Novel crispr enzymes and systems

Abstract

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. 2023241400 09 Oct 20232023241400 09 Oct 2023

Description

10001] This application is a divisional application of Australian patent application 2017257274, the entire disclosure of which is incorporated herein by this cross-reference.

[0002] Reference is made to U.S. Provisional Application Nos. 62 / 324,820 and 62 / 324,834 fded April 19, 2016, U.S. Provisional Application No. 62 / 351,558 fded June 17, 2016, U.S. Provisional Application No. 62 / 360,765 fded July 11, 2016, and U.S. Provisional Application No. 62 / 410,196, filed October 19, 2016, herein incorporated by reference. 10003] The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0004] This invention was made with government support under grant numbers MH 100706 and MH110049 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION 10005] The present invention generally relates to systems, methods and compositions used for the control of gene expression involving sequence targeting, such as perturbation of gene transcripts or nucleic acid editing, that may use vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof. 2023241400 09 Oct 2023 BACKGROUND OF THE INVENTION

[0006] Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that employ novel strategies and molecular mechanisms and are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome. This would provide a major resource for new applications in genome engineering and biotechnology. |0007] The CRISPR-Cas systems of bacterial and archaeal adaptive immunity show extreme diversity of protein composition and genomic loci architecture. The CRISPR-Cas system loci has more than 50 gene families and there is no strictly universal genes indicating fast evolution and extreme diversity of loci architecture. So far, adopting a multi-pronged approach, there is comprehensive cas gene identification of about 395 profiles for 93 Cas proteins. Classification includes signature gene profiles plus signatures of locus architecture. A new classification of CRISPR-Cas systems is proposed in which these systems are broadly divided into two classes, Class 1 with multisubunit effector complexes and Class 2 with single-subunit effector modules exemplified by the Cas9 protein. Novel effector proteins associated with Class 2 CRISPR-Cas systems may be developed as powerful genome engineering tools and the prediction of putative novel effector proteins and their engineering and optimization is important. 10008] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. SUMMARY OF THE INVENTION |0009] There exists a pressing need for alternative and robust systems and techniques for targeting nucleic acids or polynucleotides (e.g. DNA or RNA or any hybrid or derivative 2023241400   28 Apr 2026 thereof) with a wide array of applications. This invention addresses this need and provides related advantages. Adding the novel DNA or RNA-targeting systems of the present application to the repertoire of genomic and epigenomic targeting technologies may transform the study and perturbation or editing of specific target sites through direct detection, analysis and manipulation. To utilize the DNA or RNA-targeting systems of the present application effectively for genomic or epigenomic targeting without deleterious effects, it is critical to understand aspects of engineering and optimization of these DNA or RNA targeting tools.

[0010] More particularly, the present invention provides Cpf1 orthologs and uses thereof.

[0011] Even within a given type, the CRISPR-Cas orthologs and more particularly Cpf1 orthologs can differ in different aspects such as size, PAM requirements, direct repeats, specificity, and editing efficiency. The identification of additional useful orthologs allows for optimizing current applications as well as expanding the possibility for orthogonal genome editing, regulation and imaging. [0011a] In one aspect, the present invention provides an adeno-associated virus (AAV) vector comprising (a) a first regulatory element operably linked to a nucleotide sequence encoding a Cpf1 effector protein, and (b) a second regulatory element operably linked to a nucleotide sequence encoding a guide RNA comprising a guide sequence linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence 3’ of a Protospacer Adjacent Motif (PAM), wherein the guide RNA is capable of forming a complex with the Cpf1 effector protein, and wherein the Cpf1 effector protein has at least 95% sequence identity with Moraxella bovoculi AAX08_00205 Cpf1 (Mb2Cpf1) or Moraxella bovoculi AAX11_00205 Cpf1 (Mb3Cpf1). [0011b] In a further aspect the present invention provides an adeno-associated virus (AAV) vector comprising (a) a first regulatory element operably linked to a nucleotide sequence encoding a Cpf1 effector protein, and (b) a second regulatory element operably linked to a plurality of nucleotide sequences encoding a plurality of guide RNAs each comprising a guide sequence linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence 3’ of a Protospacer Adjacent Motif (PAM), wherein each guide RNA is capable of forming a complex with the Cpf1 effector protein, wherein the Cpf1 effector protein has at least 95% sequence identity with Moraxella 2023241400   22 Apr 2026 bovoculi AAX08_00205 Cpf1 (Mb2Cpf1) or Moraxella bovoculi AAX11_00205 Cpf1 (Mb3Cpf1), wherein the plurality of guide RNAs target different target sequences, and wherein the plurality of nucleotide sequences encoding the plurality of guide RNAs are operably linked to the second regulatory element in tandem.

[0012] The invention also provides a method of modifying sequences associated with or at a target locus of interest, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising a Type V CRISPR-Cas loci effector protein and one or more nucleic acid components, wherein the effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of the sequences associated with or at the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break. In a preferred embodiment, the sequences associated with or at the target locus of interest comprises DNA and the effector protein is a Cpf1 enzyme. In preferred embodiments, the effector protein is selected from a Cpf1 of Thiomicrospira sp. XS5 (TsCpf1); Prevotella bryanti B14 (25-Pb2Cpf1); Moraxella lacunata (32-MlCpf1); Lachnospiraceae bacterium MA2020 (40-Lb7Cpf1), Candidatus Methanomethylophilus alvus Mx1201 (47-CMaCpf1), Butyrivibrio sp. NC3005 (48-BsCpf1); Moraxella bovoculi AAX08_00205 (34-Mb2 Cpf1); Moraxella bovoculi AAX11_00205 (35-Mb3Cpf1) and Butivibrio fibrosolvens (49BfCpf1). In preferred embodiments, the effector protein is selected from a Cpf1 of Acidaminococcus sp. BV3L6, Thiomicrospira sp. XS5, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11_00205, Lachnospiraceae bacterium MA2020. In particular embodiments, the effector protein has a sequence homology or identity of at least 2023241400 09 Oct 2023 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with one or more of the Cpfl sequences disclosed herein, such as, but not limited to the Cpfl effector protein amino acid sequences specified herein and / or the species listed in the Figures herein. Preferred embodiments include a Cpfl effector protein and systems and methods including or involving an effector protein, having an amino acid sequence identity of at least 90%, more particularly at least 92%, 93%, 94%, 95%, 96%, 97%, 98% sequence identity with one or more of Thiomicrospira sp. XS5 (TsCpfl); Prevotella bryanti B14 (25-Pb2Cpfl); Moraxella lacunata (32-MlCpfl); Lachnospiraceae bacterium MA2020 (40-Lb7Cpfl), Candidatus Methanomethylophilus alvus Mx1201 (47-CMaCpfl), Butyrivibrio sp. NC3005 (48-BsCpfl); Moraxella bovoculi AAX08_00205 (34-Mb2 Cpfl); Moraxella bovoculi AAX11_00205 (35-Mb3Cpfl) and Butivibrio fibrosolvens (49BfCpfl), such as at least 95 sequence identity or more particularly 97% sequence identity with one or more of Thiomicrospira sp. XS5 (TsCpfl); Moraxella lacunata (32-MlCpfl); Butyrivibrio sp. NC3005 (48-BsCpfl); Moraxella bovoculi AAX08_00205 (34-Mb2 Cpfl); Moraxella bovoculi AAX11_00205 (35-Mb3Cpfl), whereby more particularly the sequences are as provided herein. In particular embodiments, the Cpfl effector protein has at least 90%, preferably at least 95% sequence identity to the Cpfl effector protein from Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAXl 1_00205. 10013J It will be appreciated that the terms Cas enzyme, CRISPR enzyme, CRISPR protein Cas protein and CRISPR Cas are generally used interchangeably and at all points of reference herein refer by analogy to novel CRISPR effector proteins further described in this application, unless otherwise apparent, such as by specific reference to Cas9. The CRISPR effector proteins described herein are preferably Cpfl effector proteins.

[0014] The invention provides a method of modifying sequences associated with or at a target locus of interest, the method comprising delivering to said sequences associated with or at the locus a non-naturally occurring or engineered composition comprising a Cpfl loci effector protein and one or more nucleic acid components, wherein the Cpfl effector protein forms a complex with the one or more nucleic acid components and upon binding of the said complex to the locus of interest the effector protein induces the modification of the sequences associated with or at the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break. In a preferred embodiment the Cpfl effector protein 2023241400 09 Oct 2023 forms a complex with one nucleic acid component; advantageously an engineered or non-naturally occurring nucleic acid component. The induction of modification of sequences associated with or at the target locus of interest can be Cpfl effector protein-nucleic acid guided. In a preferred embodiment the one nucleic acid component is a CRISPR RNA (crRNA). In a preferred embodiment the one nucleic acid component is a mature crRNA or guide RNA, wherein the mature crRNA or guide RNA comprises a spacer sequence (or guide sequence) and a direct repeat sequence or derivatives thereof. In a preferred embodiment the spacer sequence or the derivative thereof comprises a seed sequence, wherein the seed sequence is critical for recognition and / or hybridization to the sequence at the target locus. In a preferred embodiment, the seed sequence of a FnCpfl guide RNA is approximately within the first 5 nt on the 5’ end of the spacer sequence (or guide sequence). In a preferred embodiment the strand break is a staggered cut with a 5’ overhang. In a preferred embodiment, the sequences associated with or at the target locus of interest comprise linear or super coiled DNA.

[0015] Aspects of the invention relate to Cpfl effector protein complexes having one or more non-naturally occurring or engineered or modified or optimized nucleic acid components. In a preferred embodiment the nucleic acid component of the complex may comprise a guide sequence linked to a direct repeat sequence, wherein the direct repeat sequence comprises one or more stem loops or optimized secondary structures. In a preferred embodiment, the direct repeat has a minimum length of 16 nts and a single stem loop. In further embodiments the direct repeat has a length longer than 16 nts, preferrably more than 17 nts, and has more than one stem loop or optimized secondary structures. In a preferred embodiment the direct repeat may be modified to comprise one or more protein-binding RNA aptamers. In a preferred embodiment, one or more aptamers may be included such as part of optimized secondary structure. Such aptamers may be capable of binding a bacteriophage coat protein. The bacteriophage coat protein may be selected from the group comprising QP, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, Mil, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, (j)Cb5, (|)Cb8r, <^Cbl2r, (pCb23r, 7s and PRR1. In a preferred embodiment the bacteriophage coat protein is MS2. The invention also provides for the nucleic acid component of the complex being 30 or more, 40 or more or 50 or more nucleotides in length. 2023241400 09 Oct 2023 10016] The invention provides methods of genome editing wherein the method comprises two or more rounds of Cpfl effector protein targeting and cleavage. In certain embodiments, a first round comprises the Cpfl effector protein cleaving sequences associated with a target locus far away from the seed sequence and a second round comprises the Cpfl effector protein cleaving sequences at the target locus. In preferred embodiments of the invention, a first round of targeting by a Cpfl effector protein results in an indel and a second round of targeting by the Cpfl effector protein may be repaired via homology directed repair (HDR). In a most preferred embodiment of the invention, one or more rounds of targeting by a Cpfl effector protein results in staggered cleavage that may be repaired with insertion of a repair template. |0017] The invention provides methods of genome editing or modifying sequences associated with or at a target locus of interest wherein the method comprises introducing a Cpfl effector protein complex into any desired cell type, prokaryotic or eukaryotic cell, whereby the Cpfl effector protein complex effectively functions to integrate a DNA insert into the genome of the eukaryotic or prokaryotic cell. In preferred embodiments, the cell is a eukaryotic cell and the genome is a mammalian genome. In preferred embodiments the integration of the DNA insert is facilitated by non-homologous end joining (NHEJ)-based gene insertion mechanisms. In preferred embodiments, the DNA insert is an exogenously introduced DNA template or repair template. In one preferred embodiment, the exogenously introduced DNA template or repair template is delivered with the Cpfl effector protein complex or one component or a polynucleotide vector for expression of a component of the complex. In a more preferred embodiment the eukaryotic cell is a non-dividing cell (e.g. a non-dividing cell in which genome editing via HDR is especially challenging). In preferred methods of genome editing in human cells, the Cpfl effector proteins may include but are not limited to FnCpfl, AsCpfl and LbCpfl effector proteins. 10018] In such methods the target locus of interest may be comprised in a DNA molecule in vitro. In a preferred embodiment the DNA molecule is a plasmid.

[0019] In such methods the target locus of interest may be comprised in a DNA molecule within a cell. The cell may be a prokaryotic cell or a eukaryotic cell. The cell may be a mammalian cell. The mammalian cell many be a non-human primate, bovine, porcine, rodent or mouse cell. The cell may be a non-mammalian eukaryotic cell such as poultry, fish or 2023241400 09 Oct 2023 shrimp. The cell may also be a plant cell. The plant cell may be of a crop plant such as cassava, com, sorghum, wheat, or rice. The plant cell may also be of an algae, tree or vegetable. The modification introduced to the cell by the present invention may be such that the cell and progeny of the cell are altered for improved production of biologic products such as an antibody, starch, alcohol or other desired cellular output. The modification introduced to the cell by the present invention may be such that the cell and progeny of the cell include an alteration that changes the biologic product produced.

[0020] In a preferred embodiment, the target locus of interest comprises DNA.

[0021] In such methods the target locus of interest may be comprised in a DNA molecule within a cell. The cell may be a prokaryotic cell or a eukaryotic cell. The cell may be a mammalian cell. The mammalian cell many be a non-human mammal, e.g., primate, bovine, ovine, porcine, canine, rodent, Leporidae such as monkey, cow, sheep, pig, dog, rabbit, rat or mouse cell. The cell may be a non-mammalian eukaryotic cell such as poultry bird (e.g., chicken), vertebrate fish (e.g., salmon) or shellfish (e.g., oyster, claim, lobster, shrimp) cell. The cell may also be a plant cell. The plant cell may be of a monocot or dicot or of a crop or grain plant such as cassava, corn, sorghum, soybean, wheat, oat or rice. The plant cell may also be of an algae, tree or production plant, fruit or vegetable (e.g., trees such as citrus trees, e.g., orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as almond or walnut or pistachio trees; nightshade plants; plants of the genus Brassica', plants of the genus Lactuca', plants of the genus Spinacia', plants of the genus Capsicum', cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc). |0022] In any of the described methods the target locus of interest may be a genomic or epigenomic locus of interest. In any of the described methods the complex may be delivered with multiple guides for multiplexed use. In any of the described methods more than one protein(s) may be used. |0023] In preferred embodiments of the invention, biochemical or in vitro or in vivo cleavage of sequences associated with or at a target locus of interest results without a putative transactivating crRNA (tracr RNA) sequence, e.g. cleavage by an AsCpfl, LbCpfl or an FnCpfl effector protein. In other embodiments of the invention, cleavage may result with a putative transactivating crRNA (tracr RNA) sequence, e.g. cleavage by other CRISPR family 2023241400 09 Oct 2023 effector proteins, however after evaluation of the FnCpfl locus, Applicants concluded that target DNA cleavage by a Cpfl effector protein complex does not require a tracrRNA. Applicants determined that Cpfl effector protein complexes comprising only a Cpfl effector protein and a crRNA (guide RNA comprising a direct repeat sequence and a guide sequence) were sufficient to cleave target DNA. 10024] In any of the described methods the effector protein (e.g., Cpfl) and nucleic acid components may be provided via one or more polynucleotide molecules encoding the protein and / or nucleic acid component(s), and wherein the one or more polynucleotide molecules are operably configured to express the protein and / or the nucleic acid component(s). The one or more polynucleotide molecules may comprise one or more regulatory elements operably configured to express the protein and / or the nucleic acid component s). The one or more polynucleotide molecules may be comprised within one or more vectors. The invention comprehends such polynucleotide molecule(s), for instance such polynucleotide molecules operably configured to express the protein and / or the nucleic acid component(s), as well as such vector(s). 10025] In any of the described methods the strand break may be a single strand break or a double strand break.

[0026] Regulatory elements may comprise inducible promotors. Polynucleotides and / or vector systems may comprise inducible systems. ]0027] In any of the described methods the one or more polynucleotide molecules may be comprised in a delivery system, or the one or more vectors may be comprised in a delivery system. |0028] In any of the described methods the non-naturally occurring or engineered composition may be delivered via liposomes, particles (e.g. nanoparticles), exosomes, microvesicles, a gene-gun or one or more vectors, e.g., nucleic acid molecule or viral vectors. 10029] The invention also provides a non-naturally occurring or engineered composition which is a composition having the characteristics as discussed herein or defined in any of the herein described methods. |0030] The invention also provides a vector system comprising one or more vectors, the one or more vectors comprising one or more polynucleotide molecules encoding components 2023241400 09 Oct 2023 of a non-naturally occurring or engineered composition which is a composition having the characteristics as discussed herein or defined in any of the herein described methods.

[0031] The invention also provides a delivery system comprising one or more vectors or one or more polynucleotide molecules, the one or more vectors or polynucleotide molecules comprising one or more polynucleotide molecules encoding components of a non-naturally occurring or engineered composition which is a composition having the characteristics as discussed herein or defined in any of the herein described methods.

[0032] The invention also provides a non-naturally occurring or engineered composition, or one or more polynucleotides encoding components of said composition, or vector or delivery systems comprising one or more polynucleotides encoding components of said composition for use in a therapeutic method of treatment. The therapeutic method of treatment may comprise gene or genome editing, or gene therapy.

[0033] The invention also encompasses computational methods and algorithms to predict new Class 2 CRISPR-Cas systems and identify the components therein. |0034] The invention also provides for methods and compositions wherein one or more amino acid residues of the effector protein may be modified, e,g, an engineered or non-naturally-occurring effector protein or Cpfl. In an embodiment, the modification may comprise mutation of one or more amino acid residues of the effector protein. The one or more mutations may be in one or more catalytically active domains of the effector protein. The effector protein may have reduced or abolished nuclease activity compared with an effector protein lacking said one or more mutations. The effector protein may not direct cleavage of one or other DNA strand at the target locus of interest. The effector protein may not direct cleavage of either DNA strand at the target locus of interest. In a preferred embodiment, the one or more mutations may comprise two mutations. In a preferred embodiment the one or more amino acid residues are modified in a Cpfl effector protein, e,g, an engineered or non-naturally-occurring effector protein or Cpfl. In a preferred embodiment the Cpfl effector protein is an AsCpfl, LbCpfl or a FnCpfl effector protein. In a preferred embodiment, the one or more modified or mutated amino acid residues are D917A, El006A or D1255A with reference to the amino acid position numbering of the FnCpfl effector protein. In furher preferred embodiments, the one or more mutated amino acid residues are 2023241400 09 Oct 2023 D908A, E993A, D1263A with reference to the amino acid positions in AsCpfl or LbD832A, E925A, D947A or DI 180A with reference to the amino acid positions in LbCpfL

[0035] The invention also provides for the one or more mutations or the two or more mutations to be in a catalytically active domain of the effector protein comprising a RuvC domain. In some embodiments of the invention the RuvC domain may comprise a RuvCI, RuvCII or RuvCIII domain, or a catalytically active domain which is homologous to a RuvCI, RuvCII or RuvCIII domain etc or to any relevant domain as described in any of the herein described methods. The effector protein may comprise one or more heterologous functional domains. The one or more heterologous functional domains may comprise one or more nuclear localization signal (NLS) domains. The one or more heterologous functional domains may comprise at least two or more NLS domains. The one or more NLS domain(s) may be positioned at or near or in promixity to a terminus of the effector protein (e.g., Cpfl) and if two or more NLSs, each of the two may be positioned at or near or in promixity to a terminus of the effector protein (e.g., Cpfl) The one or more heterologous functional domains may comprise one or more transcriptional activation domains. In a preferred embodiment the transcriptional activation domain may comprise VP64. The one or more heterologous functional domains may comprise one or more transcriptional repression domains. In a preferred embodiment the transcriptional repression domain comprises a KRAB domain or a SID domain (e.g. SID4X). The one or more heterologous functional domains may comprise one or more nuclease domains. In a preferred embodiment a nuclease domain comprises Fokl.

