypeptide fusion protein and human cell lines stably expressing the As Cpfl polypeptide fusion protein.

[0085] The reference amino acid for AsCpfl has been published. See Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., Essletzbichler, P., 2024204029   13 Jun 2024 Volz, S.E., Joung, J., van der Oost, J., Regev, A., Koonin, E.V., and Zhang, F. (2015) Cpfl is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:1-13. A plasmid encoding human codon optimized AsCpfl, flanking nuclear localization signals (NLS) and 5’-V5 epitope tag, was generated by the Synthetic Biology department at Integrated DNA Technologies. Flanking the expression cassette was a 5’ Xhol and 3’ EcoRI restriction enzyme sites (FIG. 2). The Cpfl plasmid was digested with Xhol and EcoRI (NEB), gel purified using a column based purification system (Qiagen) and ligated using T4 DNA Ligase (NEB) into a predigested mammalian expression vector, pcDNA3.1-, from Life Technologies (FIG. 3). The resulting ligated construct was transformed into DH5a chemically competent A. coll cells. The resulting colonies were grown in LB media at 37°C overnight and subjected to DNA isolation using a Promega miniprep plasmid DNA kit. Flanking primers (T7 forward and BGH reverse) as well as 10 internal Cpfl specific primers were used for sequence verification of correct insertion using automated Sanger sequencing with BigDye Terminator reagents (ABI). The nucleic acid sequence of the Cpfl clone employed herein is shown in SEQ ID NO: 15. The amino acid sequence of the expressed recombinant protein is shown in SEQ ID NO: 16.

[0086] The AsCpfl-pcDNA3.1 vector was linearized with Pvul (NEB), which is located within the ampicillin resistance gene, and transfected into HEK293 cells. Transfection employed 500,000 HEK293 cells plated in 100mm dishes 24 hours prior to transfection. Using the transfection reagent TransIT-X2 (Mirus), the linearized vector containing AsCpfl and a neomycin-resistance gene was complexed and transfected into adherent cells. The transfection media was removed after 24 hrs and the cells were cultured in complete media for 48 hours. Using methods previously optimized for generation of stable transgenic HEK293 cells containing a stably integrated pcDNA3.1(-) vector neomycin resistance, we cultured transfected cells in the presence of the antibiotic Geneticin (G418; Gibco), which is a neomycin analog, in the complete media to select for cells that had been transfected with AsCpfl-pcDNA3.1(-) and would thus be resistant to this antibiotic. Initial G418 dosing was at 800 ug / ml with periodic media changes until the surviving cells began to recover and grow over a 10-day period. The parent HEK293 cell line was confirmed to be sensitive to the minimum dose of G418. The resulting polyclonal AsCpfl-pcDNA3.1(-) cell line, which showed G418 resistance, 2024204029   13 Jun 2024 was split using limited dilutions. The cells were trypsinized, resuspended in complete media, counted to determine concentration and diluted in 96-well plates to a concentration of theoretically less than one cell per well.

[0087] At this time, aliquots of the cells were taken and lysed with a protein lysis buffer (RIPA) to determine, via western blot, if AsCpfl was expressed. Cellular protein was quantitated using the Bio-Rad Protein Assay (Bio-Rad) and 15 ug total protein was loaded onto an SDS-PAGE Stainfree 4-20% gradient gel (Bio-Rad). As a positive control, protein from a previous cell line, SpyCas9-pcDN A3.1(-), was run in parallel for size and expression comparisons. The gel was run for 45 minutes at 180 volts and transferred to a PVDF membrane with the Bio-Rad TransBlot for 7 minutes. The blot was then blocked in SuperBlock T20 Blocking Buffer (Thermo), followed by a 1:1000 dilution of V5 primary antibody (Abeam) and 1:5000 P-actin primary antibody (Abeam) for 1 hour at room temperature. Next, the blot was washed 3 times for 15 minutes each in tris-buffered saline with Tween-20 (TBST). Goat anti-mouse HRP secondary antibody was used at a 1:3000 dilution along with the ladder specific StrepTactin secondary antibody and incubated at room temperature for 1 hour at room temperature. The blot was then washed 3 times for 15 minutes in TBST. Luminescence detection was done using the Pierce West-Femto ECL (Thermo) substrate and results are shown in FIG. 4, which confirm expression of a recombinant protein of the expected size.

[0088] Cells were continuously grown under selection in G418-containing media, and individual cells (monoclonal colonies) were allowed to expand. Viable colonies were characterized for the presence of AsCpfl by RT-qPCR, Western blotting and functional testing of crRNA guided dsDNA cleavage. Four RT-qPCR assays were designed to detect different locations within the large AsCpfl mRNA. Sequences are shown in Table 1 below.

[0089] Table 1: RT-qPCR assays in AsCpfl Assay# Location Primers and Probe SEQ ID NO 1 34-153 F34 GTGTCCAAGACCCTGAGATTC R153 GGGCTTCAGCTCTTTGTAGT P68 FAM- AGGGCAAG(ZEN)ACACTGAAGCACATCC-IBFQ 25 26 27 2 1548 1656 F1548 CAGAAACTACGCCACCAAGA R1656 GCCGTTGTTCTTCTCTTTGTTC P1590 HEX-TAAGCTGAA( ZEN)CTTCCAGATGCCCACC-IBFQ 28 29 30 2024204029   13 Jun 2024 3 2935 3037 F2 935 GTGGACCTGATGATCCACTATC R3037 GCTGGTACACGGCTTTCT P2 97 8 FAM-ACCTGAACT(ZEN)TCGGCTTCAAGAGCA-IBFQ 31 32 33 4 3827 3918 F3827 TGCTGAACCATCTGAAAGAGAG R3918 GTTCCGCAGTTCCTGGATATAG P3889 HEX-AGTCCTGGT(ZEN)TGGAGATGCCGTTC-IBFQ 34 35 36 DNA bases are shown 5’-3’ orientation. Location is specified within the AsCpfl gene construct employed herein. FAM - 6 carboxyfluorescein, HEX = hexachlorofluorescein, IBFQ = Iowa Black dark quencher, and ZEN = internal ZEN dark quencher.

[0090] Monoclonal cell lines resistant to G418 were plated in 6-well plates and cultured for 24 hrs. Cells were lysed with GITC-containing buffer and RNA was isolated using the Wizard 96-well RNA isolation binding plates (Promega) on a Corbett liquid handling robot. Liquid handling robotics (Perkin Elmer) were used to synthesize complementary DNA (cDNA) using SuperScriptll (Invitrogen) and set-up qPCR assays using Immolase (Bioline) along with 500 nmol primers and 250 nmol probes (IDT). qPCR plates were run on the AB7900-HT and analyzed using the associated software (Applied Biosystems). FIG. 5 shows the relative level of AsCpfl mRNA expression normalized to HPRT1 expression for a series of clonal lines. Not surprisingly, different clones showed different levels of AsCpfl mRNA expression.

[0091] Total protein was isolated from the same AsCpfl-expressing monoclonal cells lines in cultures grown in parallel. Cells were lysed in RIPA buffer in the presence of a proteinase inhibitor. Protein concentration in each lysate was determined by BCA assay (Pierce). Fifteen micrograms of total protein from each sample was loaded onto an SDS-PAGE stainfree 4-20% gradient gel (Bio-Rad) and run at 180V for 45 minutes in lx Tris / Glycine running buffer alongside the broad-range molecular weight marker (BioRad). Protein was transferred to a PDVF membrane using Bio-Rad TransBlot transfer unit for 7 minutes. The blot was blocked in SuperBlock T20 Blocking Buffer (Thermo), followed by incubation with a 1:1000 dilution of V5 primary antibody (Abeam) and 1:5000 P-actin primary antibody (Abeam) for 1 hour at room temperature. The blot was washed 3 times for 15 minutes each in tris-buffered saline with Tween-20 (TBST). Goat anti-mouse HRP secondary antibody was used at a 1:3000 dilution along with the ladder specific StrepTactin secondary antibody and incubated at room temperature for 1 hour at room temperature. The blot was then washed 3 times for 15 minutes in TBST. Luminescence detection was done using the Pierce West-Femto ECL (Thermo) 2024204029   13 Jun 2024 substrate. FIG. 6 shows detection of V5-tagged AsCpfl recombinant protein expression levels in 10 monoclonal cell lines. There is good concordance between observed protein levels seen in FIG. 6 and the corresponding mRNA levels from the same cell lines shown in FIG. 5.

[0092] Three monoclonal AsCpfl stable cell lines (1A1, 2A2 and 2B1) were expanded and tested for the ability to support AsCpfl-directed genome editing. Based on AsCpfl mRNA and protein levels previously determined, 1 Al is a “high” expressing line, 2A2 is a “medium” expressing line, and 2B1 is a “low” expressing line. The cell lines were transfected with 6 different crRNAs targeting different sites within an exon of the human HRPT1 gene, shown below in Table 2. The crRNAs comprise a universal 20 base Cpfl-binding domain at the 5’-end and a 24 base target-specific protospacer domain at the 3’-end.

[0093] Table 2: AsCpfl crRNAs targeting human HPRT1 Site Sequence SEQID NO: 38171_AS uaauuucuacucuuguagauuaaacacuguuucauuucauccgu 37 38254_AS uaauuucuacucuuguagauaccagcaagcuguuaauuacaaaa 38 38325_S uaauuucuacucuuguagauaccaucuuuaaccuaaaagaguuu 39 38337_AS uaauuucuacucuuguagaugguuaaagaugguuaaaugauuga 40 38351_S uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 41 38538_S uaauuucuacucuuguagauaauguaaguaauugcuucuuuuuc 42 RNA bases are shown 5’-3’ orientation, RNA bases are shown in lower case. Locations are specified within the human HPRT1 gene with orientation relative to the sense coding strand indicated (S = sense, AS = antisense).

[0094] In a reverse transfection format, arti-HPRTl crRNAs were individually mixed with Lipofectamine RNAiMAX (Life Technologies) and transfected into each of the 3 HEK-Cpfl cell lines. Transfections were done with 40,000 cells per well in 96 well plate format. RNAs were introduced at a final concentration of 30 nM in 0.75 pl of the lipid reagent. Cells were incubated at 37°C for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified with KAPA HiFi 2024204029   13 Jun 2024 DNA Polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO:394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO:395). PCR products were melted and re-annealed in NEB buffer 2 (New England Biolabs) to allow for heteroduplex formation followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. The digested products were visualized on a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of the cut products / (average molar concentration of the cut products + molar concentration of the uncut band) * 100. The cleavage efficiencies seen in the 3 cell lines are shown in Table 3 below.

[0095] Table 3: Gene targeting efficiency of 6 HPRT1 crRNAs in 3 HEK-Cpfl cell lines Site % Cleavage in T7EI assays 1A1 2A2 2B1 38171_AS 19 19.1 8.3 38254_AS 41 42.4 30.3 38325_S 27.8 26.5 14.8 38337_AS 65.3 73.7 71.6 38351S 73.3 78.6 73.4 38538_S 44.6 47.9 32.8 Locations of the crRNAs are specified within the human HPRT1 gene with orientation relative to the sense coding strand indicated (S = sense, AS = antisense). % Cleavage demonstrates alteration in the sequence of the cell line after Cpfl-mediated genome editing at the HPRT1 locus relative to wild-type.

[0096] As expected, the different crRNAs targeting different sites in HPRT1 showed different levels of gene editing activity. In cell line 1A1 this ranged from 18% to 73%. The “high” and “medium” Cpfl-expressing clones 1A1 and 2A2 showed nearly identical gene editing activity, indicating that both clones expressed Cpfl at sufficient levels to reach maximal gene editing activity at each site. Clone 2B1, the “low” expressing clone, 2024204029   13 Jun 2024 showed reduced editing activity. Clones 1 Al and 2A2 are therefore both suitable for Cpfl crRNA optimization and site screening. EXAMPLE 3

[0097] crRNA length optimization: Testing truncation of the 5’-20-base universal loop domain.

[0098] A set of 6 sites in the human HPRT1 gene were chosen to study length optimization of AsCpfl crRNAs. A series of crRNAs were synthesized all having a 3’24 base target-specific protospacer domain and having 5’-loop domains of 20, 19, 18, and 17 bases, representing a set of serial 1-base deletions from the 5’-end. A second set of crRNAs were synthesized at the same sites all having a 3’-21 base target-specific protospacer domain, likewise with 5’-loop domains of 20, 19, 18, and 17 bases.

[0099] An HEK cell line that stably expresses the AsCpfl endonuclease was employed in these studies (Example 2). In a reverse transfection format, anh-HPRTl crRNAs were individually mixed with Lipofectamine RNAiMAX (Life Technologies) and transfected into the HEK-Cpfl cell line. Transfections were done with 40,000 cells per well in 96 well plate format. RNAs were introduced at a final concentration of 30 nM in 0.75 pl of the lipid reagent. Cells were incubated at 37°C for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified with KAPA HiFi DNA Polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO:394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO:395). PCR products were melted and re-annealed in NEB buffer 2 (New England Biolabs) to allow for heteroduplex formation followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. The digested products were visualized on a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of the cut products / (average molar concentration of the cut products + molar concentration of the uncut band) * 100. Results are shown in Table 4 below and demonstrate that 5’-universal loop domains of 20 and 19 base lengths work well but a significant loss of activity is seen when 18 or 17 2024204029   13 Jun 2024 base loops domains are employed. The observations are nearly identical whether a 24 base or 21 base protospacer domain is employed.

[0100] Table 4. Effect of truncation in the 5’-loop domain with 24 or 21 base 3’- protospacer domains Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38171_AS 20-24 uaauuucuacucuuguagauuaaacacuguuucauuucauccgu 12% 37 38171-AS 19-24 aauuucuacucuuguagauuaaacacuguuucauuucauccgu 15% 43 38171-AS 18-24 auuucuacucuuguagauuaaacacuguuucauuucauccgu 4% 44 38171-AS 17-24 uuucuacucuuguagauuaaacacuguuucauuucauccgu 1% 45 38254_AS 20-24 uaauuucuacucuuguagauaccagcaagcuguuaauuacaaaa 15% 38 38254-AS 19-24 aauuucuacucuuguagauaccagcaagcuguuaauuacaaaa 36% 46 38254-AS 18-24 auuucuacucuuguagauaccagcaagcuguuaauuacaaaa 23% 47 38254-AS 17-24 uuucuacucuuguagauaccagcaagcuguuaauuacaaaa 0% 48 38325_S 20-24 uaauuucuacucuuguagauaccaucuuuaaccuaaaagaguuu 9% 39 38325-S 19-24 aauuucuacucuuguagauaccaucuuuaaccuaaaagaguuu 37% 49 38325-S 18-24 auuucuacucuuguagauaccaucuuuaaccuaaaagaguuu 27% 50 38325-S 17-24 uuucuacucuuguagauaccaucuuuaaccuaaaagaguuu 0% 51 38337_AS 20-24 uaauuucuacucuuguagaugguuaaagaugguuaaaugauuga 63% 40 38337-AS 19-24 aauuucuacucuuguagaugguuaaagaugguuaaaugauuga 65% 52 38337-AS 18-24 auuucuacucuuguagaugguuaaagaugguuaaaugauuga 46% 53 38337-AS 17-24 uuucuacucuuguagaugguuaaagaugguuaaaugauuga 4% 54 38351_S 20-24 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 57% 41 38351-S 19-24 aauuucuacucuuguagauugugaaauggcuuauaauugcuua 76% 55 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38351-S 18-24 auuucuacucuuguagauugugaaauggcuuauaauugcuua 6% 56 38351-S 17-24 uuucuacucuuguagauugugaaauggcuuauaauugcuua 0% 57 38538_S 20-24 uaauuucuacucuuguagauaauguaaguaauugcuucuuuuuc 16% 42 38538-S 19-24 aauuucuacucuuguagauaauguaaguaauugcuucuuuuuc 34% 58 38538-S 18-24 auuucuacucuuguagauaauguaaguaauugcuucuuuuuc 2% 59 38538-S 17-24 uuucuacucuuguagauaauguaaguaauugcuucuuuuuc 1% 60 38171-AS 20-21 uaauuucuacucuuguagauuaaacacuguuucauuucauc 32% 61 38171-AS 19-21 aauuucuacucuuguagauuaaacacuguuucauuucauc 44% 62 38171-AS 18-21 auuucuacucuuguagauuaaacacuguuucauuucauc 16% 63 38171-AS 17-21 uuucuacucuuguagauuaaacacuguuucauuucauc 1% 64 38254-AS 20-21 uaauuucuacucuuguagauaccagcaagcuguuaauuaca 45% 65 38254-AS 19-21 aauuucuacucuuguagauaccagcaagcuguuaauuaca 28% 66 38254-AS 18-21 auuucuacucuuguagauaccagcaagcuguuaauuaca 50% 67 38254-AS 17-21 uuucuacucuuguagauaccagcaagcuguuaauuaca 0% 68 38325-S 20-21 uaauuucuacucuuguagauaccaucuuuaaccuaaaagag 50% 69 38325-S 19-21 aauuucuacucuuguagauaccaucuuuaaccuaaaagag 49% 70 38325-S 18-21 auuucuacucuuguagauaccaucuuuaaccuaaaagag 36% 71 38325-S 17-21 uuucuacucuuguagauaccaucuuuaaccuaaaagag 0% 72 38337-AS 20-21 uaauuucuacucuuguagaugguuaaagaugguuaaaugau 72% 73 38337-AS 19-21 aauuucuacucuuguagaugguuaaagaugguuaaaugau 73% 74 38337-AS 18-21 auuucuacucuuguagaugguuaaagaugguuaaaugau 62% 75 38337-AS 17-21 uuucuacucuuguagaugguuaaagaugguuaaaugau 12% 76 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38351-S 20-21 uaauuucuacucuuguagauugugaaauggcuuauaauugc 81% 77 38351-S 19-21 aauuucuacucuuguagauugugaaauggcuuauaauugc 81% 78 38351-S 18-21 auuucuacucuuguagauugugaaauggcuuauaauugc 20% 79 38351-S 17-21 uuucuacucuuguagauugugaaauggcuuauaauugc 0% 80 38538-S 20-21 uaauuucuacucuuguagauaauguaaguaauugcuucuuu 65% 81 38538-S 19-21 aauuucuacucuuguagauaauguaaguaauugcuucuuu 41% 82 38538-S 18-21 auuucuacucuuguagauaauguaaguaauugcuucuuu 11% 83 38538-S 17-21 uuucuacucuuguagauaauguaaguaauugcuucuuu 1% 84 RNA bases are shown in lower case. Locations are specified within the human HPRT1 gene with orientation relative to the sense coding strand indicated (S = sense, AS = antisense). Sequence names include length of the 5’-universal loop domain (17-20 bases) and the 3’-target specific protospacer domain (24 or 21 bases). EXAMPLE 4