[0036] The invention also provides for the one or more heterologous functional domains to have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity. At least one or more heterologous functional domains may be at or near the amino-terminus of the effector protein and / or wherein at least one or more heterologous functional domains is at or near the carboxyterminus of the effector protein. The one or more heterologous functional domains may be fused to the effector protein. The one or more heterologous functional domains may be 2023241400 09 Oct 2023 tethered to the effector protein. The one or more heterologous functional domains may be linked to the effector protein by a linker moiety.

[0037] In some embodiments, the functional domain is a deaminase, such as a cytidine deaminase. Cytidine deaminase may be directed to a target nucleic acid to where it directs conversion of cytidine to uridine, resulting in C to T substitutions (G to A on the complementary strand). In such an embodiment, nucleotide substitutions can be effected without DNA cleavage.

[0038] In some embodiments, the invention relates to a targeted base editor comprising a Type-V CRISPR effector fused to a deaminase. Targeted base editors based on Type-II CRISPR effectors were described in Komor et al., Nature (2016) 533:420-424; Kim et al., Nature Biotechnology (2017) 35:371-376; Shimatani et al., Nature Biotechnology (2017) doi:10.1038 / nbt.3833; and Zong et al., Nature Biotechnology (2017) doi:10.1038 / nbt.3811, each of which is incorporated by reference in its entirety. |0039] In some embodiments, the targeted base editor comprises a Cpfl effector protein fused to a cytidine deaminase. In some embodiments, the cytidine deaminase is fused to the carboxy terminus of the Cpfl effector protein. In some embodiments, the Cpfl effector protein and the cytidine deaminase are fused via a linker. In various embodiments, the linker may have different length and compositions. In some embodiments, the length of the linker sequence is in the range of about 3 to about 21 amino acids residues. In some embodiments, the length of the linker sequence is over 9 amino acid residues. In some embodiments, the length of the linker sequence is about 16 amino acid residues. In some embodiments, the Cpfl effector protein and the cytidine deaminase are fused via a XTEN linker. |0040] In some embodiments, the cytidine deaminase is of eukaryotic origin, such as of human, rat or lamprey origin. In some embodiments, the cytidine deaminase is AID, APOBEC3G, APOBEC1 or CDA1. In some embodiments, the targeted base editor further comprises a domain that inhibits base excision repair (BER). In some embodiments, the targeted base editor further comprises a uracil DNA glycosylase inhibitor (UGI) fused to the Cpfl effector protein or the cytidine deaminase. ]0041] In some embodiments, the cytidine deaminase has an efficient deamination window that encloses the nucleotides susceptible to deamination editing. Accordingly, in some embodiments, the “editing window width” refers to the number of nucleotide positions 2023241400 09 Oct 2023 at a given target site for which editing efficiency of the cytidine deaminase exceeds the halfmaximal value for that target site. In some embodiments, the cytidine deaminase has an editing window width in the range of about 1 to about 6 nucleotides. In some embodiments, the editing window width of the cytidine deaminase is 1,2, 3, 4, 5, or 6 nucleotides. |0042] Not intended to be bound by theory, it is contemplated that in some embodiments, the length of the linker sequence affects the editing window width. In some embodiments, the editing window width increases from about 3 to 6 nucleotides as the linker length extends from about 3 to 21 amino acids. In some embodiments, a 16-residue linker offers an efficient deamination window of about 5 nucleotides. In some embodiments, the length of the guide RNA affects the editing window width. In some embodiments, shortening the guide RNA leads to narrowed efficient deamination window of the cytidine deaminase.

[0043] In some embodiments, mutations to the cytidine deaminase affect the editing window width. In some embodiments, the targeted base editor comprises one or more mutations that reduce the catalytic efficiency of the cytidine deaminase, such that the deaminase is prevented from deamination of multiple cytidines per DNA binding event. In some embodiments, tryptophan at residue 90 (W90) of APOBEC1 or a corresponding tryptophan residue in a homologous sequence is mutated. In some embodiments, the Cpfl effector protein is fused to an APOBEC 1 mutant that comprises a W90Y or W90F mutation. In some embodiments, tryptophan at residue 285 (W285) of APOBEC3G, or a corresponding tryptophan residue in a homologous sequence is mutated. In some embodiments, the Cpfl effector protein is fused to an APOBEC3G mutant that comprises a W285Y or W285F mutation. ]0044] In some embodiments, the targeted base editor comprises one or more mutations that reduce tolerance for non-optimal presentation of a cytidine to the deaminase active site. In some embodiments, the cytidine deaminase comprises one or more mutations that alter substrate binding activity of the deaminase active site. In some embodiments, the cytidine deaminase comprises one or more mutations that alter the conformation of DNA to be recognized and bound by the deaminase active site. In some embodiments, the cytidine deaminase comprises one or more mutations that alter the substrate accessibility to the deaminase active site. In some embodiments, arginine at residue 126 (R126) of APOBEC1 or a corresponding arginine residue in a homologous sequence is mutated. In some 2023241400 09 Oct 2023 embodiments, the Cpfl effector protein is fused to an APOBEC 1 that comprises a R126A or R126E mutation. In some embodiments, tryptophan at residue 320 (R320) of APOBEC3G, or a corresponding arginine residue in a homologous sequence is mutated. In some embodiments, the Cpfl effector protein is fused to an APOBEC3G mutant that comprises a R320A or R320E mutation. In some embodiments, arginine at residue 132 (RI32) of APOBEC 1 or a corresponding arginine residue in a homologous sequence is mutated. In some embodiments, the Cpfl effector protein is fused to an APOBEC 1 mutant that comprises aR132E mutation.

[0045] In some embodiments, the APOBEC 1 domain of the targeted base editor comprises one, two, or three mutations selected from W90Y, W90F, R126A, R126E, and R132E. In some embodiments, the APOBEC 1 domain comprises double mutations of W90Y and R126E. In some embodiments, the APOBEC 1 domain comprises double mutations of W90Y and R132E. In some embodiments, the APOBEC 1 domain comprises double mutations of R126E and R132E. In some embodiments, the APOBEC1 domain comprises three mutations of W90Y, R126E and R132E. 10046] In some embodiments, one or more mutations in the cytidine deaminase as disclosed herein reduce the editing window width to about 2 nucleotides. In some embodiments, one or more mutations in the cytidine deaminase as disclosed herein reduce the editing window width to about 1 nucleotide. In some embodiments, one or more mutations in the cytidine deaminase as disclosed herein reduce the editing window width while only minimally or modestly affecting the editing efficiency of the enzyme. In some embodiments, one or more mutations in the cytidine deaminase as disclosed herein reduce the editing window width without reducing the editing efficiency of the enzyme. In some embodiments, one or more mutations in the cytidine deaminase as disclosed herein enable discrimination of neighboring cytidine nucleotides, which would be otherwise edited with similar efficiency by the cytidine deaminase.

[0047] In some embodiments, the Cpfl effector protein is a dead Cpfl having a catalytically inactive RuvC domain (e.g., AsCpfl D908A, AsCpfl E993A, AsCpfl D1263A, LbCpfl D832A, LbCpfl E925A, LbCpfl D947A, and LbCpfl D1180A). In some embodiments, the Cpfl effector protein is a Cpfl nickase having a catalytically inactive Nuc domain (e.g., AsCpfl R1226A). 2023241400 09 Oct 2023 |0048] In some embodiments, the Cpfl effector protein recognizes a protospacer-adjacent motif (PAM) sequence on the target DNA. In some embodiments, the PAM is upstream or downstream of the target cytidine. In some embodiments, interaction between the Cpfl effector protein and the PAM sequence places the target cytidine within the efficient deamination window of the cytidine deaminase. In some embodiments, PAM specificity of the Cpfl effector protein determines the sites that can be edited by the targeted base editor. In some embodiments, the Cpfl effector protein can recognize one or more PAM sequences including but not limited to TTTV wherein V is A / C or G (e.g., wild-type AsCpfl or LbCpfl), and TTN wherein N is A / C / G or T (e.g., wild-type FnCpfl). In some embodiments, the Cpfl effector protein comprises one or more amino acid mutations resulting in altered PAM sequences. For example, the Cpfl effector protein can be an AsCpfl mutant comprising one or more amino acid mutations at S542 (e.g., S542R), K548 (e.g., K548V), N552 (e.g., N552R), or K607 (e.g., K607R), or an LbCpfl mutant comprising one or more amino acid mutations at G532 (e.g., G532R), K538 (e.g., K538V), Y542 (e.g., Y542R), or K595 (e.g., K595R). 10049] WO2016022363 also describes compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. Accordingly, similar Cpfl fusion proteins are provided herein. In particular embodiments, the Cpfl fusion protein comprises a liganddependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein

[0050] In some embodiments, the invention relates to a method of targeted base editing, comprising contacting the targeted base editor described above with a prokaryotic or eukaryotic cell, preferably a mammalian cell, simultaneously or sequentially with a guide nucleic acid, wherein the guide nucleic acid forms a complex with the Cpfl effector protein and directs the complex to bind a template strand of a target DNA in the cell, and wherein the cytidine deaminase converts a C to a U in the non-template strand of the target DNA. In some 2023241400 09 Oct 2023 embodiments, the Cpfl effector protein nicks the template / non-edited strand containing a G opposite the edited U.

[0051] The invention also provides for the effector protein (e.g., a Cpfl) comprising an effector protein (e.g., a Cpfl) from an organism from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium or Acidaminococcus.

[0052] The invention also provides for the effector protein (e.g., a Cpfl) comprising an effector protein (e.g., a Cpfl) from an organism from S. mutans, S. agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C. coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii. 10053] The effector protein may comprise a chimeric effector protein comprising a first fragment from a first effector protein (e.g., a Cpfl) ortholog and a second fragment from a second effector (e.g., a Cpfl) protein ortholog, and wherein the first and second effector protein orthologs are different. At least one of the first and second effector protein (e.g., a Cpfl) orthologs may comprise an effector protein (e.g., a Cpfl) from an organism comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium or Acidaminococcus', e.g., a chimeric effector protein comprising a first fragment and a second fragment wherein each of the first and second fragments is selected from a Cpfl of an organism comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, 2023241400 09 Oct 2023 Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Camobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium or Acidaminococcus wherein the first and second fragments are not from the same bacteria; for instance a chimeric effector protein comprising a first fragment and a second fragment wherein each of the first and second fragments is selected from a Cpfl of S. mutans, S. agalactiae, S. equisimilis, S. sanguinis, S. pneumonia; C. jejuni, C. coli; N. salsuginis, N. tergarcus; S. auricularis, S. carnosus; N. meningitides, N. gonorrhoeae; L. monocytogenes, L. ivanovii; C. botulinum, C. difficile, C. tetani, C. sordellii; Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW201 l_GWA2_33_10, Parcubacteria bacterium GW2011 _GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11 _00205, Butyrivibrio sp. NC3005, Thiomicrospira sp. XS5, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae, wherein the first and second fragments are not from the same bacteria. In particular embodiments, the chimeric effector protein is a protein comprising a first fragment and a second fragment wherein each of the first and second fragments is selected from a Cpfl of Acidaminococcus sp. BV3L6, Thiomicrospira sp. XS5, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11 _00205, Lachnospiraceae bacterium MA2020. |0054] In preferred embodiments of the invention the effector protein is derived from a Cpfl locus (herein such effector proteins are also referred to as “Cpflp”), e.g., a Cpfl protein (and such effector protein or Cpfl protein or protein derived from a Cpfl locus is also called “CRISPR enzyme”). Cpfl loci include but are not limited to the Cpfl loci of bacterial species listed in Figure 64 of EP3009511 or US2016208243. In a more preferred embodiment, the Cpflp is derived from a bacterial species selected from Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017   1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium   GW2011 _GWA2_33_10,   Parcubacteria bacterium 2023241400 09 Oct 2023 GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11 _00205, Butyrivibrio sp. NC3005, Thiomicrospira sp. XS5, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae. In certain preferred embodiments, the Cpflp is derived from a bacterial species selected from Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium ND2006, Lachnospiraceae bacterium MA2020, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11 _00205, Butyrivibrio sp. NC3005, or Thiomicrospira sp. XS5. In certain embodiments, the effector protein is derived from a subspecies of Francisella tularensis 1, including but not limited to Francisella tularensis subsp. Novicida.

[0055] In further embodiments of the invention a protospacer adjacent motif (PAM) or PAM-like motif directs binding of the effector protein complex to the target locus of interest. In a preferred embodiment of the invention, the PAM is 5’ TTN, where N is A / C / G or T and the effector protein is FnCpflp, or a Cpfl from Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAXll_00205, Butyrivibrio sp. NC3005, Thiomicrospira sp. XS5, or Lachnospiraceae bacterium MA2020. In another preferred embodiment of the invention, the PAM is 5’ TTTV, where V is A / C or G and the effector protein is AsCpfl, LbCpfl or PaCpflp. In certain embodiments, the PAM is 5’ TTN, where N is A / C / G or T, the effector protein is FnCpflp, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAXll_00205, Butyrivibrio sp. NC3005, Thiomicrospira sp. XS5, or Lachnospiraceae bacterium MA2020, and the PAM is located upstream of the 5’ end of the protospacer. In certain embodiments of the invention, the PAM is 5’ CTA, where the effector protein is FnCpflp, and the PAM is located upstream of the 5’ end of the protospacer or the target locus. In preferred embodiments, the invention provides for an expanded targeting range for RNA guided genome editing nucleases wherein the T-rich PAMs of the Cpfl family allow for targeting and editing of AT-rich genomes. |0056] In certain embodiments, the CRISPR enzyme is engineered and can comprise one or more mutations that reduce or eliminate a nuclease activity. The amino acid positions in the FnCpflp RuvC domain include but are not limited to D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A and N1257A. Applicants 2023241400 09 Oct 2023 have also identified a putative second nuclease domain which is most similar to PD-(D / E)XK nuclease superfamily and Hindi endonuclease like. The point mutations to be generated in this putative nuclease domain to substantially reduce nuclease activity include but are not limited to N580A, N584A, T587A, W609A, D610A, K613A, E614A, D616A, K624A, D625A, K627A and Y629A. In a preferred embodiment, the mutation in the FnCpflp RuvC domain is D917A or E1006A, wherein the D917A or E1006A mutation completely inactivates the DNA cleavage activity of the FnCpfl effector protein. In another embodiment, the mutation in the FnCpflp RuvC domain is D1255A, wherein the mutated FnCpfl effector protein has significantly reduced nucleolytic activity.

[0057] The amino acid positions in the AsCpflp RuvC domain include but are not limited to 908, 993, and 1263. In a preferred embodiment, the mutation in the AsCpflp RuvC domain is D908A, E993A, and DI263A, wherein the D908A, E993A, and DI263A mutations completely inactivates the DNA cleavage activity of the AsCpfl effector protein. The amino acid positions in the LbCpflp RuvC domain include but are not limited to832, 947 or 1180 . In a preferred embodiment, the mutation in the LbCpflp RuvC domain is LbD832A, E925A, D947A or DI 180A, wherein the LbD832A E925A, D947A or DI 180A mutations completely inactivates the DNA cleavage activity of the LbCpfl effector protein.

[0058] Mutations can also be made at neighboring residues, e.g., at amino acids near those indicated above that participate in the nuclease acrivity. In some embodiments, only the RuvC domain is inactivated, and in other embodiments, another putative nuclease domain is inactivated, wherein the effector protein complex functions as a nickase and cleaves only one DNA strand. In a preferred embodiment, the other putative nuclease domain is a HincII-like endonuclease domain. In some embodiments, two FnCpfl variants (each a different nickase) are used to increase specificity, two nickase variants are used to cleave DNA at a target (where both nickases cleave a DNA strand, while miminizing or eliminating off-target modifications where only one DNA strand is cleaved and subsequently repaired). In preferred embodiments the Cpfl effector protein cleaves sequences associated with or at a target locus of interest as a homodimer comprising two Cpfl effector protein molecules. In a preferred embodiment the homodimer may comprise two Cpfl effector protein molecules comprising a different mutation in their respective RuvC domains. 2023241400 09 Oct 2023 |0059] The invention contemplates methods of using two or more nickases, in particular a dual or double nickase approach. In some aspects and embodiments, a single type FnCpfl nickase may be delivered, for example a modified FnCpfl or a modified FnCpfl nickase as described herein. This results in the target DNA being bound by two FnCpfl nickases. In addition, it is also envisaged that different orthologs may be used, e.g, an FnCpfl nickase on one strand (e.g., the coding strand) of the DNA and an ortholog on the non-coding or opposite DNA strand. The ortholog can be, but is not limited to, a Cas9 nickase such as a SaCas9 nickase or a SpCas9 nickase. It may be advantageous to use two different orthologs that require different PAMs and may also have different guide requirements, thus allowing a greater deal of control for the user. In certain embodiments, DNA cleavage will involve at least four types of nickases, wherein each type is guided to a different sequence of target DNA, wherein each pair introduces a first nick into one DNA strand and the second introduces a nick into the second DNA strand. In such methods, at least two pairs of single stranded breaks are introduced into the target DNA wherein upon introduction of first and second pairs of single-strand breaks, target sequences between the first and second pairs of single-strand breaks are excised. In certain embodiments, one or both of the orthologs is controllable, i.e. inducible. 10060] In certain embodiments of the invention, the guide RNA or mature crRNA comprises, consists essentially of, or consists of a direct repeat sequence and a guide sequence or spacer sequence. In certain embodiments, the guide RNA or mature crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or spacer sequence. In certain embodiments the guide RNA or mature crRNA comprises 19 nts of partial direct repeat followed by 20-30 nt of guide sequence or spacer sequence, advantageously about 20 nt, 23-25 nt or 24 nt. In certain embodiments, the effector protein is a FnCpfl effector protein and requires at least 16 nt of guide sequence to achieve detectable DNA cleavage and a minimum of 17 nt of guide sequence to achieve efficient DNA cleavage in vitro. In certain embodiments, the direct repeat sequence is located upstream (i.e., 5’) from the guide sequence or spacer sequence. In a preferred embodiment the seed sequence (i.e. the sequence essential critical for recognition and / or hybridization to the sequence at the target locus) of the FnCpfl guide RNA is approximately within the first 5 nt on the 5’ end of the guide sequence or spacer sequence. 2023241400 09 Oct 2023 10061] In preferred embodiments of the invention, the mature crRNA comprises a stem loop or an optimized stem loop structure or an optimized secondary structure. In preferred embodiments the mature crRNA comprises a stem loop or an optimized stem loop structure in the direct repeat sequence, wherein the stem loop or optimized stem loop structure is important for cleavage activity. In certain embodiments, the mature crRNA preferably comprises a single stem loop. In certain embodiments, the direct repeat sequence preferably comprises a single stem loop. In certain embodiments, the cleavage activity of the effector protein complex is modified by introducing mutations that affect the stem loop RNA duplex structure. In preferred embodiments, mutations which maintain the RNA duplex of the stem loop may be introduced, whereby the cleavage activity of the effector protein complex is maintained. In other preferred embodiments, mutations which disrupt the RNA duplex structure of the stem loop may be introduced, whereby the cleavage activity of the effector protein complex is completely abolished. |0062] The invention also provides for the nucleotide sequence encoding the effector protein being codon optimized for expression in a eukaryote or eukaryotic cell in any of the herein described methods or compositions. In an embodiment of the invention, the codon optimized effector protein is FnCpflp and is codon optimized for operability in a eukaryotic cell or organism, e.g., such cell or organism as elsewhere herein mentioned, for instance, without limitation, a yeast cell, or a mammalian cell or organism, including a mouse cell, a rat cell, and a human cell or non-human eukaryote organism, e.g., plant.

[0063] In certain embodiments of the invention, at least one nuclear localization signal (NLS) is attached to the nucleic acid sequences encoding the Cpfl effector proteins. In preferred embodiments at least one or more C-terminal or N-terminal NLSs are attached (and hence nucleic acid molecule(s) coding for the the Cpfl effector protein can include coding for NLS(s) so that the expressed product has the NLS(s) attached or connected). In a preferred embodiment a C-terminal NLS is attached for optimal expression and nuclear targeting in eukaryotic cells, preferably human cells. In certain embodiments, the NLS sequence is heterologous to the nucleic acid sequence encoding the Cpfl effector protein. In a preferred embodiment, the codon optimized effector protein is FnCpflp and the spacer length of the guide RNA is from 15 to 35 nt. In certain embodiments, the spacer length of the guide RNA is at least 16 nucleotides, such as at least 17 nucleotides. In certain embodiments, the spacer 2023241400   22 Apr 2026 length is from 15 to 17 nt, from 17 to 20 nt, from 20 to 24 nt, eg. 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, from 27-30 nt, from 30-35 nt, or 35 nt or longer. In certain embodiments of the invention, the codon optimized effector protein is FnCpf1p and the direct repeat length of the guide RNA is at least 16 nucleotides. In certain embodiments, the codon optimized effector protein is FnCpf1p and the direct repeat length of the guide RNA is from 16 to 20 nt, e.g., 16, 17, 18, 19, or 20 nucleotides. In certain preferred embodiments, the direct repeat length of the guide RNA is 19 nucleotides.

[0064] The invention also encompasses methods for delivering multiple nucleic acid components, wherein each nucleic acid component is specific for a different target locus of interest thereby modifying multiple target loci of interest. The nucleic acid component of the complex may comprise one or more protein-binding RNA aptamers. The one or more aptamers may be capable of binding a bacteriophage coat protein. The bacteriophage coat protein may be selected from the group comprising Q0, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ^Cb5, ^Cb8r, ^Cb12r, ^Cb23r, 7s and PRR1. In a preferred embodiment the bacteriophage coat protein is MS2. The invention also provides for the nucleic acid component of the complex being 30 or more, 40 or more or 50 or more nucleotides in length.

[0065] The invention also encompasses the cells, components and / or systems of the present invention having trace amounts of cations present in the cells, components and / or systems. Advantageously, the cation is magnesium, such as Mg2+. The cation may be present in a trace amount. A preferred range may be about 1 mM to about 15 mM for the cation, which is advantageously Mg2+. A preferred concentration may be about 1 mM for human based cells, components and / or systems and about 10 mM to about 15 mM for bacteria based cells, components and / or systems. See, e.g., Gasiunas et al., PNAS, published online September 4, 2012, www.pnas.org / cgi / doi / 10.1073 / pnas.1208507109.

[0066] Accordingly, the present invention does not encompass any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement 2023241400 09 Oct 2023 requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. Nothing herein is to be construed as a promise. |0067] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of' and "consists essentially of' have the meaning ascribed to them in U.S. Patent law.

[0068] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0070] FIGS. 1A-1BB show the sequence alignment of Cas-Cpfl orthologs (SEQ ID NOS: 284-341, respectively, in order of appearance). |0071J FIGS. 2A-2B show the overview of Cpfl loci alignment.

[0072] FIGS. 3A-3X shows the PACYC184 FnCpfl (PY001) vector contruct (SEQ ID NOS: 342-364, respectively, in order of appearance). ]0073] FIGS. 4A-4I show the sequence of humanized PaCpfl, with the nucleotide sequence as SEQ ID NO: 365 and the protein sequence as SEQ ID NO: 366. 10074]    FIG. 5 depicts a PAM challenge assay. 10075] FIG. 6 depicts a schematic of an endogenous FnCpfl locus. pYOOOl is a pACY184 backbone (from NEB) with a partial FnCpfl locus. The FnCpfl locus was PCR amplified in three pieces and cloned into Xbal and Hind3 cut pACYC184 using Gibson assembly. PYOOOl contains the endogenous FnCpfl locus from 255bp of the acetyltransferase 3’ 2023241400 09 Oct 2023 sequence to the fourth spacer sequence. Only spacer 1 -3 are potentially active since space 4 is no longer flanked by direct repeats.

[0076] FIG. 7 depicts PAM libraries, which discloses discloses SEQ ID NOS: 367-370, respectively, in order of appearance. Both PAM libraries (left and right) are in pUC19. The complexity of left PAM library is 48 ~ 65k and the complexity of the right PAM library is 47 ~ 16k. Both libraries were prepared with a representation of > 500.

[0077] FIG. 8A-8E depicts FnCpfl PAM Screen Computational Analysis. After sequencing of the screen DNA, the regions corresponding to either the left PAM or the right PAM were extracted. For each sample, the number of PAMs present in the sequenced library were compared to the number of expected PAMs in the library (4A8 for the left library, 4A7 for the right). (A) The left library showed PAM depletion. To quantify this depletion, an enrichment ratio was calculated. For both conditions (control pACYC or FnCpfl containing pACYC) the ratio was calculated for each PAM in the library as .      , sample+ 0.01 ratio = — log2 . . .    -------—— initial library + 0.01 Plotting the distribution shows little enrichment in the control sample and enrichment in both bioreps. (B-D) depict PAM ratio distributions. (E) All PAMs above a ratio of 8 were collected, and the frequency distributions were plotted, revealing a 5’ YYN PAM.

[0078] FIG. 9 depicts RNAseq analysis of the Francisella tolerances Cpfl locus, which shows that the CRISPR locus is actively expressed. In addition to the Cpfl and Cas genes, two small non-coding transcript are highly transcribed, which might be the putative tracrRNAs. The CRISPR array is also expressed. Both the putative tracrRNAs and CRISPR array are transcribed in the same direction as the Cpfl and Cas genes. Here all RNA transcripts identified through the RNAseq experiment are mapped against the locus. After further evaluation of the FnCpfl locus, Applicants concluded that target DNA cleavage by a Cpfl effector protein complex does not require a tracrRNA. Applicants determined that Cpfl effector protein complexes comprising only a Cpfl effector protein and a crRNA (guide RNA comprising a direct repeat sequence and a guide sequence) were sufficient to cleave target DNA. 2023241400 09 Oct 2023

[0079] FIG. 10 depicts zooming into the Cpfl CRISPR array. Many different short transcripts can be identified. In this plot, all identified RNA transcripts are mapped against the Cpfl locus.

[0080] FIG. 11 depicts identifying two putative tracrRNAs after selecting transcripts that are less than 85 nucleotides long.

[0081] FIG. 12 depicts zooming into putative tracrRNA 1 (SEQ ID NO: 371) and the CRISPR array.

[0082] FIG. 13 depicts zooming into putative tracrRNA 2 which discloses SEQ ID NOS: 372-378, respectively, in order of appearance.

[0083] FIG. 14 depicts putative crRNA sequences (repeat in blue, spacer in black) (SEQ ID NOS: 379 and 380, respectively, in order of appearance).

[0084] FIG. 15 shows a schematic of the assay to confirm the predicted FnCpfl PAM in vivo.

[0085] FIG. 16 shows FnCpfl locus carrying cells and control cells transformed with pUC19 encoding endogenous spacer 1 with 5’ TTN PAM.

[0086] FIG. 17 shows a schematic indicating putative tracrRNA sequence positions in the FnCpfl locus, the crRNA (SEQ ID NO: 381) and the pUC protospacer vector.

[0087] FIG. 18 is a gel showing the PCR fragment with TTa PAM and proto-spacer 1 sequence incubated in cell lysate.

[0088] FIG. 19 is a gel showing the pUC-spacerl with different PAMs incubated in cell lysate.

[0089] FIG. 20 is a gel showing the BasI digestion after incubation in cell lysate.

[0090] FIG. 21 is a gel showing digestion results for three putative crRNA sequences (SEQ ID NO: 382).

[0091] FIG. 22 is a gel showing testing of different lengths of spacer against a piece of target DNA containing the target site: 5'-TTAgagaagtcatttaataaggccactgttaaaa-3' (SEQ ID NO: 119). The results show that crRNAs 1-7 mediated successful cleavage of the target DNA in vitro with FnCpfl. crRNAs 8-13 did not facilitate cleavage of the target DNA. SEQ ID NOS: 383-421 are disclosed, respectively, in order of appearance.

[0092] FIG. 23 is a schematic indicating the minimal FnCpfl locus.

[0093] FIG. 24 is a schematic indicating the minimal Cpfl guide (SEQ ID NO: 422). 2023241400 09 Oct 2023

[0094] FIG. 25A-25E depicts PaCpfl PAM Screen Computational Analysis. After sequencing of the screen DNA, the regions corresponding to either the left PAM or the right PAM were extracted. For each sample, the number of PAMs present in the sequenced library were compared to the number of expected PAMs in the library (4A7). (A) The left library showed very slight PAM depletion. To quantify this depletion, an enrichment ratio was calculated. For both conditions (control pACYC or PaCpfl containing pACYC) the ratio was calculated for each PAM in the library as ,      sample + 0.01 ratio = — log,----— ---------- 2 initial library + 0.01 Plotting the distribution shows little enrichment in the control sample and enrichment in both bioreps. (B-D) depict PAM ratio distributions. (E) All PAMs above a ratio of 4.5 were collected, and the frequency distributions were plotted, revealing a 5’ TTTV PAM, where V is A or C or G.

[0095] FIG. 26 shows a vector map of the human codon optimized PaCpfl sequence depicted as CBh-NLS-huPaCpfl-NLS-3xHA-pA.

[0096] FIGS. 27A-27B show a phylogenetic tree of 51 Cpfl loci in different bacteria. Highlighted boxes indicate Gene Reference #s: 1-17. Boxed / numbered orthologs were tested for in vitro cleavage activity with predicted mature crRNA; orthologs with boxes around their numbers showed activity in the in vitro assay.

[0097] FIGS. 28A-28H show the details of the human codon optimized sequence for Lachnospiraceae bacterium MC2017 1 Cpfl having a gene length of 3849 nts (Ref #3 in FIG. 27). FIG. 28A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 28B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 28C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 28D: Restriction Enzymes and CIS-Acting Elements. FIG. 28E: Remove Repeat Sequences. FIG. 28F-G: Optimized Sequence (Optimized Sequence Length: 3849, GC% 54.70) (SEQ ID NO: 423). FIG. 28H: Protein Sequence (SEQ ID NO: 424). 2023241400 09 Oct 2023

[0098] FIGS. 29A-29H show the details of the human codon optimized sequence for Butyrivibrio proteoclasticus Cpfl having a gene length of 3873 nts (Ref #4 in FIG. 27). FIG. 29A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 29B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 29C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 29D: Restriction Enzymes and CISActing Elements. FIG. 29E: Remove Repeat Sequences. FIG. 29F-G: Optimized Sequence (Optimized Sequence Length: 3873, GC% 54.05) (SEQ ID NO: 425). FIG. 29H: Protein Sequence (SEQ ID NO: 426).

[0099] FIGS. 30A-30H show the details of the human codon optimized sequence for Peregrinibacteria bacterium GW201 l_GWA2_33_10 Cpfl having a gene length of 4581 nts (Ref #5 in FIG. 27). FIG. 30A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 30B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 30C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 30D: Restriction Enzymes and CIS-Acting Elements. FIG. 30E: Remove Repeat Sequences. FIG. 30F-G: Optimized Sequence (Optimized Sequence Length: 4581, GC% 50.81) (SEQ ID NO: 427). FIG. 30H: Protein Sequence (SEQ ID NO: 428).

[00100] FIGS. 31A-31H show the details of the human codon optimized sequence for Parcubacteria bacterium GW2011_GWC2_44_17 Cpfl having a gene length of 4206 nts (Ref #6 in FIG. 27). FIG. 31 A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high 2023241400 09 Oct 2023 gene expression level. FIG. 3 IB: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 3 IC: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 3ID: Restriction Enzymes and CIS-Acting Elements. FIG. 3 IE: Remove Repeat Sequences. FIG. 31F-G: Optimized Sequence (Optimized Sequence Length: 4206, GC% 52.17) (SEQ ID NO: 429). FIG. 31H: Protein Sequence (SEQ ID NO: 430). 100101] FIGS. 32A-32H show the details of the human codon optimized sequence for Smithella sp. SCADC Cpfl having a gene length of 3900 nts (Ref #7 in FIG. 27). FIG. 32A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 32B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 32C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 32D: Restriction Enzymes and CISActing Elements. FIG. 69E: Remove Repeat Sequences. FIG. 32F-G: Optimized Sequence (Optimized Sequence Length: 3900, GC% 51.56) (SEQ ID NO: 431). FIG. 32H: Protein Sequence (SEQ ID NO: 432).

[00102] FIGS. 33A-33H show the details of the human codon optimized sequence for Acidaminococcus sp. BV3L6 Cpfl having a gene length of 4071 nts (Ref #8 in FIG. 27). FIG. 33A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 33B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 33C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 33D: Restriction Enzymes and CIS- 2023241400 09 Oct 2023 Acting Elements. FIG. 70E: Remove Repeat Sequences. FIG. 33F-G: Optimized Sequence (Optimized Sequence Length: 4071, GC% 54.89) (SEQ ID NO: 433). FIG. 33H: Protein Sequence (SEQ ID NO: 434).

[00103] FIGS. 34A-34H show the details of the human codon optimized sequence for Lachnospiraceae bacterium MA2020 Cpfl having a gene length of 3768 nts (Ref #9 in FIG. 27). FIG. 34A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 34B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 34C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 34D: Restriction Enzymes and CIS-Acting Elements. FIG. 7IE: Remove Repeat Sequences. FIG. 34F-G: Optimized Sequence (Optimized Sequence Length: 3768, GC% 51.53) (SEQ ID NO: 435). FIG. 34H: Protein Sequence (SEQ ID NO: 436).

[00104] FIGS. 35A-35H show the details of the human codon optimized sequence for Candidatus Methanoplasma tennitum Cpfl having a gene length of 3864 nts (Ref #10 in FIG. 27). FIG. 35A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 35B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 35C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 35D: Restriction Enzymes and CIS-Acting Elements. FIG. 35E: Remove Repeat Sequences. FIG. 35F-G: Optimized Sequence (Optimized Sequence Length: 3864, GC% 52.67) (SEQ ID NO: 437). FIG. 35H: Protein Sequence (SEQ ID NO: 438).

[00105] FIGS. 36A-36H show the details of the human codon optimized sequence for Eubacterium eligens Cpfl having a gene length of 3996 nts (Ref #11 in FIG. 27). FIG. 36A: 2023241400 09 Oct 2023 Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 36B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 36C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 36D: Restriction Enzymes and CISActing Elements. FIG. 36E: Remove Repeat Sequences. FIG. 36F-G: Optimized Sequence (Optimized Sequence Length: 3996, GC% 50.52) (SEQ ID NO: 439). FIG. 36H: Protein Sequence (SEQ ID NO: 440).

[00106] FIGS. 37A-37H show the details of the human codon optimized sequence for Moraxella bovoculi 237 Cpfl having a gene length of 4269 nts (Ref #12 in FIG. 27). FIG. 37A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 37B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 37C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 37D: Restriction Enzymes and CISActing Elements. FIG. 37E: Remove Repeat Sequences. FIG. 37F-G: Optimized Sequence (Optimized Sequence Length: 4269, GC% 53.58) (SEQ ID NO: 441). FIG. 74H: Protein Sequence (SEQ ID NO: 442).

[00107] FIGS. 38A-38H show the details of the human codon optimized sequence for Leptospira inadai Cpfl having a gene length of 3939 nts (Ref #13 in FIG. 27). FIG. 38A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 38B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage 2023241400 09 Oct 2023 frequency for a given amino acid in the desired expression organism. FIG. 38C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 38D: Restriction Enzymes and CISActing Elements. FIG. 38E: Remove Repeat Sequences. FIG. 38F-G: Optimized Sequence (Optimized Sequence Length: 3939, GC% 51.30) (SEQ ID NO: 443). FIG. 38H: Protein Sequence (SEQ ID NO: 444). [001081 FIGS. 39A-39H show the details of the human codon optimized sequence for Lachnospiraceae bacterium ND2006 Cpfl having a gene length of 3834 nts (Ref #14 in FIG. 27). FIG. 39A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 39B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 39C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 39D: Restriction Enzymes and CIS-Acting Elements. FIG. 39E: Remove Repeat Sequences. FIG. 39F-G: Optimized Sequence (Optimized Sequence Length: 3834, GC% 51.06) (SEQ ID NO: 445). FIG. 39H: Protein Sequence (SEQ ID NO: 446).

[00109] FIGS. 40A-40H show the details of the human codon optimized sequence for Porphyromonas crevioricanis 3 Cpfl having a gene length of 3930 nts (Ref #15 in FIG. 27). FIG. 40A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 40B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 40C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 40D: Restriction Enzymes and CIS-Acting Elements. FIG. 40E: Remove Repeat Sequences. FIG. 40F-G: Optimized 2023241400 09 Oct 2023 Sequence (Optimized Sequence Length: 3930, GC% 54.42) (SEQ ID NO: 447). FIG. 40H: Protein Sequence (SEQ ID NO: 448).

[00110] FIGS. 41A-41H show the details of the human codon optimized sequence for Prevotella disiens Cpfl having a gene length of 4119 nts (Ref #16 in FIG. 27). FIG. 41A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 41B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 4IC: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 4ID: Restriction Enzymes and CISActing Elements. FIG. 41E: Remove Repeat Sequences. FIG. 41F-G: Optimized Sequence (Optimized Sequence Length: 4119, GC% 51.88) (SEQ ID NO: 449). FIG. 41H: Protein Sequence (SEQ ID NO: 450).

[00111] FIGS. 42A-42H shows the details of the human codon optimized sequence for Porphyromonas macacae Cpfl having a gene length of 3888 nts (Ref #17 in FIG. 27). FIG. 42A: Codon Adaptation Index (CAI). The distribution of codon usage frequency along the length of the gene sequence. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level. FIG. 42B: Frequency of Optimal Codons (FOP). The percentage distribution of codons in computed codon quality groups. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. FIG. 42C: GC Content Adjustment. The ideal percentage range of GC content is between 30-70%. Peaks of %GC content in a 60 bp window have been removed. FIG. 79D: Restriction Enzymes and CISActing Elements. FIG. 42E: Remove Repeat Sequences. FIG. 42F-G: Optimized Sequence (Optimized Sequence Length: 3888, GC% 53.26) (SEQ ID NO: 451). FIG. 42H: Protein Sequence (SEQ ID NO: 452).