[0101] crRNA length optimization: Testing truncation of the 3’-24-base target specific protospacer domain.

[0102] The same set of 6 sites in the human HPRT1 gene was used to study the effects of truncation in the 3’-protospacer (target specific) domain. A series of AsCpfl crRNAs were synthesized all having the same 5’-20 base universal loop domain. These were paired with 3’-target specific protospacer domains of 21, 19, 18, or 17 bases, having serial deletions from the 3’-end.

[0103] An HEK cell line that stably expresses the AsCpfl endonuclease was employed in these studies (Example 2). In a reverse transfection format, anh-HPRTl AsCpfl crRNAs were individually mixed with Lipofectamine RNAiMAX (Life Technologies) and transfected into the HEK-Cpfl cell line. Transfections were done with 40,000 cells per well in 96 well plate format. RNAs were introduced at a final 2024204029   13 Jun 2024 concentration of 30 nM in 0.75 p.1 of the lipid reagent. Cells were incubated at 37°C for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified with KAPA HiFi DNA Polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO:394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO:395). PCR products were melted and re-annealed in NEB buffer 2 (New England Biolabs) to allow for heteroduplex formation followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. The digested products were visualized on a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of the cut products / (average molar concentration of the cut products + molar concentration of the uncut band) * 100. Results are shown in Table 5 below and demonstrate that a 3’-protospacer (target specific) domain of 21 base lengths work well but loss of activity is observed in a sequence / site dependent fashion as this domain is shortened. Some highly active sites (such as 38351) maintain appreciate activity even when truncated to 17 bases, however to maintain the highest likelihood of functionality at all sites a protospacer of 21 bases is recommended. Therefore, a prudent minimal length AsCpfl crRNA is 41 bases, comprising a 20-base 5’-universal loop domain and a 21-base 3’-protospacer targetspecific domain.

[0104] Table 5. Effect of truncation in the 3’-protospacer domain with a 20 base 5’-loop domain Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38171-AS 20-21 uaauuucuacucuuguagauuaaacacuguuucauuucauc 59% 61 38171-AS 20-19 uaauuucuacucuuguagauuaaacacuguuucauuuca 13% 85 38171-AS 20-18 uaauuucuacucuuguagauuaaacacuguuucauuuc 2% 86 38171-AS 20-17 uaauuucuacucuuguagauuaaacacuguuucauuu 3% 87 38254-AS 20-21 uaauuucuacucuuguagauaccagcaagcuguuaauuaca 61% 65 38254-AS 20-19 uaauuucuacucuuguagauaccagcaagcuguuaauua 5% 88 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38254-AS 20-18 uaauuucuacucuuguagauaccagcaagcuguuaauu 0% 89 38254-AS 20-17 uaauuucuacucuuguagauaccagcaagcuguuaau 0% 90 38325-S 20-21 uaauuucuacucuuguagauaccaucuuuaaccuaaaagag 70% 69 38325-S 20-19 uaauuucuacucuuguagauaccaucuuuaaccuaaaag 34% 91 38325-S 20-18 uaauuucuacucuuguagauaccaucuuuaaccuaaaa 0% 92 38325-S 20-17 uaauuucuacucuuguagauaccaucuuuaaccuaaa 0% 93 38337-AS 20-21 uaauuucuacucuuguagaugguuaaagaugguuaaaugau 80% 73 38337-AS 20-19 uaauuucuacucuuguagaugguuaaagaugguuaaaug 78% 94 38337-AS 20-18 uaauuucuacucuuguagaugguuaaagaugguuaaau 3% 95 38337-AS 20-17 uaauuucuacucuuguagaugguuaaagaugguuaaa 0% 96 38351-S 20-21 uaauuucuacucuuguagauugugaaauggcuuauaauugc 85% 77 38351-S 20-19 uaauuucuacucuuguagauugugaaauggcuuauaauu 87% 97 38351-S 20-18 uaauuucuacucuuguagauugugaaauggcuuauaau 85% 98 38351-S 20-17 uaauuucuacucuuguagauugugaaauggcuuauaa 67% 99 38538-S 20-21 uaauuucuacucuuguagauaauguaaguaauugcuucuuu 75% 81 38538-S 20-19 uaauuucuacucuuguagauaauguaaguaauugcuucu 55% 100 38538-S 20-18 uaauuucuacucuuguagauaauguaaguaauugcuuc 11% 101 38538-S 20-17 uaauuucuacucuuguagauaauguaaguaauugcuu 0% 102 RNA bases are shown in lower case. Locations are specified within the human HPRT1 gene with orientation relative to the sense-coding strand indicated (S = sense, AS = antisense). Sequence names include length of the 5’-universal loop domain (20 bases) and the 3’-protospacer target-specific domain (21, 19, 18, or 17 bases). 2024204029   13 Jun 2024 EXAMPLE 5

[0105] A single-base 2’OMe modification walk through two AsCpfl crRNAs.

[0106] Two sites in the human HPRT1 gene were chosen (38351 and 38595) to study the effects of replacement of a single RNA residue with a 2’OMe-RNA residue at every possible position within AsCpfl crRNAs. Given the possibility of sequence-specific tolerance to modification, it was necessary to perform this screening at two sites. A series of crRNAs were synthesized having a single 2’0Me residue at every possible position in single-base steps. The crRNAs were either 44 base or 41 base lengths. All had a 5’-end 20 base universal loop domain followed by a 3’-end 21 or 24 base protospacer target-specific domain.

[0107] An HEK cell line that stably expresses the AsCpfl endonuclease was employed in these studies (HEK-Cpfl) (Example 2). In a reverse transfection format, anti-HPRTl crRNAs were individually mixed with Lipofectamine RNAiMAX (Life Technologies) and transfected into the HEK-Cpfl cell line. Transfections were done with 40,000 cells per well in 96 well plate format. RNAs were introduced at a final concentration of 30 nM in 0.75 pl of the lipid reagent. Cells were incubated at 37°C for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified with KAPA HiFi DNA Polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO:394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO:395). PCR products were melted and re-annealed in NEB buffer 2 (New England Biolabs) to allow for heteroduplex formation followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. The digested products were visualized on a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of the cut products / (average molar concentration of the cut products + molar concentration of the uncut band) * 100. Results for HPRT1 site 38351 are shown in Table 6 below and for HRPT1 site 38595 in Table 7 below. The results demonstrate the locations of sites that reduce activity or totally kill activity of Cpfl to cleave dsDNA when the 2’0Me modified replaced an 2024204029   13 Jun 2024 RNA residue. The results are nearly identical whether a 24 base or 21 base protospacer domain is employed.

[0108] Sites where substitution of a 2’0Me RNA residue for an RNA residue showed loss of activity in the genome editing assay were mapped to location within the 5’-universal loop domain or the 3’-target specific protospacer domain. Results are summarized in Fig. 7. Modification of residues A2, A3, U4, UI 1, G15, and U20 within the universal loop domain leads to loss of activity; the same sites were identified for all 4 crRNA classes studied (Site 38351 44mer, Site 38351 41mer, Site 38595 44mer, and Site 38595 41mer). In contrast, the precise pattern of modification effects varied for sites within the protospacer domain, which is expected as it is common for modification tolerance to vary with sequence context and the protospacer domain has a different sequence for every target site. For the sequences studied, positions 5, 6, 13, 16, and 18 showed loss of activity with modification for all 4 crRNA classes and therefore are identified positions to avoid the 2’0Me RNA chemical modification.

[0109] Table 6: Single-base 2’OMe modification walk through HPRT1 Site 38351 AsCpfl crRNAs Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38351-44 unmod uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 77% 103 38351-44- L1 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 83% 104 38351-44- L2 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 32% 105 38351-44- L3 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 4% 106 38351-44- L4 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 2% 107 38351-44- L5 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 88% 108 38351-44- L6 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 87% 109 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38351-44- L7 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 85% 110 38351-44- L8 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 76% 111 38351-44- L9 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 89% 112 38351-44- L10 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 85% 113 38351-44- Lll uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 34% 114 38351-44- L12 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 86% 115 38351-44- L13 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 85% 116 38351-44- L14 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 86% 117 38351-44- L15 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 58% 118 38351-44- L16 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 89% 119 38351-44- L17 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 88% 120 38351-44- L18 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 82% 121 38351-44- L19 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 87% 122 38351-44- L20 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 52% 123 38351-44- T1 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 87% 124 38351-44- T2 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 79% 125 38351-44- T3 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 86% 126 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38351-44- T4 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 81% 127 38351-44- T5 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 69% 128 38351-44- T6 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 57% 129 38351-44- T7 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 84% 130 38351-44- T8 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 90% 131 38351-44- T9 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 86% 132 38351-44- T10 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 89% 133 38351-44- Tll uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 86% 134 38351-44- T12 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 90% 135 38351-44- T13 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 15% 136 38351-44- T14 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 71% 137 38351-44- T15 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 72% 138 38351-44- T16 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 68% 139 38351-44- T17 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 72% 140 38351-44- T18 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 64% 141 38351-44- T19 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 75% 142 38351-44- T20 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 71% 143 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38351-44- T21 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 72% 144 38351-44- T22 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 69% 145 38351-44- T23 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 72% 146 38351-44- T24 uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 70% 147 38351-41 unmod uaauuucuacucuuguagauugugaaauggcuuauaauugc 77% 148 38351-41- L1 uaauuucuacucuuguagauugugaaauggcuuauaauugc 87% 149 38351-41- L2 uaauuucuacucuuguagauugugaaauggcuuauaauugc 63% 150 38351-41- L3 uaauuucuacucuuguagauugugaaauggcuuauaauugc 15% 151 38351-41- L4 uaauuucuacucuuguagauugugaaauggcuuauaauugc 6% 152 38351-41- L5 uaauuucuacucuuguagauugugaaauggcuuauaauugc 88% 153 38351-41- L6 uaauuucuacucuuguagauugugaaauggcuuauaauugc 88% 154 38351-41- L7 uaauuucuacucuuguagauugugaaauggcuuauaauugc 81% 155 38351-41- L8 uaauuucuacucuuguagauugugaaauggcuuauaauugc 78% 156 38351-41- L9 uaauuucuacucuuguagauugugaaauggcuuauaauugc 90% 157 38351-41- L10 uaauuucuacucuuguagauugugaaauggcuuauaauugc 88% 158 38351-41- Lll uaauuucuacucuuguagauugugaaauggcuuauaauugc 59% 159 38351-41- L12 uaauuucuacucuuguagauugugaaauggcuuauaauugc 88% 160 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38351-41- L13 uaauuucuacucuuguagauugugaaauggcuuauaauugc 89% 161 38351-41- L14 uaauuucuacucuuguagauugugaaauggcuuauaauugc 88% 162 38351-41- L15 uaauuucuacucuuguagauugugaaauggcuuauaauugc 41% 163 38351-41- L16 uaauuucuacucuuguagauugugaaauggcuuauaauugc 90% 164 38351-41- L17 uaauuucuacucuuguagauugugaaauggcuuauaauugc 89% 165 38351-41- L18 uaauuucuacucuuguagauugugaaauggcuuauaauugc 89% 166 38351-41- L19 uaauuucuacucuuguagauugugaaauggcuuauaauugc 88% 167 38351-41- L20 uaauuucuacucuuguagauugugaaauggcuuauaauugc 77% 168 38351-41- T1 uaauuucuacucuuguagauugugaaauggcuuauaauugc 89% 169 38351-41- T2 uaauuucuacucuuguagauugugaaauggcuuauaauugc 84% 170 38351-41- T3 uaauuucuacucuuguagauugugaaauggcuuauaauugc 87% 171 38351-41- T4 uaauuucuacucuuguagauugugaaauggcuuauaauugc 86% 172 38351-41- T5 uaauuucuacucuuguagauugugaaauggcuuauaauugc 80% 173 38351-41- T6 uaauuucuacucuuguagauugugaaauggcuuauaauugc 79% 174 38351-41- T7 uaauuucuacucuuguagauugugaaauggcuuauaauugc 86% 175 38351-41- T8 uaauuucuacucuuguagauugugaaauggcuuauaauugc 89% 176 38351-41- T9 uaauuucuacucuuguagauugugaaauggcuuauaauugc 89% 177 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38351-41- T10 uaauuucuacucuuguagauugugaaauggcuuauaauugc 89% 178 38351-41- Tll uaauuucuacucuuguagauugugaaauggcuuauaauugc 89% 179 38351-41- T12 uaauuucuacucuuguagauugugaaauggcuuauaauugc 88% 180 38351-41- T13 uaauuucuacucuuguagauugugaaauggcuuauaauugc 23% 181 38351-41- T14 uaauuucuacucuuguagauugugaaauggcuuauaauugc 75% 182 38351-41- T15 uaauuucuacucuuguagauugugaaauggcuuauaauugc 77% 183 38351-41- T16 uaauuucuacucuuguagauugugaaauggcuuauaauugc 72% 184 38351-41- T17 uaauuucuacucuuguagauugugaaauggcuuauaauugc 76% 185 38351-41- T18 uaauuucuacucuuguagauugugaaauggcuuauaauugc 71% 186 38351-41- T19 uaauuucuacucuuguagauugugaaauggcuuauaauugc 77% 187 38351-41- T20 uaauuucuacucuuguagauugugaaauggcuuauaauugc 75% 188 38351-41- T21 uaauuucuacucuuguagauugugaaauggcuuauaauugc 77% 189 Oligonucleotide sequences are shown 5’-3’. Lowercase = RNA; Underlined lowercase = 2’-O-methyl RNA. The relative functional activity of each species is indicated by the % cleavage in a T7EI heteroduplex assay. The sequence name indicates if the crRNA is a 44mer with a 24 base target domain or a 41mer with a 21 base target domain. The position of the 2’OMe residue with either the loop domain (L) or target domain (T) is indicated. 2024204029   13 Jun 2024