[00112] FIG. 43A-43I shows direct repeat (DR) sequences for each ortholog (refer to numbering Ref # 3-17 in FIG. 27) and their predicted fold structure. SEQ ID NOS: 453-486, respectively, are disclosed in order of appearance. 2023241400 09 Oct 2023

[00113] FIG. 44 shows cleavage of a PCR amplicon of the human Emxl locus. SEQ ID NOS: 487-491, respectively, are disclosed in order of appearance.

[00114] FIG. 45A-45B shows the effect of truncation in 5’ DR on cleavage Activity. (A) shows a gel in which cleavage results with 5 DR truncations is indicated. (B) shows a diagram in which crDNA deltaDR5 disrupted the stem loop at the 5’ end. This indicates that the stemloop at the 5’ end is essential for cleavage activity. SEQ ID NOS: 492-497, respectively, are disclosed in order of appearance.

[00115] FIG. 46 shows the effect of crRNA-DNA target mismatch on cleavage efficiency. SEQ ID NOS: 498-508, respectively, are disclosed in order of appearance.

[00116] FIG. 47 shows the cleavage of DNA using purified Francisella and Prevotella Cpfl. SEQ ID NO: 509 is disclosed.

[00117] FIG. 48A-48B show diagrams of DR secondary structures. (A) FnCpfl DR secondary structure (SEQ ID NO: 510) (stem loop highlighted). (B) PaCpfl DR secondary structure (SEQ ID NO: 511) (stem loop highlighted, identical except for a single base difference in the loop region).

[00118] FIG. 49 shows a further depiction of the RNAseq analysis of the FnCpl locus.

[00119] FIG. 50A-50B show schematics of mature crRNA sequences. (A) Mature crRNA sequences for FnCpfl. (B) Mature crRNA sequences for PaCpfl. SEQ ID NOS: 512-515, respectively, are disclosed in order of appearance.

[00120] FIG. 51 shows cleavage of DNA using human codon optimized Francisella novicida FnCpfl. The top band corresponds to un-cleaved full length fragment (606bp). Expected cleavage product sizes of ~345bp and -261 bp are indicated by triangles.

[00121] FIG. 52 shows in vitro ortholog assay demonstrating cleavage by Cpfl orthologs.

[00122] FIGS. 53A-53C show computationally derived PAMs from the in vitro cutting assay.

[00123] FIG. 54 shows Cpfl cutting in a staggered fashion with 5’ overhangs. SEQ ID NOS: 516-518, respectively, are disclosed in order of appearance.

[00124] FIG. 55 shows effect of spacer length on cutting. SEQ ID NOS: 519-525, respectively, are disclosed in order of appearance.

[00125] FIG. 56 shows SURVEYOR data for FnCpfl mediated indels in HEK293T cells. 2023241400 09 Oct 2023

[00126] FIGS. 57A-57F show the processing of transcripts when sections of the FnCpfl locus are deleted as compared to the processing of transcripts in a wild type FnCpfl locus. FIGS. 57B, 57D and 57F zoom in on the processed spacer. SEQ ID NOS: 526-574, respectively, are disclosed in order of appearance.

[00127] FIGS. 58A-58E show the Francisella tularensis subsp. novicida U112 Cpfl CRISPR locus provides immunity against transformation of plasmids containing protospacers flanked by a 5’-TTN PAM. FIG. 58A show the organization of two CRISPR loci found in Francisella tularensis subsp. novicida UI 12 (NC_008601). The domain organization of FnCas9 and FnCpfl are compared. FIG. 58B provide a schematic illustration of the plasmid depletion assay for discovering the PAM position and identity. Competent E. coli harboring either the heterologous FnCpfl locus plasmid (pFnCpfl) or the empty vector control were transformed with a library of plasmids containing the matching protospacer flanked by randomized 5 ’ or 3 ’ PAM sequences and selected with antibiotic to deplete plasmids carrying successfully-targeted PAM. Plasmids from surviving colonies were extracted and sequenced to determine depleted PAM sequences. FIGS. 58C-58D show sequence logos for the FnCpfl PAM as determined by the plasmid depletion assay. Letter height at position is determined by information content; error bars show 95% Bayesian confidence interval. FIG. 58E shows E. coli harboring pFnCpfl demonstrate robust interference against plasmids carrying 5’-TTN PAMs (n = 3, error bars represent mean ± S.E.M.).

[00128] FIGS. 59A-59C shows heterologous expression of FnCpfl and CRISPR array in E. coli is sufficient to mediate plasmid DNA interference and crRNA maturation. Small RNA-seq of Francisella tularensis subsp. novicida U112 (FIG. 59A) reveals transcription and processing of the FnCpfl CRISPR array. The mature crRNA begins with a 19 nt partial direct repeat followed by 23-25 nt of spacer sequence. Small RNA-seq of E. coli transformed with a plasmid carrying synthetic promoter-driven FnCpfl and CRISPR array (FIG. 59B) shows crRNA processing independent of Cas genes and other sequence elements in the FnCpfl locus. FIG. 59C depicts E. coli harboring different truncations of the FnCpfl CRISPR locus and shows that only FnCpfl and the CRISPR array are required for plasmid DNA interference (n = 3, error bars show mean ± S.E.M.). SEQ ID NO: 575 is disclosed.

[00129] FIGS. 60A-60E shows FnCpfl is targeted by crRNA to cleave DNA in vitro. FIG. 60A is a schematic of the FnCpfl crRNA-DNA targeting complex. Cleavage sites are 2023241400 09 Oct 2023 indicated by red arrows (SEQ ID NOS: 576 and 577, respectively, disclosed in order of appearance). FnCpfl and crRNA alone mediated RNA-guided cleavage of target DNA in a crRNA- and Mg2+-dependent manner (FIG. 60B). FIG. 60C shows FnCpfl cleaves both linear and supercoiled DNA. FIG. 60D shows Sanger sequencing traces from FnCpfl -digested target show staggered overhangs (SEQ ID NOS: 578-580, respectively, disclosed in order of appearance). The non-templated addition of an additional adenine, denoted as N, is an artifact of the polymerase used in sequencing. Reverse primer read represented as reverse complement to aid visualization. FIG. 60E shows cleavage is dependent on base-pairing at the 5’ PAM. FnCpfl can only recognize the PAM in correctly Watson-Crick paired DNA.

[00130] FIGS. 61A-61B shows catalytic residues in the C-terminal RuvC domain of FnCpfl are necessary for DNA cleavage. FIG. 61A shows the domain structure of FnCpfl with RuvC catalytic residues highlighted. The catalytic residues were identified based on sequence homology to Thermus thermophilus RuvC (PDB ID: 4EP5). FIG. 6IB depicts a native TBE PAGE gel showing that mutation of the RuvC catalytic residues of FnCpfl (D917A and El 006A) and mutation of the RuvC (D10A) catalytic residue of SpCas9 prevents double stranded DNA cleavage. Denaturing TBE-Urea PAGE gel showing that mutation of the RuvC catalytic residues of FnCpfl (D917A and E1006A) prevents DNA nicking activity, whereas mutation of the RuvC (D10A) catalytic residue of SpCas9 results in nicking of the target site.

[00131] FIGS. 62A-62E shows crRNA requirements for FnCpfl nuclease activity in vitro. FIG. 62A shows the effect of spacer length on FnCpfl cleavage activity. FIG. 62B shows the effect of crRNA-target DNA mismatch on FnCpfl cleavage activity. FIG. 62C demonstrates the effect of direct repeat length on FnCpfl cleavage activity. FIG. 62D shows FnCpfl cleavage activity depends on secondary structure in the stem of the direct repeat RNA structure. FIG. 62E shows FnCpfl cleavage activity is unaffected by loop mutations but is sensitive to mutation in the 3’-most base of the direct repeat. SEQ ID NOS: 581-607, respectively, disclosed in order of appearance.

[00132] FIGS. 63A-63F provides an analysis of Cpfl-family protein diversity and function. FIGS. 63A-63B show a phylogenetic comparison of 16 Cpfl ortho logs selected for functional analysis. Conserved sequences are shown in dark gray. The RuvC domain, bridge helix, and zinc finger are highlighted. FIG. 63C shows an alignment of direct repeats from the 16 Cpfl- 2023241400 09 Oct 2023 family proteins. Sequences that are removed post crRNA maturation are colored gray. Nonconserved bases are colored red. The stem duplex is highlighted in gray. FIG. 63D depicts RNAfold (Lorenz et aL, 2011) prediction of the direct repeat sequence in the mature crRNA. Predictions for FnCpfl along with three less-conserved orthologs shown. FIG. 63E shows ortholog crRNAs with similar direct repeat sequences are able to function with FnCpfl to mediate target DNA cleavage. FIG. 63F shows PAM sequences for 8 Cpfl-family proteins identified using in vitro cleavage of a plasmid library containing randomized PAMs flanking the protospacer. SEQ ID NOS: 608-627, respectively, disclosed in order of appearance.

[00133] FIGS. 64A-64E shows Cpfl mediates robust genome editing in human cell lines. FIG. 64A is a schemative showing expression of individual Cpfl-family proteins in HEK 293FT cells using CMV-driven expression vectors. The corresponding crRNA is expressed using a PCR fragment containing a U6 promoter fused to the crRNA sequence. Transfected cells were analyzed using either Surveyor nuclease assay or targeted deep sequencing. FIG. 64B (top) depicts the sequence of DNMT1-targeting crRNA 3, and sequencing reads (bottom) show representative indels. IG. 64B discloses SEQ ID NOS: 628-639, respectively, in order of appearance. FIG. 64C provides a comparison of in vitro and in vivo cleavage activity. The DNMT1 target region was PCR amplified and the genomic fragment was used to test Cpfl-mediated cleavage. All 8 Cpfl-family proteins showed DNA cleavage in vitro (top). Candidates 7 - AsCpfl and 13 - Lb3Cpfl facilitated robust indel formation in human cells (bottom). FIG. 64D shows Cpfl and SpCas9 target sequences in the human DNMT1 locus (SEQ ID NOS: 640-647, respectively, disclosed in order of appearance). FIG. 64E provides a comparison of Cpfl and SpCas9 genome editing efficiency. Target sites correspond to sequences shown in FIG. 101D.

[00134] FIGS. 65A-65D shows an in vivo plasmid depletion assay for identifying FnCpfl PAM. (See also FIG. 58). FIG. 65A: Transformation of E. coli harboring pFnCpfl with a library of plasmids carrying randomized 5’ PAM sequences. A subset of plasmids were depleted. Plot shows depletion levels in ranked order. Depletion is measured as the negative log2 fold ratio of normalized abundance compared pACYC184 E. coli controls. PAMs above a threshold of 3.5 are used to generate sequence logos. FIG. 65B: Transformation of E. coli harboring pFnCpfl with a library of plasmids carrying randomized 3’ PAM sequences. A subset of plasmids were depleted. Plot shows depletion levels in ranked order. Depletion is 2023241400 09 Oct 2023 measured as the negative log2 fold ratio of normalized abundance compared pACYC184 E. coli controls and PAMs above a threshold of 3.5 are used to generate sequence logos. FIG. 65C: Input library of plasmids carrying randomized 5’ PAM sequences. Plot shows depletion levels in ranked order. Depletion is measured as the negative log2 fold ratio of normalized abundance compared pACYC184 E. coli controls. PAMs above a threshold of 3.5 are used to generate sequence logos. FIG. 65D: The number of unique PAMs passing significance threshold for pairwise combinations of bases at the 2 and 3 positions of the 5’ PAM.

[00135] FIGS. 66A-66D shows FnCpfl Protein Purification. (See also FIG. 60). FIG. 66A depicts a Coomassie blue stained acrylamide gel of FnCpfl showing stepwise purification. A band just above 160 kD eluted from the Ni-NTA column, consistent with the size of a MBP-FnCpfl fusion (189.7 kD). Upon addition of TEV protease a lower molecular weight band appeared, consistent with the size of 147 kD free FnCpfl. FIG. 66B: Size exclusion gel filtration of fnCpfl. FnCpfl eluted at a size approximately 300 kD (62.65 mL), suggesting Cpfl may exist in solution as a dimer. FIG. 66C shows protein standards used to calibrate the Superdex 200 column. BDex = Blue Dextran (void volume), Aid = Aldolase (158 kD), Ov = Ovalbumin (44 kD), RibA = Ribonuclease A (13.7 kD), Apr = Aprotinin (6.5 kD). FIG. 66D: Calibration curve of the Superdex 200 column. Ka is calculated as (elution volume - void volume) / (geometric column volume - void volume). Standards were plotted and fit to a logarithmic curve.

[00136] FIGS. 67A-67E shows cleavage patterns of FnCpfl. (See also FIG. 60). Sanger sequencing traces from FnCpfl-digested DNA targets show staggered overhangs. The non-templated addition of an additional adenine, denoted as N, is an artifact of the polymerase used in sequencing. Sanger traces are shown for different TTN PAMs with protospacer 1 (A), protospacer 2 (B), and protospacer 3 (C) and targets DNMT1 and EMX1 (D). The (-) strand sequence is reverse-complemented to show the top strand sequence. Cleavage sites are indicated by red triangles. Smaller triangles indicate putative alternative cleavage sites. Panel E shows the effect of PAM-distal crRNA-target DNA mismatch on FnCpfl cleavage activity. SEQ ID NOS: 648-687, respectively, disclosed in order of appearance.

[00137] FIGS. 68A-68B shows an amino acid sequence alignment of FnCpfl (SEQ ID NOS: 688 and 691), AsCpfl (SEQ ID NOS: 689 and 692), and LbCpfl (SEQ ID NOS: 690 and 693). (See also FIG. 63). Residues that are conserved are highlighted with a red 2023241400 09 Oct 2023 background and conserved mutations are highlighted with an outline and red font. Secondary structure prediction is highlighted above (FnCpfl) and below (LbCpfl) the alignment. Alpha helices are shown as a curly symbol and beta strands are shown as dashes. Protein domains identified in FIG. 95A are also highlighted.

[00138] FIGS. 69A-69D provides maps bacterial genomic loci corresponding to the 16 Cpfl-family proteins selected for mammalian experimentation. (See also FIG. 63). FIGS. 69A-69D disclose SEQ ID NOS: 694-709, respectively, in order of appearance.

[00139] FIGS. 70A-70E shows in vitro characterization of Cpfl-family proteins. FIG. 70A is a schematic for in vitro PAM screen using Cpfl-family proteins. A library of plasmids bearing randomized 5 ’ PAM sequences were cleaved by individual Cpfl -family proteins and their corresponding crRNAs. Uncleaved plasmid DNA was purified and sequenced to identify specific PAM motifs that were depleted. FIG. 70B indicates the number of unique sequences passing significance threshold for pairwise combinations of bases at the 2 and 3 positions of the 5’ PAM for 7 - AsCpfl. FIG. 70C indicates the number of unique PAMs passing significance threshold for triple combinations of bases at the 2, 3, and 4 positions of the 5’ PAM for 13 - LbCpfl. FIGS. 70D-70E E and F show Sanger sequencing traces from 7 -AsCpfl-digested target (E) and 13 - LbCpfl-digested target (F) and show staggered overhangs. The non-templated addition of an additional adenine, denoted as N, is an artifact of the polymerase used in sequencing. Cleavage sites are indicated by red triangles. Smaller triangles indicate putative alternative cleavage sites. FIG. 70D-E discloses SEQ ID NOS: 710715, respectively, in order of appearance.

[00140] FIGS. 71A-71F indicates human cell genome editing efficiency at additional loci. Surveyor gels show quantification of indel efficiency achieved by each Cpfl-family protein at DNMT1 target sites 1 (FIG. 71A), 2 (FIG. 71B), and 4 (FIG. 71C). FIGS. 71A-71C indicate human cell genome editing efficiency at additional loci and Sanger sequencing of cleaved of DNMT target sites. Surveyor gels show quantification of indel efficiency achieved by each Cpfl-family protein at EMX1 target sites 1 and 2. Indel distributions for AsCpfl and LbCpfl and DNMT1 target sites 2, 3, and 4. Cyan bars represent total indel coverage; blue bars represent distribution of 3’ ends of indels. For each target, PAM sequence is in red and target sequence is in light blue. FIG. 7IF discloses SEQ ID NOS: 826-831, respectively, in order of appearance. 2023241400 09 Oct 2023

[00141] FIG. 72A-72C depicts a computational analysis of the primary structure of Cpfl nucleases reveals three distinct regions. First a C-terminal RuvC like domain, which is the only functional characterized domain. Second a N-terminal alpha-helical region and thirst a mixed alpha and beta region, located between the RuvC like domain and the alpha-helical region.

[00142] FIGS. 73A-73B depicts an AsCpfl Rad50 alignment (PDB 4W9M). SEQ ID NOS: 716 and 717, respectively, disclosed in order of appearance.

[00143] FIG. 73C depicts an AsCpfl RuvC alignment (PDB 4LD0). SEQ ID NOS: 718 and 719, respectively, disclosed in order of appearance.

[00144] FIGS. 73D-73E depicts an alignment of AsCpfl and FnCpfl which identifies Rad50 domain in FnCpfl. SEQ ID NOS: 720 and 721, respectively, disclosed in order of appearance.

[00145] FIG. 74 depicts a structure of Rad50 (4W9M) in complex with DNA. DNA interacting residues are highlighted (in red).

[00146] FIG. 75 depicts a structure of RuvC (4LD0) in complex with holiday junction. DNA interacting residues are highlighted in red.

[00147] FIG. 76 depicts a blast of AsCpfl aligns to a region of the site specific recombinase XerD. An active site region of XerD is LYWTGMR (SEQ ID NO: 1) with R being a catalytic residue. SEQ ID NOS: 722-723, respectively, disclosed in order of appearance.

[00148] FIG. 77 depicts a region is conserved in Cpfl orthologs (Yellow box) and although the R is not conserved, a highly conserved aspartic acid (orange box) is just C-terminal of this region and a nearby conserved region (blue box) with an absolutely conserved arginine. The aspartic acid is D732 in LbCpfl. SEQ ID NOS: 724-776, respectively, disclosed in order of appearance.

[00149] FIG. 78A shows an experiment where 150,000 HEK293T cells were plated per 24-well 24h before transfection. Cells were transfected with 400ng huAsCpfl plasmid and lOOng of tandem guide plasmid comprising one guide sequence directed to GRIN28 and one directed to EMX1 placed in tandem behind the U6 promoter, using Lipofectamin2000. Cells were harvested 72h after transfection and AsCpfl activity mediated by tandem guides was assayed using the SURVEYOR nuclease assay. 2023241400 09 Oct 2023

[00150] FIG. 78B demonstrates INDEL formation in both the GRIN28 and the EMX1 gene.

[00151] FIG. 79 shows FnCpfl cleavage of an array with increasing concentrations of EDTA (and decreasing concentrations of Mg2+). The buffer is 20 mM TrisHCl pH 7 (room temperature), 50 mM KC1, and includes a murine RNAse inhibitor to prevent degradation of RNA due to potential trace amount of non-specific RNase carried over from protein purification.