[0110] Table 7: Single-base 2’OMe modification walk through HPRT1 Site 38595 AsCpfl crRNAs Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38595-44 unmod uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 49% 190 38595-44- L1 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 48% 191 38595-44- L2 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 34% 192 38595-44- L3 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 6% 193 38595-44- L4 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 3% 194 38595-44- L5 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 59% 195 38595-44- L6 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 54% 196 38595-44- L7 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 56% 197 38595-44- L8 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 52% 198 38595-44- L9 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 60% 199 38595-44- L10 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 56% 200 38595-44- Lll uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 23% 201 38595-44- L12 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 51% 202 38595-44- L13 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 58% 203 38595-44- L14 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 52% 204 38595-44- uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 33% 205 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: L15 38595-44- L16 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 55% 206 38595-44- L17 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 58% 207 38595-44- L18 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 61% 208 38595-44- L19 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 54% 209 38595-44- L20 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 29% 210 38595-44- T1 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 55% 211 38595-44- T2 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 53% 212 38595-44- T3 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 49% 213 38595-44- T4 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 20% 214 38595-44- T5 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 17% 215 38595-44- T6 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 23% 216 38595-44- T7 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 47% 217 38595-44- T8 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 52% 218 38595-44- T9 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 51% 219 38595-44- T10 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 55% 220 38595-44- Tll uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 53% 221 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38595-44- T12 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 58% 222 38595-44- T13 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 20% 223 38595-44- T14 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 62% 224 38595-44- T15 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 60% 225 38595-44- T16 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 15% 226 38595-44- T17 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 49% 227 38595-44- T18 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 46% 228 38595-44- T19 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 64% 229 38595-44- T20 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 57% 230 38595-44- T21 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 55% 231 38595-44- T22 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 54% 232 38595-44- T23 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 56% 233 38595-44- T24 uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 54% 234 38595-41 unmod uaauuucuacucuuguagauggaaagagaauuguuuucucc 59% 235 38595-41- L1 uaauuucuacucuuguagauggaaagagaauuguuuucucc 60% 236 38595-41- L2 uaauuucuacucuuguagauggaaagagaauuguuuucucc 49% 237 38595-41- L3 uaauuucuacucuuguagauggaaagagaauuguuuucucc 10% 238 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38595-41- L4 uaauuucuacucuuguagauggaaagagaauuguuuucucc 5% 239 38595-41- L5 uaauuucuacucuuguagauggaaagagaauuguuuucucc 63% 240 38595-41- L6 uaauuucuacucuuguagauggaaagagaauuguuuucucc 55% 241 38595-41- L7 uaauuucuacucuuguagauggaaagagaauuguuuucucc 56% 242 38595-41- L8 uaauuucuacucuuguagauggaaagagaauuguuuucucc 55% 243 38595-41- L9 uaauuucuacucuuguagauggaaagagaauuguuuucucc 63% 244 38595-41- L10 uaauuucuacucuuguagauggaaagagaauuguuuucucc 64% 245 38595-41- Lll uaauuucuacucuuguagauggaaagagaauuguuuucucc 35% 246 38595-41- L12 uaauuucuacucuuguagauggaaagagaauuguuuucucc 55% 247 38595-41- L13 uaauuucuacucuuguagauggaaagagaauuguuuucucc 56% 248 38595-41- L14 uaauuucuacucuuguagauggaaagagaauuguuuucucc 58% 249 38595-41- L15 uaauuucuacucuuguagauggaaagagaauuguuuucucc 47% 250 38595-41- L16 uaauuucuacucuuguagauggaaagagaauuguuuucucc 55% 251 38595-41- L17 uaauuucuacucuuguagauggaaagagaauuguuuucucc 64% 252 38595-41- L18 uaauuucuacucuuguagauggaaagagaauuguuuucucc 69% 253 38595-41- L19 uaauuucuacucuuguagauggaaagagaauuguuuucucc 63% 254 38595-41- L20 uaauuucuacucuuguagauggaaagagaauuguuuucucc 45% 255 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38595-41- T1 uaauuucuacucuuguagauggaaagagaauuguuuucucc 60% 256 38595-41- T2 uaauuucuacucuuguagauggaaagagaauuguuuucucc 59% 257 38595-41- T3 uaauuucuacucuuguagauggaaagagaauuguuuucucc 53% 258 38595-41- T4 uaauuucuacucuuguagauggaaagagaauuguuuucucc 21% 259 38595-41- T5 uaauuucuacucuuguagauggaaagagaauuguuuucucc 20% 260 38595-41- T6 uaauuucuacucuuguagauggaaagagaauuguuuucucc 25% 261 38595-41- T7 uaauuucuacucuuguagauggaaagagaauuguuuucucc 50% 262 38595-41- T8 uaauuucuacucuuguagauggaaagagaauuguuuucucc 64% 263 38595-41- T9 uaauuucuacucuuguagauggaaagagaauuguuuucucc 54% 264 38595-41- T10 uaauuucuacucuuguagauggaaagagaauuguuuucucc 57% 265 38595-41- Tll uaauuucuacucuuguagauggaaagagaauuguuuucucc 45% 266 38595-41- T12 uaauuucuacucuuguagauggaaagagaauuguuuucucc 52% 267 38595-41- T13 uaauuucuacucuuguagauggaaagagaauuguuuucucc 14% 268 38595-41- T14 uaauuucuacucuuguagauggaaagagaauuguuuucucc 66% 269 38595-41- T15 uaauuucuacucuuguagauggaaagagaauuguuuucucc 63% 270 38595-41- T16 uaauuucuacucuuguagauggaaagagaauuguuuucucc 16% 271 38595-41- T17 uaauuucuacucuuguagauggaaagagaauuguuuucucc 47% 272 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38595-41- T18 uaauuucuacucuuguagauggaaagagaauuguuuucucc 52% 273 38595-41- T19 uaauuucuacucuuguagauggaaagagaauuguuuucucc 64% 274 38595-41- T20 uaauuucuacucuuguagauggaaagagaauuguuuucucc 64% 275 38595-41- T21 uaauuucuacucuuguagauggaaagagaauuguuuucucc 66% 276 Oligonucleotide sequences are shown 5’-3’. Lowercase = RNA; Underlined lowercase = 2’-O-methyl RNA. The relative functional activity of each species is indicated by the % cleavage in a T7EI heteroduplex assay. The sequence name indicates if the crRNA is a 44mer with a 24 base target domain or a 41mer with a 21 base target domain. The position of the 2’OMe residue with either the loop domain (L) or target domain (T) is indicated. EXAMPLE 6

[0111] Modification of blocks of sequence in AsCpfl crRNAs.

[0112] Three sites in the human HPRT1 gene were chosen (38351, 38595, and 38104) to study the effects of replacement of a blocks of RNA residues with 2’0Me-RNA, 2’F RNA, or LNA residues within the AsCpfl crRNA. Modification of internucleotide linkages with phosphorothioate bonds (PS) as well as non-nucleotide end-modifiers were also tested. The crRNAs were either 44 base or 41 base lengths. All had a 5’-end 20 base universal loop domain followed by a 3’-end 21 or 24 base protospacer target-specific domain.

[0113] An HEK cell line that stably expresses the AsCpfl endonuclease was employed in these studies (HEK-Cpfl) (Example 2). In a reverse transfection format, anti-HPRTl crRNAs were individually mixed with Lipofectamine RNAiMAX (Life Technologies) and transfected into the HEK-Cpfl cell line. Transfections were done with 40,000 cells per well in 96 well plate format. RNAs were introduced at a final concentration of 30 nM in 0.75 pl of the lipid reagent. Cells were incubated at 37°C for 2024204029   13 Jun 2024 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified with KAPA HiFi DNA Polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO:394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO:395). PCR products were melted and re-annealed in NEB buffer 2 (New England Biolabs) to allow for heteroduplex formation followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. The digested products were visualized on a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of the cut products / (average molar concentration of the cut products + molar concentration of the uncut band) * 100. Results are shown in Table 8 below.

[0114] Large blocks of the universal 5-loop domain can be modified and retain activity (14 / 20 bases). However, the target-specific 3’-protospacer domain shows significant loss of activity when 2-3 consecutive 2’OMe residues replace RNA residues, even when those positions did not show any loss of activity in the single base walk (Example 5). Modification patterns in the protospacer domain are often expected to be impacted by sequence context, such that one modification pattern works well for one sequence but not for another sequence. The modification map shown in FIG. 7 displays modification patterns that range from minimal to high levels of modification that showed high performance at several sites and likely can be used regardless of sequence context.

[0115] 2’F residues could be placed at any position that was tolerant of 2’0Me modification. LNA residues can also be placed within the AsCpfl crRNA, and use of end-modifiers are shown below in Table 8. The phosphorothioate (PS) internucleotide linkage confers nuclease resistance and can be placed at the ends of the crRNA to block exonuclease attack or in the central regions to block endonuclease attack. Modification of large blocks of the crRNA (such as entire modification of the loop domain or the protospacer domain) with PS linkages are not compatible with crRNA function and significant loss of activity is seen when this modification pattern is employed. Limited use, such as 2-3 internucleotide linkages at each end, can be effectively employed, and such patterns are useful to block exonuclease attack. Non-base modifiers (such as a C3 2024204029   13 Jun 2024 spacer propanediol group or a ZEN modifier napthyl-azo group) can be placed at one or both ends of the crRNA without loss of activity and also block exonuclease attack.

[0116] Table 8: Functional impact of extensive modification of AsCpfl crRNAs Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38351-44-L uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 51% 277 38351-44-T uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 1% 278 38351-44- LT uaauuucuacucuuguagauugugaaauggcuuauaauugcuua 1% 279 38351-41-L uaauuucuacucuuguagauugugaaauggcuuauaauugc 53% 280 38351-41-T uaauuucuacucuuguagauugugaaauggcuuauaauugc 1% 281 38351-41- LT uaauuucuacucuuguagauugugaaauggcuuauaauugc 1% 282 38595-44-L uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 51% 283 38595-44-T uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 1% 284 38595-44- LT uaauuucuacucuuguagauggaaagagaauuguuuucuccuuc 1% 285 38595-41-L uaauuucuacucuuguagauggaaagagaauuguuuucucc 51% 286 38595-41-T uaauuucuacucuuguagauggaaagagaauuguuuucucc 1% 287 38595-41- LT uaauuucuacucuuguagauggaaagagaauuguuuucucc 1% 288 38595-41 unmod uaauuucuacucuuguagauggaaagagaauuguuuucucc 35% 235 38595-41- Tl-3 uaauuucuacucuuguagauggaaagagaauuguuuucucc 24% 289 38595-41- T7-12 uaauuucuacucuuguagauggaaagagaauuguuuucucc 2% 290 38595-41- T14-15 uaauuucuacucuuguagauggaaagagaauuguuuucucc 37% 291 38595-41- T17-21 uaauuucuacucuuguagauggaaagagaauuguuuucucc 22% 292 38595-41- uaauuucuacucuuguagauggaaagagaauuguuuucucc 1% 293 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: T6-9,18-21 38595-41- 5’C3 C3-uaauuucuacucuuguagauggaaagagaauuguuuucucc 35% 294 38595-41- 3’C3 uaauuucuacucuuguagauggaaagagaauuguuuucucc-C3 41% 295 38595-41- 2xC3 C3-uaauuucuacucuuguagauggaaagagaauuguuuucucc-C3 41% 296 38595-41- Ll-20 uaauuucuacucuuguagauggaaagagaauuguuuucucc 1% 297 38595-41- L+2 uaauuucuacucuuguagauggaaagagaauuguuuucucc 2% 298 38595-41- L+3 uaauuucuacucuuguagauggaaagagaauuguuuucucc 1% 299 38595-41- L+4 uaauuucuacucuuguagauggaaagagaauuguuuucucc 1% 300 38595-41- L+ll uaauuucuacucuuguagauggaaagagaauuguuuucucc 5% 301 38595-41- L+15 uaauuucuacucuuguagauggaaagagaauuguuuucucc 38% 302 38595-41- L+20 uaauuucuacucuuguagauggaaagagaauuguuuucucc 2% 303 38595-41-61 C3-uaauuucuacucuuguagauggaaagagaauuguuuucucc-C3 67% 304 38595-41-62 u*a*a*uuucuacucuuguagauggaaagagaauuguuuuc*u*c* c 58% 305 38595-41-63 u*a*a*uuucuacucuuguagauggaaagagaauuguuuuc*u*c* c 63% 306 38595-41-64 u*a*a*u*u*u*c*u*a*c*u*c*u*u*g*u*a*g*a*uggaaaga gaauuguuuucucc 10% 307 38595-41-65 uaauuucuacucuuguagau*g*g*a*a*a*g*a*g*a*a*u*u*g *u*u*u*u*c*u*c*c 2% 308 38595-41-66 uaauuucuacucuuguagauggaaagagaauuguuuucucc 57% 309 38595-41-67 uaauuucuacucuuguagauggaaagagaauuguuuucucc 51% 310 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38595-41-68 uaauuucuacucuuguagauggaaagagaauuguuuucucc 20% 311 38595-41-69 uaauuucuacucuuguagauggaaagagaauuguuuucucc 19% 312 38595-41-70 uaauuucuacucuuguagauggaaagagaauuguuuucucc 27% 313 38595-41-71 uaauuucuacucuuguagauggaaagagaauuguuuucucc 37% 314 38595-41-72 uaauuucuacucuuguagauggaaagagaauuguuuucucc 65% 315 38595-41-73 uaauuucuacucuuguagauggaaagagaauuguuuucucc 67% 316 38595-41-74 uaauuucuacucuuguagauggaaagagaauuguuuucucc 65% 317 38595-41-75 uaauuucuacucuuguagauggaaagagaauuguuuucucc 57% 318 38595-41-76 uaauuucuacucuuguagauggaaagagaauuguuuucucc 65% 319 38595-41-77 uaauuucuacucuuguagauggaaagagaauuguuuucucc 16% 320 38595-41-78 uaauuucuacucuuguagauggaaagagaauuguuuucucc 49% 321 38595-41-79 uaauuucuacucuuguagauggaaagagaauuguuuucucc 70% 322 38595-41-80 uaauuucuacucuuguagauggaaagagaauuguuuucucc 1% 323 38595-41-81 uaauuucuacucuuguagauggaaagagaauuguuuucucc 13% 324 38595-41-82 uaauuucuacucuuguagauggaaagagaauuguuuucucc 51% 325 38595-41-83 uaauuucuacucuuguagauggaaagagaauuguuuucucc 64% 326 38595-41-84 uaauuucuacucuuguagauggaaagagaauuguuuucucc 69% 327 38595-41-85 uaauuucuacucuuguagauggaaagagaauuguuuucucc 69% 328 38595-41-86 u*a*a*uuucuacucuuguagauggaaagagaauuguuuuc*u*c* c 61% 329 38595-41-87 +taauuucuacucuuguagauggaaagagaauuguuuucu+c+c 60% 330 38595-41-88 uaauuucuacucuuguagauggaaagagaauuguuuucucc 63% 331 38595-41-89 uaauuucuacucuuguagauggaaagagaauuguuuucucc 34% 332 38595-41-90 uaauuucuacucuuguagauggaaagagaauuguuuucucc 65% 333 38595-41-91 uaauuucuacucuuguagauggaaagagaauuguuuucucc 66% 334 38595-41-92 uaauuucuacucuuguagauggaaagagaauuguuuucucc 60% 335 38595-41-93 ZEN-uaauuucuacucuuguagauggaaagagaauuguuuucucc- 61% 336 2024204029   13 Jun 2024 Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: ZEN 38595-41-94 ZEN-uaauuucuacucuuguagauggaaagagaauuguuuucucc-C3 59% 337 38595-41-95 C3-uaauuucuacucuuguagauggaaagagaauuguuuucucc-ZEN 58% 338 38104-41-96 uaauuucuacucuuguagaucuuggguguguuaaaagugac 63% 339 38104-41-97 C3-uaauuucuacucuuguagaucuuggguguguuaaaagugac-C3 63% 340 38104-41-98 uaauuucuacucuuguagaucuuggguguguuaaaagugac 63% 341 38104-41-99 u*a*auuucuacucuuguagaucuuggguguguuaaaagu*g*a*c 67% 342 Oligonucleotide sequences are shown 5’-3’. Lowercase = RNA; Underlined lowercase = 2’-O-methyl RNA; Italics lowercase = 2’-fluoro RNA; +a, +c, +t, +g = LNA; C3 = C3 spacer (propanediol modifier); * = phosphorothioate internucleotide linkage; ZEN -napthyl-azo modifier. The relative functional activity of each species is indicated by the % cleavage in a T7EI heteroduplex assay. The sequence name indicates if the crRNA is a 44mer with a 24 base target domain or a 41mer with a 21 base target domain and the HPRT target site is indicated (38104, 38351, or 38595). EXAMPLE 7

[0117] Use of modified crRNAs with AsCpfl protein delivered as an RNP complex.

[0118] A site in the human HPRT1 gene (38104) was chosen to study the ability to use chemically modified crRNAs with AsCpfl protein to perform genome editing in HEK-293 cells using electroporation to deliver the ribonucleoprotein (RNP) complex into the cells.

[0119] Purified recombinant AsCpfl protein was employed in this example, isolated from E.coli using standard techniques. The amino-acid sequence of the recombinant protein is shown in SEQ ID NO: 12.

[0120] The AsCpfl crRNAs were heated to 95°C for 5 minutes then allowed to cool to room temperature. The crRNAs were mixed with AsCpfl protein at a molar ratio of 2024204029   13 Jun 2024 1.2:1 RNA:protein in phosphate buffered saline (PBS) (202 pmoles RNA with 168 pmoles protein in 6 pL volume, for a single transfection). The RNP complex was allowed to form at room temperature for 15 minutes. HEK293 cells were resuspended following trypsinization and washed in medium and washed a second time in PBS before use. Cells were resuspended in at a final concentration of 3.5 x 105 cells in 20 pL of Nucleofection solution. 20 pL of cell suspension was placed in the V-bottom 96-well plate and 5 pL of the Cpfl RNP complex was added to each well (5 pM final concentration) and 3 pL of Cpfl Electroporation Enhancer Solution was added to each well (Integrated DNA Technologies). 25 pL of the final mixture was transferred to each well of a 96 well Nucleocuvette electroporation module. Cells were electroporated using Amaxa 96 well shuttle protocol, program 96-DS-150. Following electroporation, 75 pL of medium was added to each well and 25 pL of the final cell mixture was transferred to 175 pL of pre-warmed medium in 96 well incubation plates (final volume 200 pL). Cells were incubated at 37°C for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified with KAPA HiFi DNA Polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID NO:394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID NO:395). PCR products were melted and re-annealed in NEB buffer 2 (New England Biolabs) to allow for heteroduplex formation followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. The digested products were visualized on a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of the cut products / (average molar concentration of the cut products + molar concentration of the uncut band) * 100. Results are shown in Table 9 below. AsCpfl crRNAs bearing low or high levels of modification, as shown below, are compatible with delivery via electroporation as an RNP complex to mediate genome editing in mammalian cells. 2024204029   13 Jun 2024

[0121] Table 9: Editing in mammalian cells using chemically-modified crRNAs with recombinant AsCpfl as RNP complexes Seq Name Sequence 5'-3' % Cleavage T7E1 Assay SEQ ID NO: 38104-41 96 uaauuucuacucuuguagaucuuggguguguuaaaagugac 57% 339 38104-41 97 C3-uaauuucuacucuuguagaucuuggguguguuaaaagugac-C3 53% 340 38104-41 98 uaauuucuacucuuguagaucuuggguguguuaaaagugac 42% 341 38104-41 99 u*a*auuucuacucuuguagaucuuggguguguuaaaagu*g*a*c 43% 342 38104-41 101 u*a*auuucuacucuuguagaucuuggguguguuaaaagug*a*c 43% 343 Oligonucleotide sequences are shown 5’-3’. Lowercase = RNA; Under ined = 2’-O- methyl RNA; C3 = C3 spacer (propanediol modifier); * = phosphorothioate internucleotide linkage. The relative functional activity of each species is indicated by the % cleavage in a T7EI heteroduplex assay. The sequence name indicates that the crRNAs are all 41mers with a 21 base target domain. EXAMPLE 8

[0122] Use of modified crRNAs with an AsCpfl expression plasmid in E. coli.

[0123] A site in the human HPRT1 gene (38346) was cloned onto an E. coli plasmid and was used to study the ability to use chemically modified crRNAs to perform sitespecific cleavage in E. coli cells. AsCpfl was expressed from a plasmid. Electroporation was used to deliver both the AsCpfl expression plasmid and chemically-synthesized crRNAs.

[0124] The AsCpfl protein was expressed from a plasmid in this example, using a phage T7 promoter and standard E. coli translation elements. The amino-acid sequence of the expression construct is shown in SEQ ID NO: 16). 2024204029   13 Jun 2024

[0125] The AsCpfl crRNAs were heated to 95°C for 5 minutes then allowed to cool to room temperature. The crRNAs and AsCpfl plasmid were mixed in TE (60 femtomoles AsCpfl plasmid with 400 pmoles RNA in 5 pL volume, for a single transformation), and added directly to 20 pL of competent A. coli cells). A bacterial strain where survival is linked to successful cleavage by Cpfl was made competent by growing cells to mid-log phase, washing 3 times in ice cold 10% glycerol, and final suspension in 1:100th volume 10% glycerol. Electroporations were performed by adding the 25 pL transformation mixture to a pre-chilled 0.1 cm electroporation cuvette and pulsing 1.8 kV exponential decay. Following electroporation, 980 pL of SOB medium was added to the electroporation cuvette with mixing and the resulting cell suspension was transferred to a sterile 15 ml culture tube. Cells were incubated with shaking (250 rpm) at 37°C for 1.5 hours, at which time IPTG was added (1 mM) followed by further shaking incubation at 37°C for 1 hour. Following incubation cells were plated on selective media to assess survival.

[0126] This example demonstrates that chemically-modified synthetic crRNAs can be used with Cpfl for gene editing in bacteria. However, high efficiency is only seen using RNAs that have been more extensively modified with exonuclease-blocking PS internucleotide linkages. The modification patterns that work best in bacterial cells perform poorly in mammalian cells (Table 10).

[0127] Table 10: Chemically-modified crRNAs compatible with Cpfl function in bacteria Seq Name Sequence 5'-3' % Cleavage Human % Cleavage Bacteria SEQ ID NO: 38346-41-1 uaauuucuacucuuguagauacauaaaacucuuu uagguua 21% 0% 344 38346-41-2 u*a*a*uuucuacucuuguagauacauaaaacuc uuuuagguua 17% 0% 345 38346-41-3 u*a*a*u*u*u*cuacucuuguagauacauaaaa cucuuuuagguua 10% 2% 346 38346-41-4 uaauuucuacucuuguagauacauaaaacucuuu u*a*g*g*u*u*a 14% 18% 347 2024204029   13 Jun 2024 38346-41-5 u*a*a*uuucuacucuuguagauacauaaaacuc uuuuagg*u*u*a 8% 5% 348 38346-41-6 u*a*a*uuucuacucuuguagauacauaaaacuc uuuu*a*g*g*u*u*a 5% 40% 349 38346-41-7 u*a*a*u*u*u*cuacucuuguagauacauaaaa cucuuuu*a*g*g*u*u*a 2% 88% 350 38346-41-8 uaauuucuacucuuguagauacauaaaacucuuu uagg*u*u*a 14% 7% 351 38346-41-9 uaauuucuacucuuguagauacauaaaacucuuu u*a*g*g*u*u*a 8% 35% 352 38346-41 10 u*a*a*uuucuacucuuguagauacauaaaacuc uuuuagg*u*u*a 12% 27% 353 38346-41 11 u*a*a*uuucuacucuuguagauacauaaaacuc uuuuag*g*u*u*a 8% 85% 354 38346-41 12 u*a*a*uuucuacucuuguagauacauaaaacuc uuuua*g*g*u*u*a 5% 92% 355 38346-41 13 u*a*a*uuucuacucuuguagauacauaaaacuc uuuu*a*g*g*u*u*a 4% 100% 356 38346-41 14 u*a*a*u*u*u*cuacucuuguagauacauaaaa cucuuuu*a*g*g*u*u*a 1% 90% 357 Oligonucleotide sequences are shown 5’-3’. Lowercase = RNA; Underlined lowercase = 2’-O-methyl RNA; C3 = C3 spacer (propanediol modifier); * = phosphorothioate internucleotide linkage. The relative functional activity in human cells is indicated by the % cleavage in a T7EI heteroduplex assay, and in bacteria is indicated by % survival in a Cpfl reporter strain. The sequence name indicates that the crRNAs are all 41mers with a 21 base target domain. EXAMPLE 9

[0128] DNA and amino acid sequences of wild type Lb Cpfl polypeptide, as encoded in isolated nucleic acid vectors

[0129] The list below shows wild type (WT) Lb Cpfl nucleases expressed as polypeptide fusion proteins as described in the present invention. It will be appreciated by one with skill in the art that many different DNA sequences can encode / express the same amino acid (AA) sequence since in many cases more than one codon can encode for the same amino acid. The DNA sequences shown below only serve as examples, and other DNA sequences that encode the same protein (e.g., same amino acid sequence) are 2024204029   13 Jun 2024 contemplated. It is further appreciated that additional features, elements or tags may be added to said sequences, such as NLS domains and the like.