[00152] FIG. 80 presents a schematic of sugar attachments for directed delivery of protein or guide, especially with GalNac.

[00153] FIG. 81 illustrates Construction of vectors for in vivo delivery. A. Cpfl Vector; B: Gene blocks encoding for U6 promoter and three Cpfl guide RNAs in tandem cloned into an AAV vector encoding for human Synapsin-GFP-KASH. C: vector for SapI cloning of annealed oligos (SEQ ID NO: 777).

[00154] FIG. 82 illustrates Validation of delivery of Cpfl construct: staining of mouse neuronal cells with anti-HA.

[00155] FIG. 83 illustrates Targeted cleavage of Macaque / human genes MecpZ, Nlgn?>, and Drdl in HEK293FT cells.

[00156] FIG. 84 illustrates Surveyor data for cleavage of Mecp2, Nlgn3, and Drdl in mouse primary cortical neurons.

[00157] FIG. 85A-85B illustrates AsCpfl efficiency in primary neurons, a) AAV 'A infected primary cortical cultures stained with anti-HA (AsCpfl), anti-GFP (GFP-KASH) and NeuN (Neuronal marker) antibodies, b) Surveyor assay 7 days post infection.

[00158] FIG. 86A-86C illustrates stereotactic AAV1 / 2 injection for AsCpfl delivery into mouse hippocampus, a) Dissected mouse brain 3 weeks after viral delivery showing GFP fluorescence in hippocampus, b) FACS histogram of sorted GFP-KASH positive cell nuclei, c) Sorted GFP-KASH nuclei co-stained with nuclear marker Ruby Dye.

[00159] FIG. 87A-87B illustrates systemic delivery of AsCpfl and GFP-KASH into adult mice using dual vector approach, a) Immunostaining 3 weeks after systemic tail vein injection showing delivery of Syn-GFP-KASH vector into neurons of various brain regions, b) NGS indel analysis of various brain regions dissected 3 weeks after systemic tail vein co-injection 2023241400 09 Oct 2023 of dual vectors. Key: OB: olfactory bulb; CTX: cortex; ST: striatum; TH: thalamus; HP: hippocampus; CB: cerebellum; SC: spinal cord.

[00160] FIG. 88A-88H illustrates stereotactic injection of AAV1 / 2 dual vectors into adult mouse hippocampus, a) Vector design, b) Immunostaining 3 weeks after stereotactic AAV1 / 2 injection, c) Quantification of double infected neurons, d) Western blot showing AsCpfl and GFP-KASH protein levels, e) NGS indel analysis 3 weeks after stereotactic injection on GFP+ sorted nuclei, f) Quantification of mono- and bi-allelic modification of Drdl in male mice. Mecp2 and Nlgn3 are x-chromosomal genes, hence only one allele can be edited, g) Quantification of multiplex editing efficiency, h) Example NGS reads showing indels in all three targeted genes (SEQ ID NOS: 778-779, respectively, in order of appearance).

[00161] FIG. 89A-89E; FIG. 89A illustrates packaging AsCpfl into a single AAV and targeting in brain by local injection. FIG. 89A: single vector design encoding AsCpfl and guide (sMeCP2 promoter: Pol II (www.ncbi.nhn.nih.gov / pmc / articles / PMC3177952 / ); short tRNA promoter (Pol III: www.ncbi.nlm.nih.gov / pmc / articles / PMC3177952 / ). FIG89B: Expression of AsCpfl in dentate gyrus upon intracranial injection of AAV 1 / 2 vector into adult mouse brain; FIG. 89C-D: Indel analysis for multiplexed editing in dentate gyrus in sorted (C) and bulk (unsorted, D) nuclei; FIG. 89E: SURVEYOR analysis of neuronal nuclei extraction shows guide RNA mediated cutting.

[00162] FIG. 90A-90C illustrates a) Schematic of pLenti-Cpfl constructs. The pLenti-Cpfl Constructs are modified from the lentiCRISPRv2 plasmids. SpCas9 was replaced by AsCpfl and the SpCas9 U6 guide expression cassette was replaced with a AsCpfl U6 guide expression cassette. Unlike lentiCRISPRv2, the U6 guide expression cassette in pLenti-Cpfl is in reverse orientation. This change was required because Cpfl recognizes its corresponding direct repeat (DR) sequence and cleaves RNA molecules that exhibit this feature. Therefore, Lenti viral RNA is susceptible for Cpfl mediated cleavage if it exhibits a direct repeat sequence. However, incorporating the U6 guide expression cassette in revers order results in a RNA molecule without the direct repeat sequence, b) Surveyor assay results from two bioreps of HEK293T cells infected with pLenti-AsCpfl carrying a single VEGFA guide and one biorep of HEK293T cells infected with pLenti-AsCpfl encoding a DNMT1-EMX1-VEGFA-GRIN2b array. Cells were analyzed 5 days after puromycin selection. Robust cutting was observed in all lenti infected cells at the targeted loci. Red triangles indicate cleavage 2023241400 09 Oct 2023 products, c) NGS results for DNMT1, EMX1, VEGFA, and GRIN2b from colonies grown for 10 days after single cell FACS sorting of HEK293T cells infected with pLenti-AsCpfl encoding a DNMTl-EMXl-VEGFA-GRIN2b array. FACS was performed after 5 days of puromycine selection. Multiplex editing was observed in a subset of examined cells. Each column represents one clonal colony, blue squares indicate editing of >30%, while squares indicate editing <30%.

[00163] FIG. 91 illustrates lentiCRISPR v2 vector as shown in “Improved vectors and genome-wide libraries for CRISPR screening” Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;l 1(8):783-4.

[00164] FIG. 92 illustrates the pYOlO (pcDNA3.1-hAsCpfl) vector as shown in “Cpfl Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System” Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3.

[00165] FIG. 93 illustrates cleavage activity of the indicated orthologues in HEK293T cells, compared to AsCpfl and LbCpfl. Cpfl and crRNA were delivered with a single plasmid (as in Fig. 100). Indels were analyzed by Surveyor nuclease assay 3 days after transfection. Cpfl orthologues: (a): Thiomicrospira sp. XS5; (b): Moraxella bovoculi AAX08_00205; (c): Moraxella bovoculi AAXll_00205; (d): Lachnospiraceae bacterium MA2020; (e): Butyrivibrio sp. NC3005.

[00166] FIG. 94A-94E illustrates PAM sequences of the indicated Cpfl orthologues as identified in a PAM screen using the cell lysate based in vitro assay published in Zetsche et aL, 2015. Cpfl orthologues: (a): Thiomicrospira sp. XS5; (b): Moraxella bovoculi AAX08 00205; (c): Moraxella bovoculi AAX11 00205; (d): Lachnospiraceae bacterium MA2020; (e): Butyrivibrio sp. NC3005.

[00167] FIG. 95A-95B shows protein sequence of Thiomicrospira sp. XS5 (SEQ ID NO: 790) (A); and the human codon optimized DNA sequence (SEQ ID NO: 791) (B).

[00168] FIG. 96A-96B shows protein sequence of Moraxella bovoculi AAX08 00205 (SEQ ID NO: 792) (A); and the human codon optimized DNA sequence (SEQ ID NO: 793) (B).

[00169] FIG. 97A-97B shows protein sequence of Moraxella bovoculi AAX11 00205 (SEQ ID NO: 794) (A); and the human codon optimized DNA sequence (SEQ ID NO: 795) (B). 2023241400 09 Oct 2023

[00170] FIG. 98A-98B shows protein sequence of Lachnospiraceae bacterium MA2020 (SEQ ID NO: 796) (A); and the human codon optimized DNA sequence (SEQ ID NO: 797) (B).

[00171] FIG. 99A-99B shows protein sequence of Butyrivibrio sp. NC3005 (SEQ ID NO: 798) (A); and the human codon optimized DNA sequence (SEQ ID NO: 799) (B).

[00172] FIG. 100A-100E shows exemplary eukaryotic expression verctors for the indicated Cpfl orthologues. (A): Thiomicrospira sp. XS5; (B): Moraxella bovoculi AAX08 00205; (C): Moraxella bovoculi AAXll_00205; (D): Lachnospiraceae bacterium MA2020; (E): Butyrivibrio sp. NC3005. These vectors were used to confirm in vivo cleavage activity of the respective Cpfl orthologues in HEK293 cells.

[00173] FIG. 101A-101C. Single AsCpfl AAV vector for multiplex targeting in brain by peripheral injection (tail vein; vector as illustrated in Fig 89); FIG 101A-B: Validation of NeuN nuclei sorting. NeuN+ nuclei population in adult mouse brain (A) but not in liver (B); FIG 10IB: Indel analysis at Drdl locus in various brain regions upon intravenous injection of AAV-PHP.B vector in adult mice (Mecp2 and Nlgn3 <1% indels N=4 replicates from 2 mice 21 d post injection).

[00174] FIG. 102A-102B: Dual AsCpfl AAV vector for multiplex targeting in brain by peripheral injection; FIG. 102A: Neuronal expression of AAV-PHP.B vector encoding sgRNA in various brain regions. FIG. 102B: Indel analysis in at Drdl locus in various brain regions upon intravenous injection of dual AAV-PHP.B vectors in adult mice. Note: same two-vector design as in Zetsche et.al. Nat. Biotech. (2016). Key: OB: olfactory bulb; CTX: cortex; ST: striatum; TH: thalamus; HP: hippocampus; CB: cerebellum; SC: spinal cord.

[00175] FIG. 103: Schematic of single AAV vector encoding AsCpfl (TYCV mutant) and single sgRNA targeting Pcsk9; Key: EFS: EFla short promoter.

[00176] FIG. 104 Precision genome deletion in vivo with single AAV AsCpfl (TYCV mutant) vector: Pcsk9 locus showing locations of sgRNA target sequence and stereotyped indel SEQ ID NOS: 800-802, respectively, in order of appearance.

[00177] FIG. 105: Precision genome deletion in vivo with single AAV AsCpfl (TYCV mutant) vector; top: Histograms showing precision stereotyped deletion in vivo (peak at -3 bp) in liver upon intravenous injection of single AAV8 AsCpfl (TYCV mutant) vector in adult mice; bottom: Stereotyped deletion absent in vitro in Neuro2a cell line. 2023241400 09 Oct 2023

[00178] FIG. 106 Precision genome deletion in vivo with single AAV AsCpfl (TYCV mutant) vector: DRD1 locus showing locations of sgRNA target sequence and stereotyped indel. SEQ ID NOS: 803-805, respectively, in order of appearance.

[00179] FIG. 107: Precision genome deletion in vivo with single AAV AsCpfl (TYCV mutant) vector; Top: DRD1 locus showing locations of sgRNA target sequence and stereotyped indel. Bottom: Histogram showing precision stereotyped deletion in vivo (peak at -3 bp) in brain.

[00180] FIG. 108A-108C. A. 108A: list of Cpfl orthologues with most active Cpfl orthologues boxed; FIG. 108B Phylogenetic tree of 17 new Cpfl ortho logs and AsCpfl, LbCpfl and FnCpfl(red). Estimated position of RuvC like domains and Nuc domain are indicated, estimation is based on the AsCpfl sequence. Alignment generated with Geneious2. FIG 108C: Alignment of Cpfl direct repeat (DR) sequences (SEQ ID NOS: 806-825, respectively, in order of appearance); high homology of sequences strongly suggest that DR sequences can be used.

[00181] FIG. 109A-109B illustrates PAM sequences of Cpfl orthologues as identified in a PAM screen using the cell lysate based in vitro assay published in Zetsche et al., 2015. FIG FIG. 109A: PAM sequences for Thiomicrospira sp. XS5 (TsCpfl); Prevotella bryanti B14 (25-Pb2Cpfl); Moraxella lacunata (32-MlCpfl); Lachnospiraceae bacterium MA2020 (40-Lb7Cpfl), Candidatus Methanomethylophilus alvus Mxl201 (47-CMaCpfl), Butyrivibrio sp. NC3005 (48-BsCpfl); Fig 109B: Moraxella bovoculi AAX08_00205 (34-Mb2 Cpfl); Moraxella bovoculi AAX11 00205 (35-Mb3Cpfl); Butivibrio fibrosolvens (49BfCpfl):

[00182] FIG 110A-110B. Cpfl ortholog activity in HEK293T cells. Briefly, 24,000 HEK cells were plated per 96-well and transfected ~24h after plating with lOOng Cpfl expression plasmid and 50ng U6-PCR fragments, encoding a guide sequence targeting VEGFA and the DR sequence corresponding to the Cpfl ortholog. Cells were harvested 3 days post 2023241400 09 Oct 2023 transfection and indel frequency was analysed by SURVEYOR assay. Ortholog 20, 34, 35 and 38 resulted in strong indel formation. Week indel frequency was observed with ortholog 32, 40, 43 and 47. Triangles In B indicate cleavage fragments.

[00183] FIG. 111. A subset of Cpfl orthologs which showed activity were tested with additional guides targeting EMX1 and DNMT1, all guides targeting TTTN PAMs. Briefly, 120,000 HEK cells were plated per 24-well. Cells were transfected ~24h post plating with 500ng plasmid expressing humanized Cpfl and crRNAs with corresponding DR sequences. Indel frequencies were analyzed by SURVEYOR assay 3 days post transfection (gel images). Plasmids were transfected before sequence confirmed and plasmid without intact guides were not included in the quantification.

[00184] FIG. 112. Quantification of gells of FIG 109.

[00185] FIG. 113A-113E. Cpfl ortholog #35(Mb3Cpfl) was tested with guides targeting NTTN PAMs. For 4 genes (A: DNMT1, B: EMX1, C:GRIN2b, D:VEGFA; E: All NTTN pooled), 16 guides targeting every possible combination of NTTN were tested. Briefly, 24,000 HEK293T cells were plated per 96-well and transfected ~24h post plating with lOOng Cpfl expression plasmid and 50ng crRNA expression plasmid. Indel frequencies were analyzed by deep sequencing (protocol as in Gao et al.BiorRxiv 2016). Mb3Cpfl has higher activity on NTTN PMAs than AsCpfl or LbCpfl, the preferred PAM motif appears to be TTTV, similar to AsCpfl and LbCpfl

[00186] FIG. 114: Mb3Cpfl (ortholog #35) was tested with RYYN PAMs (R=A or G; Y=C or T) targeting DNMT1 and EMX1. This experiment was aimed at determining if MB3Cpfl has tolerance for Cs within the PAM as predicted by the in vitro PAM screen. Briefly, 120,000 HEK cells were plated per 24-well. Cells were transfected ~24h post plating with 500g plasmid expressing humanized Cpfl and crRNAs with corresponding DR sequences. Indel frequencies were analyzed by SURVEYOR assay 3 days post transfection. MbCpfl can recognize YYN PAMs, the preferred PAM appears to be TTTV based on previous experiments. However Mb3Cpfl has a natural broad PAM recognition.

[00187] The figures herein are for illustrative purposes only and are not necessarily drawn to scale. 2023241400 09 Oct 2023 DETAILED DESCRIPTION OF THE INVENTION [001881 The present application describes novel RNA-guided endonucleases (e.g. Cpfl effector proteins) which are functionally distinct from the CRISPR-Cas9 systems described previously and hence the terminology of elements associated with these novel endonulceases are modified accordingly herein. Cpfl-associated CRISPR arrays described herein are processed into mature crRNAs without the requirement of an additional tracrRNA. The crRNAs described herein comprise a spacer sequence (or guide sequence) and a direct repeat sequence and a Cpflp-crRNA complex by itself is sufficient to efficiently cleave target DNA. The seed sequence described herein, e.g. the seed sequence of a FnCpfl guide RNA is approximately within the first 5 nt on the 5 ’ end of the spacer sequence (or guide sequence) and mutations within the seed sequence adversely affect cleavage activity of the Cpfl effector protein complex.

[00189] In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to target, e.g. have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. The section of the guide sequence through which complementarity to the target sequence is important for cleavage acitivity is referred to herein as the seed sequence. A target sequence may comprise any polynucleotide, such as DNA polynucleotides and is comprised within a target locus of interest. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. The herein described invention encompasses novel effector proteins of Class 2 CRISPR-Cas systems, of which Cas9 is an exemplary effector protein and hence terms used in this application to describe novel effector proteins, may correlate to the terms used to describe the CRISPR-Cas9 system.

[00190] The CRISPR-Cas loci has more than 50 gene families and there is no strictly universal genes. Therefore, no single evolutionary tree is feasible and a multi-pronged approach is needed to identify new families. So far, there is comprehensive cas gene identification of 395 profiles for 93 Cas proteins. Classification includes signature gene profiles plus signatures of locus architecture. Aspects of the invention relate to the 45 2023241400 09 Oct 2023 identification and engineering of novel effector proteins associated with Class 2 CRISPR-Cas systems. In a preferred embodiment, the effector protein comprises a single-subunit effector module. In a further embodiment the effector protein is functional in prokaryotic or eukaryotic cells for in vitro, in vivo or ex vivo applications. An aspect of the invention encompasses computational methods and algorithms to predict new Class 2 CRISPR-Cas systems and identify the components therein. 100191] In one embodiment, a computational method of identifying novel Class 2 CRISPR-Cas loci comprises the following steps: detecting all contigs encoding the Casl protein; identifying all predicted protein coding genes within 20kB of the casl gene; comparing the identified genes with Cas protein-specific profiles and predicting CRISPR arrays; selecting unclassified candidate CRISPR-Cas loci containing proteins larger than 500 amino acids (>500 aa); analyzing selected candidates using PSI-BLAST and HHPred, thereby isolating and identifying novel Class 2 CRISPR-Cas loci. In addition to the above mentioned steps, additional analysis of the candidates may be conducted by searching metagenomics databases for additional homologs. 100192] In one aspect the detecting all contigs encoding the Casl protein is performed by GenemarkS which a gene prediction program as further described in “GeneMarkS: a selftraining method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions.” John Besemer, Alexandre Lomsadze and Mark Borodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, herein incorporated by reference.

[00193] In one aspect the identifying all predicted protein coding genes is carried out by comparing the identified genes with Cas protein-specific profiles and annotating them according to NCBI Conserved Domain Database (CDD) which is a protein annotation resource that consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins. These are available as position-specific score matrices (PSSMs) for fast identification of conserved domains in protein sequences via RPS-BLAST. CDD content includes NCBI-curated domains, which use 3D-structure information to explicitly define domain boundaries and provide insights into sequence / structure / function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM). In a further aspect, CRISPR arrays were predicted 2023241400 09 Oct 2023 using a PILER-CR program which is a public domain software for finding CRISPR repeats as described in “PILER-CR: fast and accurate identification of CRISPR repeats”, Edgar, R.C., BMC Bioinformatics, Jan 20;8:18(2007), herein incorporated by reference.

[00194] In a further aspect, the case by case analysis is performed using PSLBLAST (Position-Specific Iterative Basic Local Alignment Search Tool). PSI-BLAST derives a position-specific scoring matrix (PSSM) or profile from the multiple sequence alignment of sequences detected above a given score threshold using protein-protein BLAST. This PSSM is used to further search the database for new matches, and is updated for subsequent iterations with these newly detected sequences. Thus, PSI-BLAST provides a means of detecting distant relationships between proteins.

[00195] In another aspect, the case by case analysis is performed using HHpred, a method for sequence database searching and structure prediction that is as easy to use as BLAST or PSI-BLAST and that is at the same time much more sensitive in finding remote homologs. In fact, HHpred’s sensitivity is competitive with the most powerful servers for structure prediction currently available. HHpred is the first server that is based on the pairwise comparison of profile hidden Markov models (HMMs). Whereas most conventional sequence search methods search sequence databases such as UniProt or the NR, HHpred searches alignment databases, like Pfam or SMART. This greatly simplifies the list of hits to a number of sequence families instead of a clutter of single sequences. All major publicly available profile and alignment databases are available through HHpred. HHpred accepts a single query sequence or a multiple alignment as input. Within only a few minutes it returns the search results in an easy-to-read format similar to that of PSI-BLAST. Search options include local or global alignment and scoring secondary structure similarity. HHpred can produce pairwise query-template sequence alignments, merged query-template multiple alignments (e.g. for transitive searches), as well as 3D structural models calculated by the MODELLER software from HHpred alignments.The term “nucleic acid-targeting system”, wherein nucleic acid is DNA or RNA, and in some aspects may also refer to DNA-RNA hybirds or derivatives thereof, refers collectively to transcripts and other elements involved in the expression of or directing the activity of DNA or RNA-targeting CRISPR-associated (“Cas”) genes, which may include sequences encoding a DNA or RNA-targeting Cas protein and a DNA or RNA-targeting guide RNA comprising a CRISPR RNA (crRNA) sequence and (in CRISPR-Cas9 2023241400 09 Oct 2023 system but not all systems) a trans-activating CRISPR-Cas system RNA (tracrRNA) sequence, or other sequences and transcripts from a DNA or RNA-targeting CRISPR locus. In the Cpfl DNA targeting RNA-guided endonuclease systems described herein, a tracrRNA sequence is not required. In general, a RNA-targeting system is characterized by elements that promote the formation of a RNA-targeting complex at the site of a target RNA sequence. In the context of formation of a DNA or RNA-targeting complex, “target sequence” refers to a DNA or RNA sequence to which a DNA or RNA-targeting guide RNA is designed to have complementarity, where hybridization between a target sequence and a RNA-targeting guide RNA promotes the formation of a RNA-targeting complex. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.

[00196] In an aspect of the invention, novel DNA targeting systems also referred to as DNA-targeting CRISPR-Cas or the CRISPR-Cas DNA-targeting system of the present application are based on identified Type V(e.g. subtype V-A and subtype V-B) Cas proteins which do not require the generation of customized proteins to target specific DNA sequences but rather a single effector protein or enzyme can be programmed by a RNA molecule to recognize a specific DNA target, in other words the enzyme can be recruited to a specific DNA target using said RNA molecule. Aspects of the invention particularly relate to DNA targeting RNA-guided Cpfl CRISPR systems.

[00197] The nucleic acids-targeting systems, the vector systems, the vectors and the compositions described herein may be used in various nucleic acids-targeting applications, altering or modifying synthesis of a gene product, such as a protein, nucleic acids cleavage, nucleic acids editing, nucleic acids splicing; trafficking of target nucleic acids, tracing of target nucleic acids, isolation of target nucleic acids, visualization of target nucleic acids, etc.

[00198] As used herein, a Cas protein or a CRISPR enzyme refers to any of the proteins presented in the new classification of CRISPR-Cas systems. In an advantageous embodiment, the present invention encompasses effector proteins identified in a Type V CRISPR-Cas loci, e.g. a Cpfl- encoding loci denoted as subtype V-A. Presently, the subtype V-A loci encompasses casl, cas2, a distinct gene denoted cpfl and a CRISPR array. Cpfl(CRISPR-associated protein Cpfl, subtype PREFRAN) is a large protein (about 1300 amino acids) that contains a RuvC-like nuclease domain homologous to the corresponding domain of Cas9 along with a counterpart to the characteristic arginine-rich cluster of Cas9. However, Cpfl 2023241400 09 Oct 2023 lacks the HNH nuclease domain that is present in all Cas9 proteins, and the RuvC-like domain is contiguous in the Cpfl sequence, in contrast to Cas9 where it contains long inserts including the HNH domain. Accordingly, in particular embodiments, the CRISPR-Cas enzyme comprises only a RuvC-like nuclease domain.

[00199] The Cpfl gene is found in several diverse bacterial genomes, typically in the same locus with casl, cas2, and cas4 genes and a CRISPR cassette (for example, FNFX1_1431-FNFX1 1428 of Francisella cf . novicida Fxl). Thus, the layout of this putative novel CRISPR-Cas system appears to be similar to that of type II-B. Furthermore, similar to Cas9, the Cpfl protein contains a readily identifiable C-terminal region that is homologous to the transposon ORF-B and includes an active RuvC-like nuclease, an arginine-rich region, and a Zn finger (absent in Cas9). However, unlike Cas9, Cpfl is also present in several genomes without a CRISPR-Cas context and its relatively high similarity with ORF-B suggests that it might be a transposon component. It was suggested that if this was a genuine CRISPR-Cas system and Cpfl is a functional analog of Cas9 it would be a novel CRISPR-Cas type, namely type V (See Annotation and Classification of CRISPR-Cas Systems. Makarova KS, Koonin EV. Methods Mol Biol. 2015;1311:47-75). However, as described herein, Cpfl is denoted to be in subtype V-A to distinguish it from C2clp which does not have an identical domain structure and is hence denoted to be in subtype V-B.

[00200] Aspects of the invention also encompass methods and uses of the compositions and systems described herein in genome engineering, e.g. for altering or manipulating the expression of one or more genes or the one or more gene products, in prokaryotic or eukaryotic cells, in vitro, in vivo or ex vivo.

[00201] In embodiments of the invention the terms mature crRNA and guide RNA and single guide RNA are used interchangeably as in foregoing cited documents such as WO 2014 / 093622 (PCT / US2013 / 074667). In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable 2023241400 09 Oct 2023 algorithm for aligning sequences, non-limiting example of which include the SmithWaterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the BurrowsWheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Preferably the guide sequence is 10-30 nucleotides long. The ability of a guide sequence to direct sequencespecific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art. A guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome.

[00202] In certain aspects the invention involves vectors. A used herein, a “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. In general, and throughout this specification, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not 2023241400 09 Oct 2023 limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Vectors for and that result in expression in a eukaryotic cell can be referred to herein as “eukaryotic expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[00203] Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). With regards to recombination and cloning methods, mention is made of U.S. patent application 10 / 815,730, published September 2, 2004 as US 2004-0171156 Al, the contents of which are herein incorporated by reference in their entirety. 2023241400 09 Oct 2023

[00204] The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissuespecific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporaldependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydro folate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit p-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion 2023241400 09 Oct 2023 proteins thereof, etc.). With regards to regulatory sequences, mention is made of U.S. patent application 10 / 491,026, the contents of which are incorporated by reference herein in their entirety. With regards to promoters, mention is made of PCT publication WO 2011 / 028929 and U.S. application 12 / 511,940, the contents of which are incorporated by reference herein in their entirety.

[00205] Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected for targeting particular types of cells.

[00206] As used herein, the term “crRNA” or “guide RNA” or “single guide RNA” or “sgRNA” or “one or more nucleic acid components” of a Type V CRISPR-Cas locus effector protein comprises any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence. In embodiments of the invention the terms mature crRNA and guide RNA and single guide RNA are used interchangeably as in foregoing cited documents such as WO 2014 / 093622 (PCT / US2013 / 074667). In some embodiments, the degree of complementarity, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). The ability of a guide sequence (within a nucleic acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence may be assessed by any suitable assay. For example, the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target 2023241400 09 Oct 2023 nucleic acid sequence (or a sequence in the vicinity thereof) may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at or in the vicinity of the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art. A guide sequence, and hence a nucleic acid-targeting guide RNA may be selected to target any target nucleic acid sequence. The target sequence may be DNA. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome.

[00207] In some embodiments, a nucleic acid-targeting guide RNA is selected to reduce the degree secondary structure within the RNA-targeting guide RNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide RNA participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A.R. Gruber et aL, 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 115162). [002081 The “tracrRNA” sequence or analogous terms includes any polynucleotide sequence that has sufficient complementarity with a crRNA sequence to hybridize. As indicated herein above, in embodiments of the present invention, the tracrRNA is not required for cleavage activity of Cpfl effector protein complexes.

[00209] Applicants also perform a challenge experiment to verily the DNA targeting and cleaving capability of a Type V protein such as Cpfl. This experiment closely parallels similar work in E. coli for the heterologous expression of StCas9 (Sapranauskas, R. et al. Nucleic Acids Res 39, 9275-9282 (2011)). Applicants introduce a plasmid containing both a PAM and a resistance gene into the heterologous E. coli, and then plate on the corresponding antibiotic. If there is DNA cleavage of the plasmid, Applicants observe no viable colonies. 2023241400 09 Oct 2023

[00210] In further detail, the assay is as follows for a DNA target. Two E.coli strains are used in this assay. One carries a plasmid that encodes the endogenous effector protein locus from the bacterial strain. The other strain carries an empty plasmid (e.g.pACYC184, control strain). All possible 7 or 8 bp PAM sequences are presented on an antibiotic resistance plasmid (pUC19 with ampicillin resistance gene). The PAM is located next to the sequence of proto-spacer 1 (the DNA target to the first spacer in the endogenous effector protein locus). Two PAM libraries were cloned. One has a 8 random bp 5’ of the proto-spacer (e.g. total of 65536 different PAM sequences = complexity). The other library has 7 random bp 3’ of the proto-spacer (e.g. total complexity is 16384 different PAMs). Both libraries were cloned to have in average 500 plasmids per possible PAM. Test strain and control strain were transformed with 5’PAM and 3’PAM library in separate transformations and transformed cells were plated separately on ampicillin plates. Recognition and subsequent cutting / interference with the plasmid renders a cell vulnerable to ampicillin and prevents growth. Approximately 12h after transformation, all colonies formed by the test and control strains where harvested and plasmid DNA was isolated. Plasmid DNA was used as template for PCR amplification and subsequent deep sequencing. Representation of all PAMs in the untransfomed libraries showed the expected representation of PAMs in transformed cells. Representation of all PAMs found in control strains showed the actual representation. Representation of all PAMs in test strain showed which PAMs are not recognized by the enzyme and comparison to the control strain allows extracting the sequence of the depleted PAM.

[00211] For minimization of toxicity and off-target effect, it will be important to control the concentration of nucleic acid-targeting guide RNA delivered. Optimal concentrations of nucleic acid-targeting guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci. The concentration that gives the highest level of on-target modification while minimizing the level of off-target modification should be chosen for in vivo delivery. The nucleic acid-targeting system is derived advantageously from a Type V CRISPR system. In some embodiments, one or more elements of a nucleic acid-targeting system is derived from a particular organism comprising an endogenous RNA-targeting system. In preferred embodiments of the invention, the RNA- 2023241400 09 Oct 2023 targeting system is a Type V CRISPR system. In particular embodiments, the Type V RNA-targeting Cas enzyme is Cpfl. The terms “orthologue” (also referred to as “ortholog” herein) and “homologue” (also referred to as “homolog” herein) are well known in the art. By means of further guidance, a “homologue” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related, or are only partially structurally related. An “orthologue” of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of. Orthologous proteins may but need not be structurally related, or are only partially structurally related. Homologs and orthologs may be identified by homology modelling (see, e.g., Greer, Science vol. 228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or "structural BLAST" (Dey F, Cliff Zhang Q, Petrey D, Honig B. Toward a "structural BLAST": using structural relationships to infer function. Protein Sci. 2013 Apr;22(4):359-66. doi: 10.1002 / pro.2225.). See also Shmakov et al. (2015) for application in the field of CRISPR-Cas loci. Homologous proteins may but need not be structurally related, or are only partially structurally related. In particular embodiments, the homologue or orthologue of Cpfl as referred to herein has a sequence homology or identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with Cpfl. In further embodiments, the homologue or orthologue of Cpfl as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with the wild type Cpfl. Where the Cpfl has one or more mutations (mutated), the homologue or orthologue of said Cpfl as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95% with the mutated Cpfl.

[00212] In an embodiment, the Type V DNA-targeting Cas protein may be a Cpfl ortholog of an organism of a genus which includes but is not limited to Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter. Species of organism of such a genus can be as otherwise herein discussed.

[00213] It will be appreciated that any of the functionalities described herein may be engineered into CRISPR enzymes from other orthologs, incuding chimeric enzymes 2023241400 09 Oct 2023 comprising fragments from multiple orthologs. Examples of such orthologs are described elsewhere herein. Thus, chimeric enzymes may comprise fragments of CRISPR enzyme orthologs of organisms of a genus which includes but is not limited to Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter. A chimeric enzyme can comprise a first fragment and a second fragment, and the fragrments can be of CRISPR enzyme orthologs of organisms of genuses herein mentioned or of species herein mentioned; advantageously the fragments are from CRISPR enzyme orthologs of different species.

[00214] In embodiments, the Type V DNA-targeting effector protein, in particular the Cpfl protein as referred to herein also encompasses a functional variant of Cpfl or a homologue or an orthologue thereof. A “functional variant” of a protein as used herein refers to a variant of such protein which retains at least partially the activity of that protein. Functional variants may include mutants (which may be insertion, deletion, or replacement mutants), including polymorphs, etc. Also included within functional variants are fusion products of such protein with another, usually unrelated, nucleic acid, protein, polypeptide or peptide. Functional variants may be naturally occurring or may be man-made. Advantageous embodiments can involve engineered or non-naturally occurring Type V DNA-targeting effector protein, e.g., Cpfl or an ortholog or homolog thereof.

[00215] In an embodiment, nucleic acid molecule(s) encoding the Type V DNA-targeting effector protein, in particular Cpfl or an ortholog or homolog thereof, may be codon-optimized for expression in a eukaryotic cell. A eukaryote can be as herein discussed. Nucleic acid molecule(s) can be engineered or non-naturally occurring.

[00216] In an embodiment, the Type V DNA-targeting effector protein, in particular Cpfl or an ortholog or homolog thereof, may comprise one or more mutations (and hence nucleic acid molecule(s) coding for same may have mutation(s)). The mutations may be artificially introduced mutations and may include but are not limited to one or more mutations in a catalytic domain. Examples of catalytic domains with reference to a Cas9 enzyme may include but are not limited to RuvC I, RuvC II, RuvC III and HNH domains. 2023241400 09 Oct 2023

[00217] In an embodiment, the Type V protein such as Cpfl or an ortholog or homolog thereof, may be used as a generic nucleic acid binding protein with fusion to or being operably linked to a functional domain. Exemplary functional domains may include but are not limited to translational initiator, translational activator, translational repressor, nucleases, in particular ribonucleases, a spliceosome, beads, a light inducible / controllable domain or a chemically inducible / controllable domain.

[00218] In some embodiments, the unmodified nucleic acid-targeting effector protein may have cleavage activity. In some embodiments, the DNA-targeting effector protein may direct cleavage of one or both nucleic acid (DNA or RNA) strands at the location of or near a target sequence, such as within the target sequence and / or within the complement of the target sequence or at sequences associated with the target sequence. In some embodiments, the nucleic acid-targeting effector protein may direct cleavage of one or both DNA or RNA strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, the cleavage may be staggered, i.e. generating sticky ends. In some embodiments, the cleavage is a staggered cut with a 5’ overhang. In some embodiments, the cleavage is a staggered cut with a 5’ overhang of 1 to 5 nucleotides, preferably of 4 or 5 nucleotides. In some embodiments, the cleavage site is distant from the PAM, e.g., the cleavage occurs after the 18th nucleotide on the non-target strand and after the 23rd nucleotide on the targeted strand . In some embodiments, the cleavage site occurs after the 18th nucleotide (counted from the PAM) on the non-target strand and after the 23rd nucleotide (counted from the PAM) on the targeted strand . In some embodiments, a vector encodes a nucleic acid-targeting effector protein that may be mutated with respect to a corresponding wild-type enzyme such that the mutated nucleic acid-targeting effector protein lacks the ability to cleave one or both DNA or RNA strands of a target polynucleotide containing a target sequence. As a further example, two or more catalytic domains of a Cas protein (e.g. RuvC I, RuvC II, and RuvC III or the HNH domain of a Cas9 protein) may be mutated to produce a mutated Cas protein substantially lacking all DNA cleavage activity. As described herein, corresponding catalytic domains of a Cpfl effector protein may also be mutated to produce a mutated Cpfl effector protein lacking all DNA cleavage activity or having substantially reduced DNA cleavage activity. In some embodiments, a nucleic acid-targeting effector protein may be considered to substantially lack 2023241400 09 Oct 2023 all RNA cleavage activity when the RNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavage activity of the non-mutated form of the enzyme; an example can be when the nucleic acid cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form. An effector protein may be identified with reference to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the Type V CRISPR system. Most preferably, the effector protein is a Type V protein such as Cpfl. By derived, Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as known in the art or as described herein.

[00219] Again, it will be appreciated that the terms Cas and CRISPR enzyme and CRISPR protein and Cas protein are generally used interchangeably and at all points of reference herein refer by analogy to novel CRISPR effector proteins further described in this application, unless otherwise apparent, such as by specific reference to Cas9. As mentioned above, many of the residue numberings used herein refer to the effector protein from the Type V CRISPR locus. However, it will be appreciated that this invention includes many more effector proteins from other species of microbes. In certain embodiments, effector proteins may be constitutively present or inducibly present or conditionally present or administered or delivered. Effector protein optimization may be used to enhance function or to develop new functions, one can generate chimeric effector proteins. And as described herein effector proteins may be modified to be used as a generic nucleic acid binding proteins.

[00220] Typically, in the context of a nucleic acid-targeting system, formation of a nucleic acid-targeting complex (comprising a guide RNA hybridized to a target sequence and complexed with one or more nucleic acid-targeting effector proteins) results in cleavage of one or both DNA strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. As used herein the term “sequence(s) associated with a target locus of interest” refers to sequences near the vicinity of the target sequence (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence, wherein the target sequence is comprised within a target locus of interest).

[00221] An example of a codon optimized sequence, is in this instance a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in 2023241400 09 Oct 2023 humans), or for another eukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 human codon optimized sequence in WO 2014 / 093622 (PCT / US2013 / 074667) as an example of a codon optimized sequence (from knowledge in the art and this disclosure, codon optimizing coding nucleic acid molecule(s), especially as to effector protein (e.g., Cpfl) is within the ambit of the skilled artisan). Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known. In some embodiments, an enzyme coding sequence encoding a DNA / RNA-targeting Cas protein is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. In some embodiments, processes for modifying the germ line genetic identity of human beings and / or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes, may be excluded. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp / codon / and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon 2023241400 09 Oct 2023 optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a DNA / RNA-targeting Cas protein corresponds to the most frequently used codon for a particular amino acid. As to codon usage in yeast, reference is made to the online Yeast Genome database available at http: / / www.yeastgenome.org / community / codon_usage.shtml, or Codon selection in yeast, Bennetzen and Hall, J Biol Chern. 1982 Mar 25;257(6):3026-31. As to codon usage in plants including algae, reference is made to Codon usage in higher plants, green algae, and cyanobacteria, Campbell and Gowri, Plant Physiol. 1990 Jan; 92(1): 1-11.; as well as Codon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan 25;17(2):477-98; or Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton BR, J Mol Evol. 1998 Apr;46(4):449-59.