[0130] Examples are shown for WT LbCpfl showing amino acid and DNA sequences for those proteins as LbCpfl alone and LbCpfl fused to an N-terminal V5-tag, an N-terminal SV40 NLS domain, a C-terminal SV40 NLS domain, and a C-terminal 6xHis-tag. SEQ ID NO :3 LbCpfl Native DNA Sequence ATGAGCAAACTGGAAAAATTTACGAATTGTTATAGCCTGTCCAAGACCCTGCGTTTCAAAGC CATCCCCGTTGGCAAAACCCAGGAGAATATTGATAATAAACGTCTGCTGGTTGAGGATGAAA AAAGAGCAGAAGACTATAAGGGAGTCAAAAAACTGCTGGATCGGTACTACCTGAGCTTTATA AATGACGTGCTGCATAGCATTAAACTGAAAAATCTGAATAACTATATTAGTCTGTTCCGCAA GAAAACCCGAACAGAGAAAGAAAATAAAGAGCTGGAAAACCTGGAGATCAATCTGCGTAAAG AGATCGCAAAAGCTTTTAAAGGAAATGAAGGTTATAAAAGCCTGTTCAAAAAAGACATTATT GAAACCATCCTGCCGGAATTTCTGGATGATAAAGACGAGATAGCGCTCGTGAACAGCTTCAA CGGGTTCACGACCGCCTTCACGGGCTTTTTCGATAACAGGGAAAATATGTTTTCAGAGGAAG C CAAAAGCACC T CGATAGCGTTCCGTTGCAT TAAT GAAAAT T T GACAAGATATAT CAGCAAC ATGGATATTTTC GAGAAAGT T GAT GC GAT C T T T GACAAACAT GAAGT GCAGGAGAT TAAGGA AAAAATTCTGAACAGCGATTATGATGTTGAGGATTTTTTCGAGGGGGAATTTTTTAACTTTG TACTGACACAGGAAGGTATAGATGTGTATAATGCTATTATCGGCGGGTTCGTTACCGAATCC GGCGAGAAAATTAAGGGTCTGAATGAGTACATCAATCTGTATAACCAAAAGACCAAACAGAA ACTGCCAAAATTCAAACCGCTGTACAAGCAAGTCCTGAGCGATCGGGAAAGCTTGAGCTTTT ACGGTGAAGGTTATACCAGCGACGAGGAGGTACTGGAGGTCTTTCGCAATACCCTGAACAAG AACAGC GAAAT TTTCAGCTCCAT TAAAAAGC T GGAGAAAC T GT T TAAGAAT T T T GAC GAGTA CAGCAGCGCAGGTATTTTTGTGAAGAACGGACCTGCCATAAGCACCATTAGCAAGGATATTT TTGGAGAGTGGAATGTTATCCGTGATAAATGGAACGCGGAATATGATGACATACACCTGAAA AAGAAGGCTGTGGTAACTGAGAAATATGAAGACGATCGCCGCAAAAGCTTTAAAAAAATCGG CAGCTTTAGCCTGGAGCAGCTGCAGGAATATGCGGACGCCGACCTGAGCGTGGTCGAGAAAC T GAAGGAAAT TAT TATCCAAAAAGT GGAT GAGAT T TACAAGGTATAT GGTAGCAGCGAAAAA CTGTTTGATGCGGACTTCGTTCTGGAAAAAAGCCTGAAAAAAAATGATGCTGTTGTTGCGAT CATGAAAGACCTGCTCGATAGCGTTAAGAGCTTTGAAAATTACATTAAAGCATTCTTTGGCG AGGGCAAAGAAACAAACAGAGACGAAAGCTTTTATGGCGACTTCGTCCTGGCTTATGACATC CTGTTGAAGGTAGATCATATATATGATGCAATTCGTAATTACGTAACCCAAAAGCCGTACAG CAAAGATAAGTTCAAACTGTATTTCCAGAACCCGCAGTTTATGGGTGGCTGGGACAAAGACA AGGAGACAGACTATCGCGCCACTATTCTGCGTTACGGCAGCAAGTACTATCTCGCCATCATG GACAAAAAATAT GCAAAGT GT C T GCAGAAAAT C GATAAAGAC GAC GT GAAC GGAAAT TAC GA AAAGATTAATTATAAGCTGCTGCCAGGGCCCAACAAGATGTTACCGAAAGTATTTTTTTCCA AAAAATGGATGGCATACTATAACCCGAGCGAGGATATACAGAAGATTTACAAAAATGGGACC TTCAAAAAGGGGGATATGTTCAATCTGAATGACTGCCACAAACTGATCGATTTTTTTAAAGA TAGCATCAGCCGTTATCCTAAATGGTCAAACGCGTATGATTTTAATTTCTCCGAAACGGAGA AATATAAAGACATTGCTGGTTTCTATCGCGAAGTCGAAGAACAGGGTTATAAAGTTAGCTTT GAATCGGCCAGCAAGAAAGAGGTTGATAAACTGGTGGAGGAGGGTAAGCTGTATATGTTTCA GATTTATAACAAAGACTTTAGCGACAAAAGCCACGGTACTCCTAATCTGCATACGATGTACT TTAAACTGCTGTTTGATGAGAATAACCACGGCCAAATCCGTCTCTCCGGTGGAGCAGAACTT TTTATGCGGCGTGCGAGCCTAAAAAAGGAAGAACTGGTGGTGCATCCCGCCAACAGCCCGAT TGCTAACAAAAATCCAGATAATCCTAAGAAGACCACCACACTGTCGTACGATGTCTATAAGG ATAAACGTTTCTCGGAAGACCAGTATGAATTGCATATACCGATAGCAATTAATAAATGCCCA AAAAACAT T T T CAAAAT CAACAC T GAAGT TCGTGTGCTGCT GAAACAT GAT GATAAT CC GTA 2024204029   13 Jun 2024 TGTGATCGGAATTGACCGTGGGGAGAGAAATCTGCTGTATATTGTAGTCGTTGATGGCAAGG GCAACATCGTTGAGCAGTATAGCCTGAATGAAATAATTAATAATTTTAACGGTATACGTATT AAAACCGACTATCATAGCCTGCTGGATAAAAAGGAGAAAGAGCGTTTTGAGGCACGCCAAAA TTGGACGAGCATCGAAAACATCAAGGAACTGAAGGCAGGATATATCAGCCAAGTAGTCCATA AAATCTGTGAACTGGTGGAGAAGTACGACGCTGTCATTGCCCTGGAAGACCTCAATAGCGGC TTTAAAAACAGCCGGGTGAAGGTGGAGAAACAGGTATACCAAAAGTTTGAAAAGATGCTCAT TGATAAGCTGAACTATATGGTTGATAAAAAGAGCAACCCGTGCGCCACTGGCGGTGCACTGA AAGGGTACCAAATTACCAATAAATTTGAAAGCTTTAAAAGCATGAGCACGCAGAATGGGTTT ATTTTTTATATACCAGCATGGCTGACGAGCAAGATTGACCCCAGCACTGGTTTTGTCAATCT GCTGAAAACCAAATACACAAGCATTGCGGATAGCAAAAAATTTATTTCGAGCTTCGACCGTA TTATGTATGTTCCGGAGGAAGATCTGTTTGAATTTGCCCTGGATTATAAAAACTTCAGCCGC ACCGATGCAGATTATATCAAAAAATGGAAGCTGTACAGTTATGGTAATCGTATACGTATCTT CCGTAATCCGAAGAAAAACAATGTGTTCGATTGGGAAGAGGTCTGTCTGACCAGCGCGTATA AAGAACTGTTCAACAAGTACGGAATAAATTATCAGCAAGGTGACATTCGCGCACTGCTGTGT GAACAGTCAGATAAAGCATTTTATAGCAGCTTTATGGCGCTGATGAGCCTGATGCTCCAGAT GCGCAACAGCATAACCGGTCGCACAGATGTTGACTTTCTGATCAGCCCTGTGAAGAATAGCG ACGGCATCTTCTACGATTCCAGGAACTATGAAGCACAGGAAAACGCTATTCTGCCTAAAAAT GCCGATGCCAACGGCGCCTATAATATTGCACGGAAGGTTCTGTGGGCGATTGGACAGTTCAA GAAAGCGGAAGATGAGAAGCTGGATAAGGTAAAAATTGCTATTAGCAATAAGGAATGGCTGG AGTACGCACAGACATCGGTTAAACACGCGGCCGCTTCCCTGCAGGTAATTAAATAA SEQ ID NO:4 LbCpfl Native Protein Sequence MLKNVGIDRLDVEKGRKNMSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRA EDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIA KAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKS TSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLT QEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGE GYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGE WNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKE IIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGK ETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKET DYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKW MAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYK DIAGFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKL LFDENNHGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKR FSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNI VEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKIC ELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGY 2024204029   13 Jun 2024 QITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMY VPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKEL FNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGI FYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYA QTSVKH SEQ ID NO:6 E.coli optimized Lb Cpfl DNA ATGCTGAAAAACGTGGGTATTGATCGTCTGGATGTTGAAAAAGGTCGCAAAAATATGAGCAAACT GGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTGCGTTTTAAAGCAATTCCGGTTGGTA AAACC CAAGAGAACAT T GATAATAAAC GCCTGCTGGTC GAAGAT GAAAAAC GC GC T GAAGAT TAT AAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTCATTAACGATGTGCTGCACAGCAT TAAACTGAAGAACCTGAACAACTATATCAGCCTGTTTCGTAAAAAAACCCGCACCGAAAAAGAAA ACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGAAATCGCCAAAGCGTTTAAAGGTAAC GAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAACCATTCTGCCGGAATTTCTGGATGA TAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTTACCACCGCATTTACCGGCTTTTTTG ATAAT C GC GAAAACAT GT T CAGC GAAGAAGCAAAAAGCACCAGCAT T GCAT T T C GC T GCAT TAAT GAAAAT C T GAC CC GC TACAT TAGCAACAT GGATAT C T T T GAAAAAGT GGAC GC GAT C T T C GATAA ACACGAAGTGCAAGAGATCAAAGAGAAAATCCTGAACAGCGATTATGACGTCGAAGATTTTTTTG AAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTATCGACGTTTATAACGCAATTATTGGT GGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGAATGAATATATCAACCTGTATAACCA GAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTATAAACAGGTTCTGAGCGATCGTGAAA GCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGAGGTTCTGGAAGTTTTTCGTAACACC C T GAATAAAAACAGC GAGAT C T T TAGCAGCAT CAAAAAGC T T GAGAAAC T GT T CAAAAAC T T T GA T GAGTATAGCAGC GCAGGCAT CT T T GT TAAAAAT GGT CC GGCAAT TAGCACCATCAGCAAAGATA TTTTTGGCGAATGGAATGTGATCCGCGATAAATGGAATGCCGAATATGATGATATCCACCTGAAA AAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTCGTAAAAGCTTCAAGAAAATTGGTAG CTTTAGCCT GGAACAGC T GCAAGAATAT GCAGAT GCAGAT C T GAGC GT T GT GGAAAAAC T GAAAG AAAT CAT CATT CAGAAGGT GGAC GAGAT C TATAAAGT T TAT GGTAGCAGC GAAAAAC T GT T C GAT GCAGATTTTGTTC T GGAAAAAAGCC T GAAAAAGAAT GAT GCCGTTGTGGCCATTAT GAAAGAT C T GCTGGATAGCGTTAAGAGCTTCGAGAATTACATCAAAGCCTTTTTTGGTGAGGGCAAAGAAACCA ATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTATGATATTCTGCTGAAAGTGGACCAT ATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGTATAGCAAAGACAAGTTCAAACTGTA CTTTCAGAACCCGCAGTTTATGGGTGGTTGGGATAAAGATAAAGAAACCGATTATCGTGCCACCA TCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGGACAAAAAATACGCAAAATGCCTGCAG AAAATCGACAAAGATGATGTGAATGGCAACTATGAAAAAATCAACTACAAACTGCTGCCTGGTCC GAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGGATGGCCTATTATAACCCGAGCGAGG ATAT T CAAAAGAT C TACAAAAAT GGCACC T T TAAAAAGGGC GACAT GT T CAAT C T GAAC GAT T GC CACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTTATCCGAAATGGTCCAATGCCTATGA T T T TAAC T T TAGC GAAACC GAAAAATACAAAGACAT TGCCGGTTTTTAT C GC GAAGT GGAAGAAC AGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGAGGTTGATAAGCTGGTTGAAGAGGGC AAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCGACAAAAGCCATGGCACCCCGAATCT GCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAACCATGGTCAGATTCGTCTGAGCGGTG GTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGAAGAACTGGTTGTTCATCCGGCAAAT AGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAACCACGACACTGAGCTATGATGTGTA 2024204029   13 Jun 2024 TAAAGACAAAC GT T T TAGC GAGGAT CAGTAT GAAC TGCATATCCCGATTGC CAT CAATAAAT GCC CGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCTGCTGAAACATGATGATAATCCGTAT GTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATATTGTTGTTGTTGATGGTAAAGGCAA CATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAACTTTAACGGCATCCGCATCAAAACCG AC TAT CATAGC C T GC T GGACAAGAAAGAAAAAGAAC GT T T T GAAGCAC GT CAGAAC T GGAC CAGT ATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCCAGGTGGTTCATAAAATCTGTGAGCT GGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTGAATAGCGGTTTCAAAAATAGCCGTG TGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTGAACTACATG GTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCACTGAAAGGTTATCAGATTACCAACAA ATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATCTTCTATATTCCGGCATGGCTGA CCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCTGAAAACAAAATATACCTCCATTGCC GACAGCAAGAAGTTTATTAGCAGCTTTGATCGCATTATGTATGTTCCGGAAGAGGACCTGTTTGA ATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCCGACTACATCAAAAAATGGAAACTGT ACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAAGAAAAACAATGTGTTCGATTGGGAA GAAGT T T GT C T GACCAGC GCATATAAAGAAC T T T T CAACAAATAC GGCAT CAAC TAT CAGCAGGG TGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCGTTTTATAGCAGTTTTATGGCACTGA TGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCGCACCGATGTGGATTTTCTGATTAGT CCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCAATTACGAAGCACAAGAAAATGCAAT TCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATTGCACGTAAAGTTCTGTGGGCAATTG GCCAGTTTAAGAAAGCAGAAGATGAGAAGCTGGACAAAGTGAAAATTGCGATCAGCAATAAAGAG T GGCT GGAATACGCACAGACCAGCGTTAAACAT TGA SEQ ID NO:7 E.coli optimized Lb Cpfl AA MLKNVGIDRLDVEKGRKNMSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDY KGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGN EGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCIN ENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIG GFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNT LNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFD ADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDH IYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQ KIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDC HKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEG KLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPAN SPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPY VIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTS IENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYM VDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIA DSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWE EVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLIS PVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKE WLEYAQTSVKH SEQ ID NO :9 Hs optimized Lb Cpfl DNA 2024204029   13 Jun 2024 ATGCTGAAGAACGTGGGCATCGACCGGCTGGACGTGGAAAAGGGCAGAAAGAACATGAGCAAGCT CGAGAAGTTCACCAACTGCTACAGCCTGAGCAAGACCCTGCGGTTCAAGGCCATTCCTGTGGGCA AGACCCAAGAGAACATCGACAACAAGCGGCTGCTGGTGGAAGATGAGAAGAGAGCCGAGGACTAC AAGGGCGTGAAGAAGCTGCTGGACCGGTACTACCTGAGCTTCATCAACGACGTGCTGCACAGCAT CAAGCTCAAGAACCTGAACAACTACATCAGCCTGTTCCGGAAGAAAACCCGGACCGAGAAAGAGA ACAAAGAGCTGGAAAACCTCGAGATCAACCTGCGGAAAGAGATCGCCAAGGCCTTCAAGGGCAAC GAGGGCTACAAGAGCCTGTTCAAGAAGGACATCATCGAGACAATCCTGCCTGAGTTCCTGGACGA CAAGGACGAGATCGCCCTGGTCAACAGCTTCAACGGCTTCACAACCGCCTTCACCGGCTTTTTCG ACAACCGCGAGAATATGTTCAGCGAGGAAGCCAAGAGCACCTCTATCGCCTTCCGGTGCATCAAC GAGAATCTGACCCGGTACATCAGCAACATGGATATCTTCGAGAAGGTGGACGCCATCTTCGACAA GCACGAGGTGCAAGAGATCAAAGAAAAGATCCTGAACAGCGACTACGACGTCGAGGACTTCTTCG AGGGCGAGTTCTTCAACTTCGTGCTGACACAAGAGGGCATCGATGTGTACAACGCCATCATCGGC GGCTTCGTGACAGAGAGCGGCGAGAAGATCAAGGGCCTGAACGAGTACATCAACCTCTACAACCA GAAAACGAAGCAGAAGCTGCCCAAGTTCAAGCCCCTGTACAAACAGGTGCTGAGCGACAGAGAGA GCCTGTCCTTTTACGGCGAGGGCTATACCAGCGACGAAGAGGTGCTGGAAGTGTTCAGAAACACC CTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAGCTCGAAAAGCTGTTTAAGAACTTCGA CGAGTACAGCAGCGCCGGCATCTTCGTGAAGAATGGCCCTGCCATCAGCACCATCTCCAAGGACA TCTTCGGCGAGTGGAACGTGATCCGGGACAAGTGGAACGCCGAGTACGACGACATCCACCTGAAG