[00222] In some embodiments, a vector encodes a nucleic acid-targeting effector protein such as the Type V DNA-targeting effector protein, in particular Cpfl or an ortholog or homolog thereof comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the RNA-targeting effector protein comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g., zero or at least one or more NLS at the amino-terminus and zero or at one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 2); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 3)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 4) or RQRRNELKRSP (SEQ ID NO: 5); the hRNPAl M9 NLS having the sequence 2023241400 09 Oct 2023 NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 6); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 7) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 8) and PPKKARED (SEQ ID NO: 9) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 10) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 11) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 12) and PKQKKRK (SEQ ID NO: 13) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 14) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 15) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 16) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 17) of the steroid hormone receptors (human) glucocorticoid. In general, the one or more NLSs are of sufficient strength to drive accumulation of the DNA-targeting Cas protein in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the nucleic acid-targeting effector protein, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the nucleic acid-targeting protein, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g., a stain specific for the nucleus such as DAP I). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of nucleic acid-targeting complex formation (e.g., assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by DNA-targeting complex formation and / or DNA-targeting Cas protein activity), as compared to a control not exposed to the nucleic acidtargeting Cas protein or nucleic acid-targeting complex, or exposed to a nucleic acid-targeting Cas protein lacking the one or more NLSs. In preferred embodiments of the herein described Cpfl effector protein complexes and systems the codon optimized Cpfl effector proteins comprise an NLS attached to the C-terminal of the protein. In certain embodiments, the NLS sequence is heterologous to the nucleic acid sequence encoding the Cpfl effector protein. 2023241400 09 Oct 2023

[00223] In some embodiments, one or more vectors driving expression of one or more elements of a nucleic acid-targeting system are introduced into a host cell such that expression of the elements of the nucleic acid-targeting system direct formation of a nucleic acidtargeting complex at one or more target sites. For example, a nucleic acid-targeting effector enzyme and a nucleic acid-targeting guide RNA could each be operably linked to separate regulatory elements on separate vectors. RNA(s) of the nucleic acid-targeting system can be delivered to a transgenic nucleic acid-targeting effector protein animal or mammal, e.g., an animal or mammal that constitutively or inducibly or conditionally expresses nucleic acidtargeting effector protein; or an animal or mammal that is otherwise expressing nucleic acidtargeting effector proteins or has cells containing nucleic acid-targeting effector proteins, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo nucleic acid-targeting effector proteins. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the nucleic acidtargeting system not included in the first vector, nucleic acid-targeting system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a nucleic acid-targeting effector protein and the nucleic acid-targeting guide RNA, embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the nucleic acid-targeting effector protein and the nucleic acid-targeting guide RNA may be operably linked to and expressed from the same promoter. Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a nucleic acid-targeting system are as used in the foregoing documents, such as WO 2014 / 093622 (PCT / US2013 / 074667). In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more 2023241400 09 Oct 2023 sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct may be used to target nucleic acid-targeting activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell. In some embodiments, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a a nucleic acid-targeting effector protein. Nucleic acidtargeting effector protein or nucleic acid-targeting guide RNA or RNA(s) can be delivered separately; and advantageously at least one of these is delivered via a particle complex, nucleic acid-targeting effector protein mRNA can be delivered prior to the nucleic acidtargeting guide RNA to give time for nucleic acid-targeting effector protein to be expressed. Nucleic acid-targeting effector protein mRNA might be administered 1-12 hours (preferably around 2-6 hours) prior to the administration of nucleic acid-targeting guide RNA. Alternatively, nucleic acid-targeting effector protein mRNA and nucleic acid-targeting guide RNA can be administered together. Advantageously, a second booster dose of guide RNA can be administered 1-12 hours (preferably around 2-6 hours) after the initial administration of nucleic acid-targeting effector protein mRNA + guide RNA. Additional administrations of nucleic acid-targeting effector protein mRNA and / or guide RNA might be useful to achieve the most efficient levels of genome modification. 100224] In one aspect, the invention provides methods for using one or more elements of a nucleic acid-targeting system. The nucleic acid-targeting complex of the invention provides an effective means for modifying a target DNA (single or double stranded, linear or supercoiled). The nucleic acid-targeting complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target DNA in a multiplicity of cell types. As such the nucleic acid-targeting complex of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary nucleic acid-targeting complex comprises a DNA-targeting effector protein complexed with a guide RNA hybridized to a target sequence within the target locus of interest. 2023241400 09 Oct 2023

[00225] In one aspect, the invention provides for methods of modifying a target polynucleotide. In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme (including any of the modified enzymes, such as deadCpfl or Cpfl nickase, etc.) as described herein) complexed with a guide sequence (including any of the modified guides of guide sequences as described herein) hybridized to a target sequence within said target polynucleotide, preferably wherein said guide sequence is linked to a direct repeat sequence. In one aspect, the invention provides a method of modifying expression of DNA in a eukaryotic cell, such that said binding results in increased or decreased expression of said DNA. In some embodiments, the method comprises allowing a nucleic acid-targeting complex to bind to the DNA such that said binding results in increased or decreased expression of said DNA; wherein the nucleic acid-targeting complex comprises a nucleic acid-targeting effector protein complexed with a guide RNA. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors drive expression of one or more of: the Cpfl, and the (multiple) guide sequence linked to the DR sequence. Similar considerations and conditions apply as above for methods of modifying a target DNA. In fact, these sampling, culturing and re-introduction options apply across the aspects of the present invention. In one aspect, the invention provides for methods of modifying a target DNA in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non-human animal, and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant. For re-introduced cells it is particularly preferred that the cells are stem cells. The cells can be modified according to the invention to produce gene products, for example in controlled amounts, which may be increased or decreased, depending on use, and / or mutated. In certain embodiments, a genetic locus of the cell is repaired.

[00226] Indeed, in any aspect of the invention, the nucleic acid-targeting complex may comprise a nucleic acid-targeting effector protein complexed with a guide RNA hybridized to a target sequence. 2023241400 09 Oct 2023

[00227] The invention relates to the engineering and optimization of systems, methods and compositions used for the control of gene expression involving DNA sequence targeting, that relate to the nucleic acid-targeting system and components thereof. In advantageous embodiments, the effector enzyme is a Type V protein such as Cpfl. An advantage of the present methods is that the CRISPR system minimizes or avoids off-target binding and its resulting side effects. This is achieved using systems arranged to have a high degree of sequence specificity for the target DNA. [002281 In relation to a nucleic acid-targeting complex or system preferably, the crRNA sequence has one or more stem loops or hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the crRNA sequence is between 10 to 30 nucleotides in length, the nucleic acid-targeting effector protein is a Type V Cas enzyme. In certain embodiments, the crRNA sequence is between 42 and 44 nucleotides in length, and the nucleic acid-targeting Cas protein is Cpfl of Francisella tularensis subsp.novocida UI 12. In certain embodiments, the crRNA comprises, consists essentialy of, or consists of 19 nucleotides of a direct repeat and between 23 and 25 nucleotides of spacer sequence, and the nucleic acid-targeting Cas protein is Cpfl of Francisella tularensis subsp.novocida U112.

[00229] The use of two different aptamers (each associated with a distinct nucleic acidtargeting guide RNAs) allows an activator-adaptor protein fusion and a repressor-adaptor protein fusion to be used, with different nucleic acid-targeting guide RNAs, to activate expression of one DNA, whilst repressing another. They, along with their different guide RNAs can be administered together, or substantially together, in a multiplexed approach. A large number of such modified nucleic acid-targeting guide RNAs can be used all at the same time, for example 10 or 20 or 30 and so forth, whilst only one (or at least a minimal number) of effector protein molecules need to be delivered, as a comparatively small number of effector protein molecules can be used with a large number modified guides. The adaptor protein may be associated (preferably linked or fused to) one or more activators or one or more repressors. For example, the adaptor protein may be associated with a first activator and a second activator. The first and second activators may be the same, but they are preferably different activators. Three or more or even four or more activators (or repressors) may be used, but package size may limit the number being higher than 5 different functional domains. 2023241400 09 Oct 2023 Linkers are preferably used, over a direct fusion to the adaptor protein, where two or more functional domains are associated with the adaptor protein. Suitable linkers might include the GlySer linker.

[00230] It is also envisaged that the nucleic acid-targeting effector protein-guide RNA complex as a whole may be associated with two or more functional domains. For example, there may be two or more functional domains associated with the nucleic acid-targeting effector protein, or there may be two or more functional domains associated with the guide RNA (via one or more adaptor proteins), or there may be one or more functional domains associated with the nucleic acid-targeting effector protein and one or more functional domains associated with the guide RNA (via one or more adaptor proteins).

[00231] The fusion between the adaptor protein and the activator or repressor may include a linker. For example, GlySer linkers GGGS (SEQ ID NO: 18) can be used. They can be used in repeats of 3 ((GGGGS)3 (SEQ ID NO: 19)) or 6 (SEQ ID NO: 20), 9 (SEQ ID NO: 21) or even 12 (SEQ ID NO: 22) or more, to provide suitable lengths, as required. Linkers can be used between the guide RNAs and the functional domain (activator or repressor), or between the nucleic acid-targeting Cas protein (Cas) and the functional domain (activator or repressor). The linkers the user to engineer appropriate amounts of “mechanical flexibility”.

[00232] The invention comprehends a nucleic acid-targeting complex comprising a nucleic acid-targeting effector protein and a guide RNA, wherein the nucleic acid-targeting effector protein comprises at least one mutation, such that the nucleic acid-targeting effector protein has no more than 5% of the activity of the nucleic acid-targeting effector protein not having the at least one mutation and, optional, at least one or more nuclear localization sequences; the guide RNA comprises a guide sequence capable of hybridizing to a target sequence in a RNA of interest in a cell; and wherein: the nucleic acid-targeting effector protein is associated with two or more functional domains; or at least one loop of the guide RNA is modified by the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins, and wherein the adaptor protein is associated with two or more functional domains; or the nucleic acidtargeting Cas protein is associated with one or more functional domains and at least one loop of the guide RNA is modified by the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains. 2023241400 09 Oct 2023

[00233] In one aspect, the invention provides a method of generating a model eukaryotic cell comprising a mutated disease gene. In some embodiments, a disease gene is any gene associated an increase in the risk of having or developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotic cell, wherein the one or more vectors drive expression of one or more of: a Cpfl enzyme and a protected guide RNA comprising a guide sequence linked to a direct repeat sequence; and (b) allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said disease gene, wherein the CRISPR complex comprises the Cpfl enzyme complexed with the guide RNA comprising the sequence that is hybridized to the target sequence within the target polynucleotide, thereby generating a model eukaryotic cell comprising a mutated disease gene. In some embodiments, said cleavage comprises cleaving one or two strands at the location of the target sequence by said Cpfl enzyme. In some embodiments, said cleavage results in decreased transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by non-homologous end joining (NHEJ)-based gene insertion mechanisms with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid changes in a protein expression from a gene comprising the target sequence.

[00234] In an aspect the invention provides methods as herein discussed wherein the host is a eukaryotic cell. In an aspect the invention provides a method as herein discussed wherein the host is a mammalian cell. In an aspect the invention provides a method as herein discussed, wherein the host is a non-human eukaryote cell. In an aspect the invention provides a method as herein discussed, wherein the non-human eukaryote cell is a non-human mammal cell. In an aspect the invention provides a method as herein discussed, wherein the non-human mammal cell may be including, but not limited to, primate bovine, ovine, procine, canine, rodent, Leporidae such as monkey, cow, sheep, pig, dog, rabbit, rat or mouse cell. In an aspect the invention provides a method as herein discussed, the cell may be a a nonmammalian eukaryotic cell such as poultry bird (e.g., chicken), vertebrate fish (e.g., salmon) or shellfish (e.g., oyster, claim, lobster, shrimp) cell. In an aspect the invention provides a method as herein discussed, the non-human eukaryote cell is a plant cell. The plant cell may 2023241400 09 Oct 2023 be of a monocot or dicot or of a crop or grain plant such as cassava, com, sorghum, soybean, wheat, oat or rice. The plant cell may also be of an algae, tree or production plant, fruit or vegetable (e.g., trees such as citrus trees, e.g., orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as almond or walnut or pistachio trees; nightshade plants; plants of the genus Brassica', plants of the genus Lactuca', plants of the genus Spinacia', plants of the genus Capsicum', cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc).

[00235] In one aspect, the invention provides a method for developing a biologically active agent that modulates a cell signaling event associated with a disease gene. In some embodiments, a disease gene is any gene associated an increase in the risk of having or developing a disease. In some embodiments, the method comprises (a) contacting a test compound with a model cell of any one of the above-described embodiments; and (b) detecting a change in a readout that is indicative of a reduction or an augmentation of a cell signaling event associated with said mutation in said disease gene, thereby developing said biologically active agent that modulates said cell signaling event associated with said disease gene.

[00236] In one aspect the invention provides for a method of selecting one or more cell(s) by introducing one or more mutations in a gene in the one or more cell (s), the method comprising: introducing one or more vectors into the cell (s), wherein the one or more vectors drive expression of one or more of: Cpf 1, a guide sequence linked to a direct repeat sequence, and an editing template; wherein the editing template comprises the one or more mutations that abolish Cpfl cleavage; allowing homologous recombination of the editing template with the target polynucleotide in the cell(s) to be selected; allowing a Cpfl CRISPR-Cas complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the Cpfl CRISPR-Cas complex comprises the Cpfl complexed with (1) the guide sequence that is hybridized to the target sequence within the target polynucleotide, and (2) the direct repeat sequence, wherein binding of the Cpfl CRISPR-Cas complex to the target polynucleotide induces cell death, thereby allowing one or more cell(s) in which one or more mutations have been introduced to be selected; this includes the present split Cpfl. In another preferred embodiment of the invention the cell to be selected may be a eukaryotic 2023241400 09 Oct 2023 cell. Aspects of the invention allow for selection of specific cells without requiring a selection marker or a two-step process that may include a counter-selection system.

[00237] In one aspect, the invention provides a recombinant polynucleotide comprising a guide sequence downstream of a direct repeat sequence, wherein the guide sequence when expressed directs sequence-specific binding of a Cpfl CRISPR-Cas complex to a corresponding target sequence present in a eukaryotic cell. In some embodiments, the target sequence is a viral sequence present in a eukaryotic cell. In some embodiments, the target sequence is a proto-oncogene or an oncogene.

[00238] In one aspect, the invention provides a vector system or eukaryotic host cell comprising (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences (including any of the modified guide sequences as described herein) downstream of the DR sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a Cpfl CRISPR-Cas complex to a target sequence in a eukaryotic cell, wherein the Cpfl CRISPR-Cas complex comprises Cpfl (including any of the modified enzymes as described herein) complexed with the guide sequence that is hybridized to the target sequence (and optionally the DR sequence); and / or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cpfl enzyme comprising a nuclear localization sequence and / or NES. In some embodiments, the host cell comprises components (a) and (b). In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into a genome of the host eukaryotic cell. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a Cpfl CRISPR-Cas complex to a different target sequence in a eukaryotic cell. . In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences and / or nuclear export sequences or NES of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in and / or out of the nucleus of a eukaryotic cell.

[00239] The present invention provides Cpfl orthologues of particular interest. Indeed, it has been found that while Cpfl orthologues from various species are capable of forming a CRISPR-Cas complex with a target sequence of interest, some Cpfl orthologues have particular advantages in that they have one or more advantages selected from higher 2023241400 09 Oct 2023 specificity, lower PAM requirements, higher cleavage activity, ... etc. In some embodiments, the Cpfl enzyme is derived from Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW201 l_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11 00205, Butyrivibrio sp. NC3005, Thiomicrospira sp. XS5, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae Cpfl, including any of the modified enzymes as described herein, and may include further alteration or mutation of the Cpfl, and can be a chimeric Cpfl. A number of Cpfl orthologues have been identified as being of particular interest for applications described herein, such as but not limited to Moraxella bovoculi AAX08 00205 or Moraxella bovoculi AAX11 00205. Accordingly, in particular embodiments, the Cpfl protein is derived from Moraxella bovoculi AAX08 00205 or Moraxella bovoculi AAX11 00205, more particularly has at least 90%, or even more preferably 95% sequence identity with a wild-type Cpfl sequence from Moraxella bovoculi AAX08 00205 or Moraxella bovoculi AAX11 00205, more particularly the wild-type sequences of AAX08 00205 or Moraxella bovoculi AAX11 00205 provided herein as SEQ ID NO: 55 and SEQ ID NO: 56, respectively. Such Cpfl effector sequences include Cpfl effector sequences which are mutated compared to the wild-type sequence. In some embodiments, the CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In a preferred embodiment, the strand break is a staggered cut with a 5’ overhang. In some embodiments, the Cpfl lacks DNA strand cleavage activity (e.g., no more than 5% nuclease activity as compared with a wild type enzyme or enzyme not having the mutation or alteration that decreases nuclease activity). In particular embodiments, the Cpfl enzyme lacking the ability to cleave one or both DNA strands is a mutated Cpfl. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the direct repeat has a minimum length of 16 nts and a single stem loop. In 2023241400 09 Oct 2023 further embodiments the direct repeat has a length longer than 16 nts, preferably more than 17 nts, and has more than one stem loop or optimized secondary structures. In some embodiments, the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or between 1630, or between 16-25, or between 16-20 nucleotides in length.

[00240] In one aspect, the invention provides a kit comprising one or more of the components described herein. In some embodiments, the kit comprises a vector system or host cell as described herein and instructions for using the kit. Modified Cpfl enzymes

[00241] Computational analysis of the primary structure of Cpfl nucleases reveals three distinct regions. First a C-terminal RuvC like domain, which is the only functional characterized domain. Second a N-tenninal alpha-helical region and thirst a mixed alpha and beta region, located between the RuvC like domain and the alpha-helical region.

[00242] Several small stretches of unstructured regions are predicted within the Cpfl primary structure. Unstructured regions, which are exposed to the solvent and not conserved within different Cpfl orthologs, are preferred sides for splits and insertions of small protein sequences . In addition, these sides can be used to generate chimeric proteins between Cpfl orthologs.

[00243] Based on the above information, mutants can be generated which lead to inactivation of the enzyme or which modify the double strand nuclease to nickase activity. In alternative embodiments, this information is used to develop enzymes with reduced off-target effects (described elsewhere herein)

[00244] In certain of the above-described Cpfl enzymes, the enzyme is modified by mutation of one or more residues including but not limited to positions D917, E1006, E1028, D1227, D1255A, N1257, according to FnCpfl protein or any corresponding ortholog. In an aspect the invention provides a herein-discussed composition wherein the Cpfl enzyme is an inactivated enzyme which comprises one or more mutations selected from the group consisting of D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A and N1257A according to FnCpfl protein or corresponding positions in a Cpfl ortholog. In an aspect the invention provides a herein-discussed composition, wherein the CRISPR enzyme comprises D917, or El006 and D917, or D917 and DI255, according to FnCpfl protein or a corresponding position in a Cpfl ortholog. 2023241400 09 Oct 2023

[00245] In certain of the above-described Cpfl enzymes, the enzyme is modified by mutation of one or more residues (in the RuvC domain) including but not limited to positions R909, R912, R930, R947, K949, R951, R955, K965, K968, K1000, K1002, R1003, K1009, K1017, K1022, K1029, K1035, K1054, K1072, K1086, R1094, K1095, KI 109, KI 118, KI 142, KI 150, KI 158, KI 159, R1220, R1226, R1242, and / or R1252 with reference to amino acid position numbering of AsCpfl (Acidaminococcus sp. BV3L6).