AAAAAGGCCGTGGTCACCGAGAAGTACGAGGACGACAGAAGAAAGAGCTTCAAGAAGATCGGCAG CTTCAGCCTGGAACAGCTGCAAGAGTACGCCGACGCCGATCTGAGCGTGGTGGAAAAGCTGAAAG AGATTATCATCCAGAAGGTCGACGAGATCTACAAGGTGTACGGCAGCAGCGAGAAGCTGTTCGAC GCCGACTTTGTGCTGGAAAAGAGCCTCAAAAAGAACGACGCCGTGGTGGCCATCATGAAGGACCT GCTGGATAGCGTGAAGTCCTTCGAGAACTATATTAAGGCCTTCTTTGGCGAGGGCAAAGAGACAA ACCGGGACGAGAGCTTCTACGGCGATTTCGTGCTGGCCTACGACATCCTGCTGAAAGTGGACCAC ATCTACGACGCCATCCGGAACTACGTGACCCAGAAGCCTTACAGCAAGGACAAGTTTAAGCTGTA CTTCCAGAATCCGCAGTTCATGGGCGGCTGGGACAAAGACAAAGAAACCGACTACCGGGCCACCA TCCTGAGATACGGCTCCAAGTACTATCTGGCCATTATGGACAAGAAATACGCCAAGTGCCTGCAG AAGATCGATAAGGACGACGTGAACGGCAACTACGAGAAGATTAACTACAAGCTGCTGCCCGGACC TAACAAGATGCTGCCTAAGGTGTTCTTTAGCAAGAAATGGATGGCCTACTACAACCCCAGCGAGG ATATCCAGAAAATCTACAAGAACGGCACCTTCAAGAAAGGCGACATGTTCAACCTGAACGACTGC CACAAGCTGATCGATTTCTTCAAGGACAGCATCAGCAGATACCCCAAGTGGTCCAACGCCTACGA CTTCAATTTCAGCGAGACAGAGAAGTATAAGGATATCGCCGGGTTCTACCGCGAGGTGGAAGAAC AGGGCTATAAGGTGTCCTTTGAGAGCGCCAGCAAGAAAGAGGTGGACAAGCTGGTCGAAGAGGGC AAGCTGTACATGTTCCAGATCTATAACAAGGACTTCTCCGACAAGAGCCACGGCACCCCTAACCT GCACACCATGTACTTTAAGCTGCTGTTCGATGAGAACAACCACGGCCAGATCAGACTGTCTGGCG GAGCCGAGCTGTTTATGAGAAGGGCCAGCCTGAAAAAAGAGGAACTGGTCGTTCACCCCGCCAAC TCTCCAATCGCCAACAAGAACCCCGACAATCCCAAGAAAACCACCACACTGAGCTACGACGTGTA CAAGGATAAGCGGTTCTCCGAGGACCAGTACGAGCTGCACATCCCTATCGCCATCAACAAGTGCC C CAAGAATAT C T T CAAGAT CAACAC C GAAGT GCGGGTGCTGCT GAAGCAC GAC GACAAC CC T TAC GTGATCGGCATCGACAGAGGCGAGCGGAACCTGCTGTATATCGTGGTGGTGGACGGCAAGGGCAA TATCGTGGAACAGTACTCCCTGAATGAGATCATCAACAACTTCAATGGCATCCGGATCAAGACGG AC TAC CACAGC C T GC T GGACAAAAAAGAGAAAGAAC GC T T C GAGGCC C GGCAGAAC T GGAC CAGC ATCGAGAACATCAAAGAACTGAAGGCCGGCTACATCTCCCAGGTGGTGCACAAGATCTGCGAGCT GGTTGAGAAGTATGACGCCGTGATTGCCCTGGAAGATCTGAATAGCGGCTTTAAGAACAGCCGCG TGAAGGTCGAGAAACAGGTGTACCAGAAATTCGAGAAGATGCTGATCGACAAGCTGAACTACATG GTCGACAAGAAGTCTAACCCCTGCGCCACAGGCGGAGCCCTGAAGGGATATCAGATCACCAACAA GTTCGAGTCCTTCAAGAGCATGAGCACCCAGAATGGCTTCATCTTCTACATCCCCGCCTGGCTGA 2024204029   13 Jun 2024 CCAGCAAGATCGATCCTAGCACCGGATTCGTGAACCTGCTCAAGACCAAGTACACCAGCATTGCC GACAGCAAGAAGTTCATCTCCAGCTTCGACCGGATTATGTACGTGCCCGAAGAGGACCTGTTCGA ATTCGCCCTGGATTACAAGAACTTCAGCCGGACCGATGCCGACTATATCAAGAAGTGGAAGCTGT ATAGCTACGGCAACCGCATCCGCATCTTCAGAAACCCGAAGAAAAACAACGTGTTCGACTGGGAA GAAGTGTGCCTGACCAGCGCCTACAAAGAACTCTTCAACAAATACGGCATCAACTACCAGCAGGG CGACATCAGAGCCCTGCTGTGCGAGCAGAGCGACAAGGCCTTTTACAGCTCCTTCATGGCCCTGA TGTCCCTGATGCTGCAGATGCGGAATAGCATCACCGGCAGGACCGACGTGGACTTCCTGATCAGC CCTGTGAAGAATTCCGACGGGATCTTCTACGACAGCAGAAACTACGAGGCTCAAGAGAACGCCAT CCTGCCTAAGAACGCCGATGCCAACGGCGCCTATAATATCGCCAGAAAGGTGCTGTGGGCCATCG GCCAGTTTAAGAAGGCCGAGGACGAGAAACTGGACAAAGTGAAGATCGCCATCTCTAACAAAGAG TGGCTGGAATACGCCCAGACCAGCGTGAAACAC SEQ ID NO:10 Hs optimized Lb Cpfl AA MLKNVGIDRLDVEKGRKNMSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDY KGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGN EGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCIN ENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIG GFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNT LNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFD ADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDH IYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQ KIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDC HKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEG KLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPAN SPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPY VIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTS IENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYM VDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIA DSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWE EVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLIS PVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKE WLEYAQTSVKH SEQ ID NO:13 E.coli optimized Lb Cpfl with flanking NLS's, V5 tag and 6x His - DNA ATGGGTAAACCGATTCCGAATCCGCTGCTGGGTCTGGATAGCACCGCACCGAAAAAAAAACGTAA AGTTGGTATTCATGGTGTTCCGGCAGCACTGAAAAACGTGGGTATTGATCGTCTGGATGTTGAAA AAGGTCGCAAAAATATGAGCAAACTGGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTG CGTTTTAAAGCAATTCCGGTTGGTAAAACCCAAGAGAACATTGATAATAAACGCCTGCTGGTCGA AGATGAAAAACGCGCTGAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCT T CAT TAAC GAT GT GC T GCACAGCAT TAAAC T GAAGAACC T GAACAAC TATATCAGCCTGTTTCGT AAAAAAACCCGCACCGAAAAAGAAAACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGA AATCGCCAAAGCGTTTAAAGGTAACGAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAA CCATTCTGCCGGAATTTCTGGATGATAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTT 2024204029   13 Jun 2024 ACCACCGCATTTACCGGCTTTTTTGATAATCGCGAAAACATGTTCAGCGAAGAAGCAAAAAGCAC CAGCATTGCATTTCGCTGCATTAATGAAAATCTGACCCGCTACATTAGCAACATGGATATCTTTG AAAAAGT GGAC GC GAT C T T C GATAAACAC GAAGT GCAAGAGAT CAAAGAGAAAAT CC T GAACAGC GATTATGACGTCGAAGATTTTTTTGAAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTAT CGACGTTTATAACGCAATTATTGGTGGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGA ATGAATATATCAACCTGTATAACCAGAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTAT AAACAGGTTCTGAGCGATCGTGAAAGCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGA GGTTCTGGAAGTTTTTCGTAACACCCTGAATAAAAACAGCGAGATCTTTAGCAGCATCAAAAAGC TTGAGAAACTGTTCAAAAACTTTGATGAGTATAGCAGCGCAGGCATCTTTGTTAAAAATGGTCCG GCAAT TAGCAC CAT CAGCAAAGATAT T T T T GGC GAAT GGAAT GT GAT CC GC GATAAAT GGAAT GC CGAATATGATGATATCCACCTGAAAAAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTC GTAAAAGCT TCAAGAAAAT T GGTAGCT T TAGCC T GGAACAGCT GCAAGAATAT GCAGAT GCAGAT CTGAGCGTTGTGGAAAAACTGAAAGAAATCATCATTCAGAAGGTGGACGAGATCTATAAAGTTTA TGGTAGCAGCGAAAAACTGTTCGATGCAGATTTTGTTCTGGAAAAAAGCCTGAAAAAGAATGATG CCGTTGTGGCCATTATGAAAGATCTGCTGGATAGCGTTAAGAGCTTCGAGAATTACATCAAAGCC TTTTTTGGTGAGGGCAAAGAAACCAATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTA TGATATTCTGCTGAAAGTGGACCATATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGT ATAGCAAAGACAAGTTCAAACTGTACTTTCAGAACCCGCAGTTTATGGGTGGTTGGGATAAAGAT AAAGAAACCGATTATCGTGCCACCATCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGGA CAAAAAATACGCAAAAT GCCT GCAGAAAATC GACAAAGAT GAT GT GAAT GGCAAC TAT GAAAAAA TCAACTACAAACTGCTGCCTGGTCCGAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGG ATGGCCTATTATAACCCGAGCGAGGATATTCAAAAGATCTACAAAAATGGCACCTTTAAAAAGGG CGACATGTTCAATCTGAACGATTGCCACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTT ATCCGAAATGGTCCAATGCCTATGATTTTAACTTTAGCGAAACCGAAAAATACAAAGACATTGCC GGTTTTTATCGCGAAGTGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGA GGTTGATAAGCTGGTTGAAGAGGGCAAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCG ACAAAAGCCATGGCACCCCGAATCTGCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAAC CATGGTCAGATTCGTCTGAGCGGTGGTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGA AGAACTGGTTGTTCATCCGGCAAATAGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAA CCACGACACTGAGCTATGATGTGTATAAAGACAAACGTTTTAGCGAGGATCAGTATGAACTGCAT ATCCCGATTGCCATCAATAAATGCCCGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCT GCTGAAACATGATGATAATCCGTATGTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATA TTGTTGTTGTTGATGGTAAAGGCAACATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAAC TTTAACGGCATCCGCATCAAAACCGACTATCATAGCCTGCTGGACAAGAAAGAAAAAGAACGTTT TGAAGCACGTCAGAACTGGACCAGTATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCC AGGTGGTTCATAAAATCTGTGAGCTGGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTG AATAGCGGTTTCAAAAATAGCCGTGTGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAAT GCTGATCGACAAACTGAACTACATGGTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCAC T GAAAGGT TAT CAGAT TAC CAACAAAT T T GAAAGC T T TAAAAGCAT GAGCACC CAGAAC GGC T T T ATCTTCTATATTCCGGCATGGCTGACCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCT GAAAACAAAATATACCT CCAT T GCC GACAGCAAGAAGT T TAT TAGCAGC T T T GAT CGCAT TAT GT ATGTTCCGGAAGAGGACCTGTTTGAATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCC GACTACATCAAAAAATGGAAACTGTACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAA GAAAAACAATGTGTTCGATTGGGAAGAAGTTTGTCTGACCAGCGCATATAAAGAACTTTTCAACA AATACGGCATCAACTATCAGCAGGGTGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCG TTTTATAGCAGTTTTATGGCACTGATGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCG CACCGATGTGGATTTTCTGATTAGTCCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCA 2024204029   13 Jun 2024 ATTACGAAGCACAAGAAAATGCAATTCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATT GCACGTAAAGTTCTGTGGGCAATTGGCCAGTTTAAGAAAGCAGAAGATGAGAAGCTGGACAAAGT GAAAATTGCGATCAGCAATAAAGAGTGGCTGGAATACGCACAGACCAGCGTTAAACATCCGAAAA AAAAAC GCAAAGT GC T C GAGCAC CACCAC CACCAC CAC T GA SEQ ID NO:14 Amino acid sequence for LbCpfl fusion, with 5'-and 3'-flanking NLS's, 5'-V5 tag and 3'-6x His, used for gene editing in both E. coll and human cells MGKPIPNPLLGLDSTAPKKKRKVGIHGVPAALKNVGIDRLDVEKGRKNMSKLEKFTNCYSLSKTL RFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFR KKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGF TTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNS DYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLY KQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGP AISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADAD LSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKA FFGEGKETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKD KETDYRATILRYGSKYYLAIMDKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKW MAYYNPSEDIQKIYKNGTFKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIA GFYREVEEQGYKVSFESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENN HGQIRLSGGAELFMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELH IPIAINKCPKNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINN FNGIRIKTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDL NSGFKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDA DYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKA FYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNI ARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHPKKKRKVLEHHHHHH SEQ ID NO:17 Hs optimized Lb Cpfl with flanking NLS's, V5 tag and 6x His - DNA ATGGGCAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACAGCACAGCCCCTAAGAAAAAGCGGAA AGTGGGCATCCATGGCGTGCCAGCCGCTCTGAAGAATGTGGGCATCGACAGACTGGACGTGGAAA AGGGCAGAAAGAACATGAGCAAGCTCGAGAAGTTCACCAACTGCTACAGCCTGAGCAAGACCCTG CGGTTCAAGGCCATTCCTGTGGGCAAGACCCAAGAGAACATCGACAACAAGCGGCTGCTGGTGGA AGATGAGAAGAGAGCCGAGGACTACAAGGGCGTGAAGAAGCTGCTGGACCGGTACTACCTGAGCT T CAT CAAC GAC GT GC T GCACAGCAT CAAGC T GAAGAACC T GAACAAC TACAT CAGCC T GT T CC GG AAGAAAACCCGGACCGAGAAAGAGAACAAAGAGCTGGAAAACCTCGAGATCAACCTGCGGAAAGA GATCGCCAAGGCCTTCAAGGGCAACGAGGGCTACAAGAGCCTGTTCAAGAAGGACATCATCGAGA CAATCCTGCCTGAGTTCCTGGACGACAAGGACGAGATCGCCCTGGTCAACAGCTTCAACGGCTTC ACAACCGCCTTCACCGGCTTTTTCGACAACCGCGAGAATATGTTCAGCGAGGAAGCCAAGAGCAC CTCTATCGCCTTCCGGTGCATCAACGAGAATCTGACCCGGTACATCAGCAACATGGATATCTTCG AGAAGGT GGAC GC CAT C T T C GACAAGCAC GAGGT GCAAGAGAT CAAAGAAAAGAT CC T GAACAGC GACTACGACGTCGAGGACTTCTTCGAGGGCGAGTTCTTCAACTTCGTGCTGACACAAGAGGGCAT CGATGTGTACAACGCCATCATCGGCGGCTTCGTGACAGAGAGCGGCGAGAAGATCAAGGGCCTGA 2024204029   13 Jun 2024 ACGAGTACATCAACCTCTACAACCAGAAAACGAAGCAGAAGCTGCCCAAGTTCAAGCCCCTGTAC AAACAGGTGCTGAGCGACAGAGAGAGCCTGTCCTTTTACGGCGAGGGCTATACCAGCGACGAAGA GGTGCTGGAAGTGTTCAGAAACACCCTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAGC TCGAAAAGCTGTTTAAGAACTTCGACGAGTACAGCAGCGCCGGCATCTTCGTGAAGAATGGCCCT GCCATCAGCACCATCTCCAAGGACATCTTCGGCGAGTGGAACGTGATCCGGGACAAGTGGAACGC CGAGTACGACGACATCCACCTGAAGAAAAAGGCCGTGGTCACCGAGAAGTACGAGGACGACAGAA GAAAGAGCTTCAAGAAGATCGGCAGCTTCAGCCTGGAACAGCTGCAAGAGTACGCCGACGCCGAT CTGAGCGTGGTGGAAAAGCTGAAAGAGATTATCATCCAGAAGGTCGACGAGATCTACAAGGTGTA CGGCAGCAGCGAGAAGCTGTTCGACGCCGACTTTGTGCTGGAAAAGAGCCTCAAAAAGAACGACG CCGTGGTGGCCATCATGAAGGACCTGCTGGATAGCGTGAAGTCCTTCGAGAACTATATTAAGGCC TTCTTTGGCGAGGGCAAAGAGACAAACCGGGACGAGAGCTTCTACGGCGATTTCGTGCTGGCCTA CGACATCCTGCTGAAAGTGGACCACATCTACGACGCCATCCGGAACTACGTGACCCAGAAGCCTT ACAGCAAGGACAAGTTTAAGCTGTACTTCCAGAATCCGCAGTTCATGGGCGGCTGGGACAAAGAC AAAGAAACCGACTACCGGGCCACCATCCTGAGATACGGCTCCAAGTACTATCTGGCCATTATGGA CAAGAAATACGCCAAGTGCCTGCAGAAGATCGATAAGGACGACGTGAACGGCAACTACGAGAAGA TTAACTACAAGCTGCTGCCCGGACCTAACAAGATGCTGCCTAAGGTGTTCTTTAGCAAGAAATGG ATGGCCTACTACAACCCCAGCGAGGATATCCAGAAAATCTACAAGAACGGCACCTTCAAGAAAGG CGACATGTTCAACCTGAACGACTGCCACAAGCTGATCGATTTCTTCAAGGACAGCATCAGCAGAT ACCCCAAGTGGTCCAACGCCTACGACTTCAATTTCAGCGAGACAGAGAAGTATAAGGATATCGCC GGGTTCTACCGCGAGGTGGAAGAACAGGGCTATAAGGTGTCCTTTGAGAGCGCCAGCAAGAAAGA GGTGGACAAGCTGGTCGAAGAGGGCAAGCTGTACATGTTCCAGATCTATAACAAGGACTTCTCCG ACAAGAGCCACGGCACCCCTAACCTGCACACCATGTACTTTAAGCTGCTGTTCGATGAGAACAAC CACGGCCAGATCAGACTGTCTGGCGGAGCCGAGCTGTTTATGAGAAGGGCCAGCCTGAAAAAAGA GGAACTGGTCGTTCACCCCGCCAACTCTCCAATCGCCAACAAGAACCCCGACAATCCCAAGAAAA CCACCACACTGAGCTACGACGTGTACAAGGATAAGCGGTTCTCCGAGGACCAGTACGAGCTGCAC ATCCCTATCGCCATCAACAAGTGCCCCAAGAATATCTTCAAGATCAACACCGAAGTGCGGGTGCT GCTGAAGCACGACGACAACCCTTACGTGATCGGCATCGATCGGGGCGAGAGAAACCTGCTGTATA TCGTGGTGGTGGACGGCAAGGGCAATATCGTGGAACAGTACTCCCTGAATGAGATCATCAACAAC T T CAAT GGCAT CC GGAT CAAGAC GGAC TACCACAGCC T GC T GGACAAAAAAGAGAAAGAAC GC T T CGAGGCCAGGCAGAACTGGACCAGCATCGAGAACATCAAAGAACTGAAGGCCGGCTACATCTCCC AGGTGGTGCACAAGATCTGCGAGCTGGTTGAGAAGTATGACGCCGTGATTGCCCTGGAAGATCTG AATAGCGGCTTTAAGAACAGCCGCGTGAAGGTCGAGAAACAGGTGTACCAGAAATTCGAGAAGAT GCTGATCGACAAGCTGAACTACATGGTCGACAAGAAGTCTAACCCCTGCGCCACAGGCGGAGCCC TGAAGGGATATCAGATCACCAACAAGTTCGAGTCCTTCAAGAGCATGAGCACCCAGAATGGCTTC ATCTTCTACATCCCCGCCTGGCTGACCAGCAAGATCGATCCTAGCACCGGATTCGTGAACCTGCT CAAGACCAAGTACACCAGCATTGCCGACAGCAAGAAGTTCATCTCCAGCTTCGACCGGATTATGT ACGTGCCCGAAGAGGACCTGTTCGAATTCGCCCTGGATTACAAGAACTTCAGCCGGACCGATGCC GACTATATCAAGAAGTGGAAGCTGTATAGCTACGGCAACCGCATCCGCATCTTCAGAAACCCGAA GAAAAACAACGTGTTCGACTGGGAAGAAGTGTGCCTGACCAGCGCCTACAAAGAACTCTTCAACA AATACGGCATCAACTACCAGCAGGGCGACATCAGAGCCCTGCTGTGCGAGCAGAGCGACAAGGCC TTTTACAGCTCCTTCATGGCCCTGATGAGCCTGATGCTGCAGATGCGGAATAGCATCACCGGCAG AACCGACGTGGACTTCCTGATCAGCCCCGTGAAAAACTCCGACGGCATCTTTTACGACAGCCGGA ATTACGAGGCTCAAGAGAACGCCATCCTGCCTAAGAACGCCGATGCCAACGGCGCCTATAATATC GCCAGAAAGGTGCTGTGGGCCATCGGCCAGTTTAAGAAGGCCGAGGACGAGAAACTGGACAAAGT GAAGATCGCCATCTCTAACAAAGAGTGGCTGGAATACGCCCAGACCAGCGTGAAGCACCCCAAAA AGAAACGGAAAGTGCTGGAACACCACCACCATCACCAC 2024204029   13 Jun 2024 SEQ ID NO:20 E.coli optimized Lb Cpfl with OpT NLS and 6x His -AA MGDPLKNVGIDRLDVEKGRKNMSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRA EDYKGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAF KGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFR CINENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNA IIGGFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVF RNTLNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDI HLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEK LFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLK VDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAK CLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNL NDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLV EEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVH PANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDD NPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQN WTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKL NYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYT SIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVF DWEEVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDF LISPVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAIS NKEWLEYAQTSVKHGRSSDDEATADSQHAAPPKKKRKVLEHHHHHH SEQ ID NO:23 E.coli optimized Lb Cpfl with OpT NLS and 6x His -DNA ATGGGGGATCCACTGAAAAACGTGGGTATTGATCGTCTGGATGTTGAAAAAGGTCGCAAAAATAT GAGCAAACTGGAAAAGTTCACCAACTGTTATAGCCTGAGCAAAACCCTGCGTTTTAAAGCAATTC CGGTTGGTAAAACCCAAGAGAACATTGATAATAAACGCCTGCTGGTCGAAGATGAAAAACGCGCT GAAGATTATAAAGGCGTGAAAAAACTGCTGGATCGCTATTATCTGAGCTTCATTAACGATGTGCT GCACAGCATTAAACTGAAGAACCTGAACAACTATATCAGCCTGTTTCGTAAAAAAACCCGCACCG AAAAAGAAAACAAAGAGCTGGAAAACCTGGAAATCAATCTGCGTAAAGAAATCGCCAAAGCGTTT AAAGGTAACGAGGGTTATAAAAGCCTGTTCAAGAAAGACATCATCGAAACCATTCTGCCGGAATT TCTGGATGATAAAGATGAAATTGCCCTGGTGAATAGCTTTAATGGCTTTACCACCGCATTTACCG GCTTTTTTGATAATCGCGAAAACATGTTCAGCGAAGAAGCAAAAAGCACCAGCATTGCATTTCGC TGCATTAATGAAAATCTGACCCGCTACATTAGCAACATGGATATCTTTGAAAAAGTGGACGCGAT C T T CGATAAACAC GAAGT GCAAGAGAT CAAAGAGAAAAT CC T GAACAGC GAT TAT GACGTC GAAG ATTTTTTTGAAGGCGAGTTCTTTAACTTCGTTCTGACCCAAGAAGGTATCGACGTTTATAACGCA ATTATTGGTGGTTTTGTTACCGAAAGCGGTGAGAAAATCAAAGGCCTGAATGAATATATCAACCT GTATAACCAGAAAACCAAACAGAAACTGCCGAAATTCAAACCGCTGTATAAACAGGTTCTGAGCG ATCGTGAAAGCCTGAGCTTTTATGGTGAAGGTTATACCAGTGATGAAGAGGTTCTGGAAGTTTTT CGTAACACCCTGAATAAAAACAGCGAGATCTTTAGCAGCATCAAAAAGCTTGAGAAACTGTTCAA AAACTTTGATGAGTATAGCAGCGCAGGCATCTTTGTTAAAAATGGTCCGGCAATTAGCACCATCA GCAAAGATAT T T T T GGC GAAT GGAAT GT GAT CC GC GATAAAT GGAAT GC C GAATAT GAT GATAT C CACCTGAAAAAAAAGGCCGTGGTGACCGAGAAATATGAAGATGATCGTCGTAAAAGCTTCAAGAA AATTGGTAGCTTTAGCCTGGAACAGCTGCAAGAATATGCAGATGCAGATCTGAGCGTTGTGGAAA 2024204029   13 Jun 2024 AACTGAAAGAAATCATCATTCAGAAGGTGGACGAGATCTATAAAGTTTATGGTAGCAGCGAAAAA CTGTTCGATGCAGATTTTGTTCTGGAAAAAAGCCTGAAAAAGAATGATGCCGTTGTGGCCATTAT GAAAGAT C T GC T GGATAGC GT TAAGAGC T T C GAGAAT TACAT CAAAGCC T T T T T TGGT GAGGGCA AAGAAACCAATCGTGATGAAAGTTTCTATGGCGATTTTGTGCTGGCCTATGATATTCTGCTGAAA GTGGACCATATTTATGATGCCATTCGCAATTATGTTACCCAGAAACCGTATAGCAAAGACAAGTT CAAACTGTACTTTCAGAACCCGCAGTTTATGGGTGGTTGGGATAAAGATAAAGAAACCGATTATC GTGCCACCATCCTGCGTTATGGTAGTAAATACTATCTGGCCATCATGGACAAAAAATACGCAAAA TGCCTGCAGAAAATCGACAAAGATGATGTGAATGGCAACTATGAAAAAATCAACTACAAACTGCT GCCTGGTCCGAATAAAATGCTGCCGAAAGTGTTCTTTAGCAAGAAATGGATGGCCTATTATAACC CGAGCGAGGATATTCAAAAGATCTACAAAAATGGCACCTTTAAAAAGGGCGACATGTTCAATCTG AACGATTGCCACAAACTGATCGATTTCTTCAAAGATTCAATTTCGCGTTATCCGAAATGGTCCAA TGCCTATGATTTTAACTTTAGCGAAACCGAAAAATACAAAGACATTGCCGGTTTTTATCGCGAAG TGGAAGAACAGGGCTATAAAGTGAGCTTTGAAAGCGCAAGCAAAAAAGAGGTTGATAAGCTGGTT GAAGAGGGCAAACTGTATATGTTCCAGATTTACAACAAAGATTTTAGCGACAAAAGCCATGGCAC CCCGAATCTGCATACCATGTACTTTAAACTGCTGTTCGACGAAAATAACCATGGTCAGATTCGTC TGAGCGGTGGTGCCGAACTGTTTATGCGTCGTGCAAGTCTGAAAAAAGAAGAACTGGTTGTTCAT CCGGCAAATAGCCCGATTGCAAACAAAAATCCGGACAATCCGAAAAAAACCACGACACTGAGCTA TGATGTGTATAAAGACAAACGTTTTAGCGAGGATCAGTATGAACTGCATATCCCGATTGCCATCA ATAAATGCCCGAAAAACATCTTTAAGATCAACACCGAAGTTCGCGTGCTGCTGAAACATGATGAT AATCCGTATGTGATTGGCATTGATCGTGGTGAACGTAACCTGCTGTATATTGTTGTTGTTGATGG TAAAGGCAACATCGTGGAACAGTATAGTCTGAACGAAATTATCAACAACTTTAACGGCATCCGCA T CAAAAC C GAC TATCATAGCCTGCT GGACAAGAAAGAAAAAGAAC GT T T T GAAGCAC GT CAGAAC TGGACCAGTATTGAAAACATCAAAGAACTGAAAGCCGGTTATATTAGCCAGGTGGTTCATAAAAT CTGTGAGCTGGTAGAAAAATACGATGCAGTTATTGCACTGGAAGATCTGAATAGCGGTTTCAAAA ATAGCCGTGTGAAAGTCGAAAAACAGGTGTATCAGAAATTCGAGAAAATGCTGATCGACAAACTG AACTACATGGTCGACAAAAAAAGCAATCCGTGTGCAACCGGTGGTGCACTGAAAGGTTATCAGAT TACCAACAAATTTGAAAGCTTTAAAAGCATGAGCACCCAGAACGGCTTTATCTTCTATATTCCGG CATGGCTGACCAGCAAAATTGATCCGAGCACCGGTTTTGTGAACCTGCTGAAAACAAAATATACC TCCATTGCCGACAGCAAGAAGTTTATTAGCAGCTTTGATCGCATTATGTATGTTCCGGAAGAGGA CCTGTTTGAATTCGCACTGGATTACAAAAATTTCAGCCGTACCGATGCCGACTACATCAAAAAAT GGAAACTGTACAGCTATGGTAACCGCATTCGCATTTTTCGCAACCCGAAGAAAAACAATGTGTTC GATTGGGAAGAAGTTTGTCTGACCAGCGCATATAAAGAACTTTTCAACAAATACGGCATCAACTA TCAGCAGGGTGATATTCGTGCACTGCTGTGTGAACAGAGCGATAAAGCGTTTTATAGCAGTTTTA TGGCACTGATGAGCCTGATGCTGCAGATGCGTAATAGCATTACCGGTCGCACCGATGTGGATTTT CTGATTAGTCCGGTGAAAAATTCCGATGGCATCTTTTATGATAGCCGCAATTACGAAGCACAAGA AAATGCAATTCTGCCGAAAAACGCAGATGCAAATGGTGCATATAACATTGCACGTAAAGTTCTGT GGGCAAT T GGC CAGT T TAAGAAAGCAGAAGAT GAGAAGC T GGACAAAGT GAAAAT T GC GAT CAGC AATAAAGAGTGGCTGGAATACGCACAGACCAGCGTTAAACATGGTCGTAGCAGTGATGATGAAGC AACCGCAGATAGCCAGCATGCAGCACCGCCGAAAAAAAAACGCAAAGTGCTCGAGCACCACCACC ACCACCACTGA SEQ ID NO:396 Hs optimized Lb Cpfl with OpT NLS and 6x His -DNA ATGCTGAAGAACGTGGGCATCGACCGGCTGGACGTGGAAAAGGGCAGAAAGAACATGAGCAAGCT CGAGAAGTTCACCAACTGCTACAGCCTGAGCAAGACCCTGCGGTTCAAGGCCATTCCTGTGGGCA AGACCCAAGAGAACATCGACAACAAGCGGCTGCTGGTGGAAGATGAGAAGAGAGCCGAGGACTAC 2024204029   13 Jun 2024 AAGGGCGTGAAGAAGCTGCTGGACCGGTACTACCTGAGCTTCATCAACGACGTGCTGCACAGCAT CAAGCTCAAGAACCTGAACAACTACATCAGCCTGTTCCGGAAGAAAACCCGGACCGAGAAAGAGA ACAAAGAGCTGGAAAACCTCGAGATCAACCTGCGGAAAGAGATCGCCAAGGCCTTCAAGGGCAAC GAGGGCTACAAGAGCCTGTTCAAGAAGGACATCATCGAGACAATCCTGCCTGAGTTCCTGGACGA CAAGGACGAGATCGCCCTGGTCAACAGCTTCAACGGCTTCACAACCGCCTTCACCGGCTTTTTCG ACAACCGCGAGAATATGTTCAGCGAGGAAGCCAAGAGCACCTCTATCGCCTTCCGGTGCATCAAC GAGAATCTGACCCGGTACATCAGCAACATGGATATCTTCGAGAAGGTGGACGCCATCTTCGACAA GCACGAGGTGCAAGAGATCAAAGAAAAGATCCTGAACAGCGACTACGACGTCGAGGACTTCTTCG AGGGCGAGTTCTTCAACTTCGTGCTGACACAAGAGGGCATCGATGTGTACAACGCCATCATCGGC GGCTTCGTGACAGAGAGCGGCGAGAAGATCAAGGGCCTGAACGAGTACATCAACCTCTACAACCA GAAAACGAAGCAGAAGCTGCCCAAGTTCAAGCCCCTGTACAAACAGGTGCTGAGCGACAGAGAGA GCCTGTCCTTTTACGGCGAGGGCTATACCAGCGACGAAGAGGTGCTGGAAGTGTTCAGAAACACC CTGAACAAGAACAGCGAGATCTTCAGCTCCATCAAGAAGCTCGAAAAGCTGTTTAAGAACTTCGA CGAGTACAGCAGCGCCGGCATCTTCGTGAAGAATGGCCCTGCCATCAGCACCATCTCCAAGGACA TCTTCGGCGAGTGGAACGTGATCCGGGACAAGTGGAACGCCGAGTACGACGACATCCACCTGAAG AAAAAGGCCGTGGTCACCGAGAAGTACGAGGACGACAGAAGAAAGAGCTTCAAGAAGATCGGCAG CTTCAGCCTGGAACAGCTGCAAGAGTACGCCGACGCCGATCTGAGCGTGGTGGAAAAGCTGAAAG AGATTATCATCCAGAAGGTCGACGAGATCTACAAGGTGTACGGCAGCAGCGAGAAGCTGTTCGAC GCCGACTTTGTGCTGGAAAAGAGCCTCAAAAAGAACGACGCCGTGGTGGCCATCATGAAGGACCT GCTGGATAGCGTGAAGTCCTTCGAGAACTATATTAAGGCCTTCTTTGGCGAGGGCAAAGAGACAA ACCGGGACGAGAGCTTCTACGGCGATTTCGTGCTGGCCTACGACATCCTGCTGAAAGTGGACCAC ATCTACGACGCCATCCGGAACTACGTGACCCAGAAGCCTTACAGCAAGGACAAGTTTAAGCTGTA CTTCCAGAATCCGCAGTTCATGGGCGGCTGGGACAAAGACAAAGAAACCGACTACCGGGCCACCA TCCTGAGATACGGCTCCAAGTACTATCTGGCCATTATGGACAAGAAATACGCCAAGTGCCTGCAG AAGATCGATAAGGACGACGTGAACGGCAACTACGAGAAGATTAACTACAAGCTGCTGCCCGGACC TAACAAGATGCTGCCTAAGGTGTTCTTTAGCAAGAAATGGATGGCCTACTACAACCCCAGCGAGG ATATCCAGAAAATCTACAAGAACGGCACCTTCAAGAAAGGCGACATGTTCAACCTGAACGACTGC CACAAGCTGATCGATTTCTTCAAGGACAGCATCAGCAGATACCCCAAGTGGTCCAACGCCTACGA CTTCAATTTCAGCGAGACAGAGAAGTATAAGGATATCGCCGGGTTCTACCGCGAGGTGGAAGAAC AGGGCTATAAGGTGTCCTTTGAGAGCGCCAGCAAGAAAGAGGTGGACAAGCTGGTCGAAGAGGGC AAGCTGTACATGTTCCAGATCTATAACAAGGACTTCTCCGACAAGAGCCACGGCACCCCTAACCT GCACACCATGTACTTTAAGCTGCTGTTCGATGAGAACAACCACGGCCAGATCAGACTGTCTGGCG GAGCCGAGCTGTTTATGAGAAGGGCCAGCCTGAAAAAAGAGGAACTGGTCGTTCACCCCGCCAAC TCTCCAATCGCCAACAAGAACCCCGACAATCCCAAGAAAACCACCACACTGAGCTACGACGTGTA CAAGGATAAGCGGTTCTCCGAGGACCAGTACGAGCTGCACATCCCTATCGCCATCAACAAGTGCC C CAAGAATAT C T T CAAGAT CAACAC C GAAGT GCGGGTGCTGCT GAAGCAC GAC GACAAC CC T TAC GTGATCGGCATCGACAGAGGCGAGCGGAACCTGCTGTATATCGTGGTGGTGGACGGCAAGGGCAA TATCGTGGAACAGTACTCCCTGAATGAGATCATCAACAACTTCAATGGCATCCGGATCAAGACGG AC TAC CACAGC C T GC T GGACAAAAAAGAGAAAGAAC GC T T C GAGGCC C GGCAGAAC T GGAC CAGC ATCGAGAACATCAAAGAACTGAAGGCCGGCTACATCTCCCAGGTGGTGCACAAGATCTGCGAGCT GGTTGAGAAGTATGACGCCGTGATTGCCCTGGAAGATCTGAATAGCGGCTTTAAGAACAGCCGCG TGAAGGTCGAGAAACAGGTGTACCAGAAATTCGAGAAGATGCTGATCGACAAGCTGAACTACATG GTCGACAAGAAGTCTAACCCCTGCGCCACAGGCGGAGCCCTGAAGGGATATCAGATCACCAACAA GTTCGAGTCCTTCAAGAGCATGAGCACCCAGAATGGCTTCATCTTCTACATCCCCGCCTGGCTGA CCAGCAAGATCGATCCTAGCACCGGATTCGTGAACCTGCTCAAGACCAAGTACACCAGCATTGCC GACAGCAAGAAGTTCATCTCCAGCTTCGACCGGATTATGTACGTGCCCGAAGAGGACCTGTTCGA ATTCGCCCTGGATTACAAGAACTTCAGCCGGACCGATGCCGACTATATCAAGAAGTGGAAGCTGT 2024204029   13 Jun 2024 ATAGCTACGGCAACCGCATCCGCATCTTCAGAAACCCGAAGAAAAACAACGTGTTCGACTGGGAA GAAGTGTGCCTGACCAGCGCCTACAAAGAACTCTTCAACAAATACGGCATCAACTACCAGCAGGG CGACATCAGAGCCCTGCTGTGCGAGCAGAGCGACAAGGCCTTTTACAGCTCCTTCATGGCCCTGA TGTCCCTGATGCTGCAGATGCGGAATAGCATCACCGGCAGGACCGACGTGGACTTCCTGATCAGC CCTGTGAAGAATTCCGACGGGATCTTCTACGACAGCAGAAACTACGAGGCTCAAGAGAACGCCAT CCTGCCTAAGAACGCCGATGCCAACGGCGCCTATAATATCGCCAGAAAGGTGCTGTGGGCCATCG GCCAGTTTAAGAAGGCCGAGGACGAGAAACTGGACAAAGTGAAGATCGCCATCTCTAACAAAGAG TGGCTGGAATACGCCCAGACCAGCGTGAAGCACGGCAGATCTAGTGACGATGAGGCCACCGCCGA TAGCCAGCATGCAGCCCCTCCAAAGAAAAAGCGGAAAGTGCTGGAACACCACCACCATCACCAC SEQ ID NO:24 Hs optimized Lb Cpfl with OpT NLS and 6x His - AA MLKNVGIDRLDVEKGRKNMSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDY KGVKKLLDRYYLSFINDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGN EGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCIN ENLTRYISNMDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIG GFVTESGEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNT LNKNSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFD ADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDILLKVDH IYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIMDKKYAKCLQ KIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGTFKKGDMFNLNDC HKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSFESASKKEVDKLVEEG KLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAELFMRRASLKKEELVVHPAN SPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCPKNIFKINTEVRVLLKHDDNPY VIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRIKTDYHSLLDKKEKERFEARQNWTS IENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSGFKNSRVKVEKQVYQKFEKMLIDKLNYM VDKKSNPCATGGALKGYQITNKFESFKSMSTQNGFIFYIPAWLTSKIDPSTGFVNLLKTKYTSIA DSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWE EVCLTSAYKELFNKYGINYQQGDIRALLCEQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLIS PVKNSDGIFYDSRNYEAQENAILPKNADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKE WLEYAQTSVKHGRSSDDEATADSQHAAPPKKKRKVLEHHHHHH EXAMPLE 10

[0131] Use of modified crRNAs with LbCpfl protein delivered as an RNP complex.

[0132] Twelve sites in the human HPRT1 gene, 38094-S (SEQ ID No. 358), 38104-S (SEQ ID No. 361), 38115-AS (SEQ ID No. 364), 38146-AS (SEQ ID No. 367), 38164AS (SEQ ID No. 370), 38164-S (SEQ ID No. 372), 38186-S (SEQ ID No. 376), 38228-S (SEQ ID No. 379), 38330-AS (SEQ ID No. 382), 38343-S (SEQ ID No. 385), 38455-S (SEQ ID No. 388) and 38486-S (SEQ ID No. 391) (where A and AS represent the sense and antisense strand, respectively), were chosen to study the target editing activity of 2024204029   13 Jun 2024 LbCpfl, as compared to that of AsCpfl and SpyCas9. Studies were done comparing the ability to use chemically modified crRNAs with LbCpfl protein to perform genome editing in HEK-293 cells using electroporation to deliver the ribonucleoprotein protein (RNP) complexes into cells.

[0133] Purified recombinant LbCpfl protein was employed in this example, isolated from E.coli using standard techniques. The amino-acid sequence of the recombinant protein is shown in SEQ ID NO: 14.

[0134] The LbCpfl crRNAs, and AsCpfl control crRNAs, were heated to 95°C for 5 minutes then allowed to cool to room temperature. The crRNAs were mixed with LbCpfl, or AsCpfl, at a molar ratio of 1:1 RNA:protein in PBS (5 pM RNP complex in 10 pL volume, for a single transfection). The RNP complex was allowed to form at room temperature for 15 minutes. HEK293 cells were resuspended following trypsinization and washed in medium and washed a second time in PBS before use. Cells were resuspended in at a final concentration of 3.5 x 105 cells in 20 pL of Nucleofection solution. 20 pL of cell suspension was placed in the V-bottom 96-well plate and 5 pL of the Cpfl RNP complex was added to each well (5 pM final concentration) and 3 pM of Cpfl Electroporation Enhancer Solution was added to each well (Integrated DNA Technologies). 25 pL of the final mixture was transferred to each well of a 96 well Nucleocuvette electroporation module. Cells were electroporated using Amaxa 96 well shuttle protocol, program 96-DS-150. Following electroporation, 75 pL of medium was added to each well and 25 pL of the final cell mixture was transferred to 175 pL of pre-warmed medium in 96 well incubation plates (final volume 200 pL). Cells were incubated at 37°C for 48 hours. Genomic DNA was isolated using QuickExtract solution (Epicentre). Genomic DNA was amplified with KAPA HiFi DNA Polymerase (Roche) and primers targeting the HPRT region of interest (HPRT-low forward primer: AAGAATGTTGTGATAAAAGGTGATGCT (SEQ ID No. 394); HPRT-low reverse primer: ACACATCCATGGGACTTCTGCCTC (SEQ ID No. 395)). PCR products were melted and re-annealed in NEB buffer 2 (New England Biolabs) to allow for heteroduplex formation followed by digestion with 2 units of T7 endonuclease 1 (T7EI; New England Biolabs) for 1 hour at 37°C. The digested products were visualized on a Fragment Analyzer (Advanced Analytical Technologies). Percent cleavage of targeted DNA was calculated as the average molar concentration of the cut products / (average molar concentration of the cut products + molar concentration of the 2024204029   13 Jun 2024 uncut band) * 100. The sequences are shown in Table 10, and the results are graphically represented in FIG. 9.

[0135] Table 10: Sequences of modified AsCpfl and LbCpfl crRNAs tested Seq Name Sequence 5'-3' SEQ ID NO: 38094-S-Control C3-uaauuucuacucuuguagauauagucuuuccuugggugugu-C3 358 38094-S-21 C3-uaauuucuacuaaguguagauauagucuuuccuugggugugu-C3 359 38094-S-23 C3-uaauuucuacuaaguguagauauagucuuuccuuggguguguua-C3 360 38104-S-Cpfl C3-uaauuucuacucuuguagaucuuggguguguuaaaagugac-C3 361 38104-S-41- 97 C3-uaauuucuacuaaguguagaucuuggguguguuaaaagugac-C3 362 38104-S-23 C3-uaauuucuacuaaguguagaucuuggguguguuaaaagugacca-C3 363 38115-AS-Cpfl C3-uaauuucuacucuuguagauacacacccaaggaaagacuau-C3 364 38115- AS- 21 C3-uaauuucuacuaaguguagauacacacccaaggaaagacuau-C3 365 38115- AS- 23 C3-uaauuucuacuaaguguagauacacacccaaggaaagacuauga-C3 366 38146- AS-Cpfl C3-uaauuucuacucuuguagauauccgugcugaguguaccaug-C3 367 38146- AS- 21 C3-uaauuucuacuaaguguagauauccgugcugaguguaccaug-C3 368 38146- AS- 23 C3-uaauuucuacuaaguguagauauccgugcugaguguaccaugca-C3 369 38164- AS-Cpfl C3-uaauuucuacucuuguagauuaaacacuguuucauuucauc-C3 370 38164- AS- 21 C3-uaauuucuacuaaguguagauuaaacacuguuucauuucauc-C3 371 38164- AS- 23 C3-uaauuucuacuaaguguagauuaaacacuguuucauuucauccg-C3 372 38164- S-Cpfl C3-uaauuucuacucuuguagaugaaacgucagucuucucuuuu-C3 373 38164-S-21 C3-uaauuucuacuaaguguagaugaaacgucagucuucucuuuu-C3 374 38164-S-23 C3-uaauuucuacuaaguguagaugaaacgucagucuucucuuuugu-C3 375 38186-S-Cpfl C3-uaauuucuacucuuguagauuaaugcccuguagucucucug-C3 376 38186-S-21 C3-uaauuucuacuaaguguagauuaaugcccuguagucucucug-C3 377 38186-S-23 C3-uaauuucuacuaaguguagauuaaugcccuguagucucucugua-C3 378 38228-S-Cpfl C3-uaauuucuacucuuguagauuaauuaacagcuugcugguga-C3 379 38228-S-21 C3-uaauuucuacuaaguguagauuaauuaacagcuugcugguga-C3 380 38228-S-23 C3-uaauuucuacuaaguguagauuaauuaacagcuugcuggugaaa-C3 381 2024204029   13 Jun 2024 Seq Name Sequence 5’-3’ SEQ ID NO: 38330-AS-Cpfl C3-uaauuucuacucuuguagaugguuaaagaugguuaaaugau-C3 382 38330-AS-2 1 C3-uaauuucuacuaaguguagaugguuaaagaugguuaaaugau-C3 383 38330-AS -23 C3-uaauuucuacuaaguguagaugguuaaagaugguuaaaugauug-C3 384 38343-S-Cpfl C3-uaauuucuacucuuguagauugugaaauggcuuauaauugc-C3 385 38343-S-2 1 C3-uaauuucuacuaaguguagauugugaaauggcuuauaauugc-C3 386 38343-S-23 C3-uaauuucuacuaaguguagauugugaaauggcuuauaauugcuu-C3 387 38455-S-Cpfl C3-uaauuucuacucuuguagauguuguuggauuugaaauucca-C3 388 38455-S-21 C3-uaauuucuacuaaguguagauguuguuggauuugaaauucca-C3 389 38455-S-23 C3-uaauuucuacuaaguguagauguuguuggauuugaaauuccaga-C3 390 38486-S-Cpfl C3-uaauuucuacucuuguagauuuguaggauaugcccuugacu-C3 391 38486-S-21 C3-uaauuucuacuaaguguagauuuguaggauaugcccuugacu-C3 392 38486-S-23 C3-uaauuucuacuaaguguagauuuguaggauaugcccuugacuau-C3 393 RNA bases are shown 5'-3' orientation, RNA bases are shown in lower case. Locations are specified within the human HPRT1 gene with orientation relative to the sense coding strand indicated (S = sense, AS= antisense). C3 = C3 spacer (propanediol modifier). Cpfl = Cpfl crRNA control. 21 and 23 represent the length of the 3' protospacer for each crRNA.

[0136] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0137] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly 2024204029   13 Jun 2024 contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0138] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. An isolated nucleic acid, wherein the isolated nucleic acid encodes an As Cpfl polypeptide codon optimized for expression in H. sapiens.

2. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid comprises SEQIDNO:15.

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

4. The isolated polypeptide of claim 3, wherein the isolated polypeptide comprises SEQ ID NO: 12.

5. An isolated expression vector encoding SEQ ID NO: 15.

6. A host cell comprising an isolated expression vector encoding SEQ ID NO: 15, wherein the isolated expression vector encoding SEQ ID NO: 15 is operably linked to a suitable promoter to permit expression of a polypeptide comprising SEQ ID NO: 12.

7. The host cell of claim 6, wherein host cell comprises a human cell.

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

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

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

11. An isolated CRISPR / Cpfl endonuclease system, comprising: an AsCpfl polypeptide, and a suitable AsCpfl crRNA.

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

13. The isolated CRISPR / Cpfl endonuclease system of claim 11, wherein the suitable AsCpfl crRNA is selected from a length-truncated AsCpfl crRNA or a2024204029   13 Jun 2024chemically-modified AsCpfl crRNA, or an AsCpfl crRNA comprising both length truncations and chemical modifications.

14. An isolated CRISPR / Cpfl endonuclease system, comprising:a human cell line expressing a AsCpfl polypeptide and a suitable AsCpfl crRNA.

15. The isolated CRISPR / Cpfl endonuclease system of claim 14, wherein the AsCpfl polypeptide comprises SEQ ID NO: 12.

16. The isolated CRISPR / Cpfl endonuclease system of claim 14, wherein the suitable AsCpfl crRNA is selected from a length-truncated AsCpfl crRNA or a chemically-modified AsCpfl crRNA, or an AsCpfl crRNA comprising both length truncations and chemical modifications.

17. An isolated AsCpfl crRNA, wherein the isolated AsCpfl crRNA is active in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated protein endonuclease system.

18. The isolated AsCpfl crRNA of claim 17, wherein the isolated AsCpfl crRNA is selected from length-truncated AsCpfl crRNA, a chemically-modified AsCpfl crRNA, or an AsCpfl crRNA comprising both length truncations and chemical modifications.

19. The isolated AsCpfl crRNA of claim 17, wherein the isolated AsCpfl crRNA is a length-truncated AsCpfl crRNA comprising a 5’-universal loop domain of 19 to 20 nucleotides in length and a 3’-target specific protospacer domain of 19 to 21 nucleotides in length.

20. The isolated AsCpfl crRNA of claim 17, wherein the isolated AsCpfl crRNA comprises both a length truncation and a chemical modification.

21. The isolated AsCpfl crRNA of claim 20, wherein the chemical modification is selected from the group consisting of an end-group modification (e.g., C3 spacer), 2’0Me modification, 2’-fluoro modification and LNA modification.

22. A method of performing gene editing, comprising:contacting a candidate editing target site locus with an active CRISPR / Cpfl endonuclease system having a wild-type AsCpfl polypeptide and a suitable AsCpfl crRNA.2024204029   13 Jun 202423. The method of claim 22, wherein the wild-type AsCpfl polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 12, SEQ ID NO: 16 and SEQ ID NO:19.

24. The method of claim 22, wherein the suitable AsCpfl crRNA is selected from a length-truncated AsCpfl crRNA, a chemically-modified AsCpfl crRNA, or an AsCpfl crRNA comprising both length truncations and chemical modifications.

25. An isolated nucleic acid, wherein the isolated nucleic acid encodes an Lb Cpfl polypeptide codon optimized for expression in H. sapiens.

26. The isolated nucleic acid of claim 25, wherein the isolated nucleic acid comprises SEQ ID NO: 17 or SEQ ID NO:396.

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

28. The isolated polypeptide of claim 27, wherein the isolated polypeptide comprises SEQ ID NO: 14 or SEQ ID NO:24.

29. An isolated expression vector encoding SEQ ID NO: 17 or SEQ ID NO:396.

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

31. The host cell of claim 30, wherein host cell comprises a human cell.

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

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

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

35. An isolated CRISPR / Cpfl endonuclease system, comprising: an Lb Cpfl polypeptide, and2024204029   13 Jun 2024a suitable Cpfl crRNA.

36. The isolated CRISPR / Cpfl endonuclease system of claim 35, wherein the Lb Cpfl polypeptide comprises SEQ ID NO: 14.

37. The isolated CRISPR / Cpfl endonuclease system of claim 35, wherein the suitable Cpfl crRNA is selected from a length-truncated Cpfl crRNA or a chemically-modified Cpfl crRNA, or a Cpfl crRNA comprising both length truncations and chemical modifications.

38. An isolated CRISPR / Cpfl endonuclease system, comprising:a human cell line expressing a Lb Cpfl polypeptide and a suitable Cpfl crRNA.

39. The isolated CRISPR / Cpfl endonuclease system of claim 38, wherein the Lb Cpfl polypeptide comprises SEQ ID NO: 14 or SEQ ID NO:24.

40. The isolated CRISPR / Cpfl endonuclease system of claim 38, wherein the suitable Cpfl crRNA is selected from a length-truncated Cpfl crRNA or a chemically-modified Cpfl crRNA, or an Cpfl crRNA comprising both length truncations and chemical modifications.

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

42. The method of claim 41, wherein the wild-type Lb Cpfl polypeptide selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 14, SEQ ID NO:20 and SEQ ID NO:24.

43. The method of claim 41, wherein the suitable Cpfl crRNA is selected from a length-truncated Cpfl crRNA, a chemically-modified Cpfl crRNA, or an Cpfl crRNA comprising both length truncations and chemical modifications.

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

45. The method of claim 41, wherein the suitable Cpfl crRNA comprises both a length truncation and a chemical modification.2024204029   13 Jun 202446. The method of claim 45, wherein the chemical modification is selected from the group consisting of an end-group modification (e.g., C3 spacer), 2’OMe modification, 2’-fluoro modification and LNA modification.

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