[00246] In certain of the above-described non-naturally-occurring CRISPR enzymes, the enzyme is modified by mutation of one or more residues (in the RAD50) domain including but not limited positions K324, K335, K337, R331, K369, K370, R386, R392, R393, K400, K404, K406, K408, K414, K429, K436, K438, K459, K460, K464, R670, K675, R681, K686, K689, R699, K705, R725, K729, K739, K748, and / or K752 with reference to amino acid position numbering of AsCpfl (Acidaminococcus sp. BV3L6).

[00247] In certain of the Cpfl enzymes, the enzyme is modified by mutation of one or more residues including but not limited positions R912, T923, R947, K949, R951, R955, K965, K968, K1000, R1003, K1009, K1017, K1022, K1029, K1072, K1086, Fl 103, R1226, and / or R1252 with reference to amino acid position numbering of AsCpfl (Acidaminococcus sp. BV3L6).

[00248] In certain embodiments, the Cpfl enzyme is modified by mutation of one or more residues including but not limited positions R833, R836, K847, K879, K881, R883, R887, K897, K900, K932, R935, K940, K948, K953, K960, K984, K1003, K1017, R1033, RI 138, RI 165, and / or R1252 with reference to amino acid position numbering of LbCpfl (Lachnospiraceae bacterium ND2006).

[00249] In certain embodiments, the Cpfl enzyme is modified by mutation of one or more residues including but not limited positions K15, R18, K26, Q34, R43, K48, K51, R56, R84, K85, K87, N93, R103, N104, T118, K123, K134, R176, K177, R192, K200, K226, K273, K275, T291, R301, K307, K369, S404, V409, K414, K436, K438, K468, D482, K516, R518, K524, K530, K532, K548, K559, K570, R574, K592, D596, K603, K607, K613, C647, R681, K686, H720, K739, K748, K757, T766, K780, R790, P791, K796, K809, K815, T816, K860, R862, R863, K868, K897, R909, R912, T923, R947, K949, R951, R955, K965, K968, K1000, R1003, K1009, K1017, K1022, K1029, A1053, K1072, K1086, Fl 103, S1209, 2023241400 09 Oct 2023 R1226, R1252, K1273, K1282, and / or K1288 with reference to amino acid position numbering of AsCpfl (Acidaminococcus sp. BV3L6).

[00250] In certain embodiments, the enzyme is modified by mutation of one or more residues including but not limited positions K15, R18, K26, R34, R43, K48, K51, K56, K87, K88, D90, K96, K106, K107, K120, Q125, K143, R186, K187, R202, K210, K235, K296, K298, K314, K320, K326, K397, K444, K449, E454, A483, E491, K527, K541, K581, R583, K589, K595, K597, K613, K624, K635, K639, K656, K660, K667, K671, K677, K719, K725, K730, K763, K782, K791, R800, K809, K823, R833, K834, K839, K852, K858, K859, K869, K871, R872, K877, K905, R918, R921, K932, I960, K962, R964, R968, K978, K981, K1013, R1016, K1021, K1029, K1034, K1041, K1065, K1084, and / or K1098 with reference to amino acid position numbering of FnCpfl (Francisella novicida UI 12).

[00251] In certain embodiments, the enzyme is modified by mutation of one or more residues including but not limited positions K15, R18, K26, K34, R43, K48, K51, R56, K83, K84, R86, K92, R102, K103, KI 16, K121, R158, E159, R174, R182, K206, K251, K253, K269, K271, K278, P342, K380, R385, K390, K415, K421, K457, K471, A506, R508, K514, K520, K522, K538, Y548, K560, K564, K580, K584, K591, K595, K601, K634, K640, R645, K679, K689, K707, T716, K725, R737, R747, R748, K753, K768, K774, K775, K785, K787, R788, Q793, K821, R833, R836, K847, K879, K881, R883, R887, K897, K900, K932, R935, K940, K948, K953, K960, K984, K1003, K1017, R1033, KI 121, RI 138, RI 165, KI 190, KI 199, and / or K1208 with reference to amino acid position numbering of LbCpfl (Lachnospiraceae bacterium ND2006).

[00252] In certain embodiments, the enzyme is modified by mutation of one or more residues including but not limited positions K14, R17, R25, K33, M42, Q47, K50, D55, K85, N86, K88, K94, R104, K105, KI 18, K123, K131, R174, K175, R190, R198, 1221, K267, Q269, K285, K291, K297, K357, K403, K409, K414, K448, K460, K501, K515, K550, R552, K558, K564, K566, K582, K593, K604, K608, K623, K627, K633, K637, E643, K780, Y787, K792, K830, Q846, K858, K867, K876, K890, R900, K901, M906, K921, K927, K928, K937, K939, R940, K945, Q975, R987, R990, K1001, R1034, 11036, R1038, R1042, K1052, K1055, K1087, R1090, K1095, N1103, KI 108, KI 115, KI 139, KI 158, RI 172, KI 188, K1276, R1293, A1319, K1340, K1349, and / or K1356 with reference to amino acid position numbering of MbCpfl (Moraxella bovoculi 237). 2023241400 09 Oct 2023 Deactivated / inactivated Cpfl protein

[00253] Where the Cpfl protein has nuclease activity, the Cpfl protein may be modified to have diminished nuclease activity e.g., nuclease inactivation of at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% as compared with the wild type enzyme; or to put in another way, a Cpfl enzyme having advantageously about 0% of the nuclease activity of the non-mutated or wild type Cpfl enzyme or CRISPR enzyme, or no more than about 3% or about 5% or about 10% of the nuclease activity of the non-mutated or wild type Cpfl enzyme, e.g. of the non-mutated or wild type Francisella novicida UI 12 (FnCpfl), Acidaminococcus sp. BV3L6 (AsCpfl), Lachnospiraceae bacterium ND2006 (LbCpfl) or Moraxella bovoculi 237 (MbCpfl Cpfl enzyme or CRISPR enzyme, or Lachnospiraceae bacterium MA2020 Cpfl enzyme or, Moraxella bovoculi AAX08_00205 Cpfl enzyme or CRISPR enzyme, Moraxella bovoculi AAX11_00205 Cpfl enzyme or CRISPR enzyme, Butyrivibrio sp. NC3005 Cpfl enzyme or CRISPR enzyme, Thiomicrospira sp. XS5 Cpfl enzyme or CRISPR enzyme. This is possible by introducing mutations into the nuclease domains of the Cpfl and orthologs thereof.

[00254] More particularly, the inactivated Cpfl enzymes include enzymes mutated in amino acid positions As908, As993, Asl263 of AsCpfl or corresponding positions in Cpfl orthologs. Additionally, the inactivated Cpfl enzymes include enzymes mutated in amino acid position Lb832, 925, 947 or 1180 of LbCpfl or corresponding positions in Cpfl orthologs. More particularly, the inactivated Cpfl enzymes include enzymes comprising one or more of mutations AsD908A, AsE993A, AsD1263A of AsCpfl or corresponding mutations in Cpfl orthologs. Additionally, the inactivated Cpfl enzymes include enzymes comprising one or more of mutations LbD832A, E925A, D947A or D1180A of LbCpfl or corresponding mutations in Cpfl ortho logs.

[00255] The inactivated Cpfl CRISPR enzyme may have associated (e.g., via fusion protein) one or more functional domains, including for example, one or more domains from the group comprising, consisting essentially of, or consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g., light inducible). Preferred domains are Fokl, VP64, P65, HSF1, MyoDl. In the event that Fokl is 2023241400 09 Oct 2023 provided, it is advantageous that multiple Fokl functional domains are provided to allow for a functional dimer and that gRNAs are designed to provide proper spacing for functional use (Fokl) as specifically described in Tsai et al. Nature Biotechnology, Vol. 32, Number 6, June 2014). The adaptor protein may utlilize known linkers to attach such functional domains. In some cases it is advantageous that additionally at least one NLS is provided. In some instances, it is advantageous to position the NLS at the N terminus. When more than one functional domain is included, the functional domains may be the same or different. [002561 In general, the positioning of the one or more functional domain on the inactivated Cpfl enzyme is one which allows for correct spatial orientation for the functional domain to affect the target with the attributed functional effect. For example, if the functional domain is a transcription activator (e.g., VP64 or p65), the transcription activator is placed in a spatial orientation which allows it to affect the transcription of the target. Likewise, a transcription repressor will be advantageously positioned to affect the transcription of the target, and a nuclease (e.g., Fokl) will be advantageously positioned to cleave or partally cleave the target. This may include positions other than the N- / C- terminus of the CRISPR enzyme. Enzymes according to the invention can be applied in optimized functional CRISPR-Cas systems which are of interest for functional screening

[00257] In an aspect the invention provides non-naturally occurring or engineered composition comprising a Type V, more particularly Cpfl CRISPR guide RNAs comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein the guide RNA is modified by the insertion of distinct RNA sequence(s) that bind to two or more adaptor proteins (e.g. aptamers), and wherein each adaptor protein is associated with one or more functional domains; or, wherein the guide RNA is modified to have at least one non-coding functional loop. In particular embodiments, the guide RNA is modified by the insertion of distinct RNA sequence(s) 5’ of the direct repeat, within the direct repeat, or 3’ of the guide sequence. When there is more than one functional domain, the functional domains can be same or different, e.g., two of the same or two different activators or repressors. In an aspect the invention provides non-naturally occurring or engineered CRISPR-Cas complex composition comprising the guide RNA as herein-discussed and a CRISPR enzyme which is a Cpfl enzyme, wherein optionally the Cpfl enzyme comprises at least one mutation, such that the Cpfl enzyme has no more than 5% of the nuclease activity of 2023241400 09 Oct 2023 the Cpfl enzyme not having the at least one mutation, and optionally one or more comprising at least one or more nuclear localization sequences. In an aspect the invention provides a herein-discussed Cpfl CRISPR guide RNA or the Cpfl CRISPR-Cas complex including a non-naturally occurring or engineered composition comprising two or more adaptor proteins, wherein each protein is associated with one or more functional domains and wherein the adaptor protein binds to the distinct RNA sequence(s) inserted into the guide RNA. In particular embodiments, the guide RNA is additionally or alternatively modified so as to still ensure binding of the Cpfl CRISPR complex but to prevent cleavage by the Cpfl enzyme (as detailed elsewhere herein).

[00258] In an aspect the invention provides a non-naturally occurring or engineered composition comprising a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, a Cpfl enzyme comprising at least one or more nuclear localization sequences, wherein the Cpfl enzyme comprises at least one mutation, such that the Cpfl enzyme has no more than 5% of the nuclease activity of the Cpfl enzyme not having the at least one mutation, wherein the guide RNA is modified by the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains; or, wherein the guide RNA is modified to have at least one non-coding functional loop, and wherein the composition comprises two or more adaptor proteins, wherein the each protein is associated with one or more functional domains. In an aspect the invention provides a herein-discussed composition, wherein the Cpfl enzyme has a diminished nuclease activity of at least 97%, or 100% as compared with the Cpfl enzyme not having the at least one mutation. In an aspect the invention provides a herein-discussed composition, wherein the Cpfl enzyme comprises two or more mutations. The mutations may be selected from D917A, E1006, E1028, D1227, D1255A, N1257, according to FnCpfl protein or a corresponding position in an ortholog. The amino acid mutations in may be selected from D908A, E993A, DI263A according to AsCpfl protein or a corresponding position in an ortholog. The amino acid mutations may be selected from D832A, E925A, D947A or D1180A according to LbCpfl protein or a corresponding position in an ortholog. In an aspect the invention provides a herein-discussed composition wherein the Cpfl enzyme comprises two or more mutations selected from the group consisting of D917A, E1006A, El028A, D1227A, 2023241400 09 Oct 2023 D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A and N1257A according to FnCpfl protein or any corresponding ortholog or D908A, E993A, DI263A according to AsCpfl protein or a corresponding position in an ortholog or D832A, E925A, D947A or DI 180A according to LbCpfl protein or a corresponding position in an ortholog. In an aspect the invention provides a herein-discussed composition, wherein the CRISPR enzyme comprises D917, or E1006 and D917, or D917 and D1255, according to FnCpfl protein or any corresponding ortholog or D908, E993, D1263 according to AsCpfl protein or a corresponding position in an ortholog or D832, E925, D947 or D1180A according to LbCpfl protein or a corresponding position in an ortholog. In an aspect the invention provides a herein-discussed composition, wherein the Cpfl enzyme is associated with one or more functional domains. In an aspect the invention provides a herein-discussed composition, wherein the two or more functional domains associated with the adaptor protein are each a heterologous functional domain. In an aspect the invention provides a herein-discussed composition, wherein the one or more functional domains associated with the Cpfl enzyme are each a heterologous functional domain. In an aspect the invention provides a herein-discussed composition, wherein the adaptor protein is a fusion protein comprising the functional domain, the fusion protein optionally comprising a linker between the adaptor protein and the functional domain, the linker optionally including a GlySer linker. In an aspect the invention provides a herein-discussed composition, wherein the gRNA is not modified by the insertion of distinct RNA sequence(s) that bind to the two or more adaptor proteins. In an aspect the invention provides a herein-discussed composition, wherein the one or more functional domains associated with the adaptor protein is a transcriptional activation domain. In an aspect the invention provides a herein-discussed composition, wherein the one or more functional domains associated with the Cpfl enzyme is a transcriptional activation domain. In an aspect the invention provides a herein-discussed composition, wherein the one or more functional domains associated with the adaptor protein is a transcriptional activation domain comprising VP64, p65, MyoDl, HSF1, RTA or SET7 / 9. In particular embodiments, the functional domain is the catalytic histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300 (aa 1048-1664). The p300 histone acetyltransferase protein catalyzes acetylation of histone H3 lysine 27 at its target sites and releases the DNA from its heterochromatin state so as to facilitate transcription thereof (Hilton et al. 2015, 2023241400 09 Oct 2023 Nature Nature Biotechnology, 33: 510-517). In an aspect the invention provides a herein-discussed composition, wherein the one or more functional domains associated with the Cpfl enzyme is a transcriptional activation domain comprises VP64, p65, MyoDl, HSF1, RTA, SET7 / 9 or core protein p300. In an aspect the invention provides a herein-discussed composition, wherein the one or more functional domains associated with the adaptor protein is a transcriptional repressor domain. In an aspect the invention provides a herein-discussed composition, wherein the one or more functional domains associated with the Cpfl enzyme is a transcriptional repressor domain. In an aspect the invention provides a herein-discussed composition, wherein the transcriptional repressor domain is a KRAB domain. In an aspect the invention provides a herein-discussed composition, wherein the transcriptional repressor domain is a NuE domain, NcoR domain, SID domain or a SID4X domain. In an aspect the invention provides a herein-discussed composition, wherein at least one of the one or more functional domains associated with the adaptor protein have one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, DNA integration activity RNA cleavage activity, DNA cleavage activity or nucleic acid binding activity. In an aspect the invention provides a herein-discussed composition, wherein the one or more functional domains associated with the Cpfl enzyme have one or more activities comprising methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, DNA integration activity RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, or molecular switch activity or chemical inducibility or light inducibility. In an aspect the invention provides a herein-discussed composition, wherein the DNA cleavage activity is due to a Fokl nuclease. In an aspect the invention provides a herein-discussed composition, wherein the one or more functional domains is attached to the Cpfl enzyme so that upon binding to the gRNA and target the functional domain is in a spatial orientation allowing for the functional domain to function in its attributed function; or, optionally,wherein the one or more functional domains is attached to the Cpfl enzyme via a linker, optionally a GlySer linker. In an aspect the invention provides a herein-discussed composition, wherein the gRNA is modified so that, after gRNA binds the adaptor protein and further binds to the Cpfl enzyme and target, the functional domain is in a spatial orientation 2023241400 09 Oct 2023 allowing for the functional domain to function in its attributed function. In an aspect the invention provides a herein-discussed composition, wherein the one or more functional domains associated with the Cpfl enzyme is attached to the RuvC domain of Cpfl .. In an aspect the invention provides a herein-discussed composition, wherein the direct repeat of the guide RNA is modified by the insertion of the distinct RNA sequence(s). In an aspect the invention provides a herein-discussed composition, wherein the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins is an aptamer sequence. In an aspect the invention provides a herein-discussed composition, wherein the aptamer sequence is two or more aptamer sequences specific to the same adaptor protein. In an aspect the invention provides a herein-discussed composition, wherein the aptamer sequence is two or more aptamer sequences specific to different adaptor protein. In an aspect the invention provides a herein-discussed composition, wherein the adaptor protein comprises MS2, PP7, Qp, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, Mil, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, 4>Cb5, (|)Cb8r, 4>Cbl2r, (|)Cb23r, 7s, PRR1.Accordingly, in particular embodiments, the aptamer is selected from a binding protein specifically binding any one of the adaptor proteins listed above. In an aspect the invention provides a herein-discussed composition, wherein the cell is a eukaryotic cell. In an aspect the invention provides a herein-discussed composition, wherein the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell, whereby the mammalian cell is optionally a mouse cell. In an aspect the invention provides a herein-discussed composition, wherein the mammalian cell is a human cell. In an aspect the invention provides a herein-discussed composition, wherein a first adaptor protein is associated with a p65 domain and a second adaptor protein is associated with a HSF1 domain. In an aspect the invention provides a herein-discussed composition, wherein the composition comprises a CRISPR-Cas complex having at least three functional domains, at least one of which is associated with the Cpfl enzyme and at least two of which are associated with gRNA.

[00259] In an aspect there is more than one gRNA, and the gRNAs target different sequences whereby when the composition is employed, there is multiplexing. In an aspect the invention provides a composition wherein there is more than one gRNA modified by the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins. 2023241400 09 Oct 2023

[00260] In an aspect one or more adaptor proteins associated with one or more functional domains is present and bound to the distinct RNA sequence(s) inserted into the guide RNA.

[00261] In an aspect the target sequence(s) are non-coding or regulatory sequences. The regulatory sequences can be promoter, enhancer or silencer sequence(s).

[00262] In an aspect the guide RNA is modified to have at least one non-coding functional loop; e.g., wherein the at least one non-coding functional loop is repressive; for instance, wherein at least one non-coding functional loop comprises Alu.

[00263] In an aspect the invention provides a method of screening for gain of function (GOF) or loss of function (LOF) or for screen non-coding RNAs or potential regulatory regions (e.g. enhancers, repressors) comprising the cell line of as herein-discussed or cells of the model herein-discussed containing or expressing Cpfl and introducing a composition as herein-discussed into cells of the cell line or model, whereby the gRNA includes either an activator or a repressor, and monitoring for GOF or LOF respectively as to those cells as to which the introduced gRNA includes an activator or as to those cells as to which the introduced gRNA includes a repressor. The screening of the instant invention is referred to as a SAM screen.

[00264] In an aspect the invention provides a genome wide library comprising a plurality of Cpfl guide RNAs (gRNAs) comprising guide sequences, each of which is capable of hybridizing to a target sequence in a genomic locus of interest in a cell and whereby the library is capable of targeting a plurality of target sequences in a plurality of genomic loci in a population of eukaryotic cells, wherein each gRNA is modified by the insertion of distinct RNA sequence(s) that binds to one or more or two or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains; or, wherein the gRNA is modified to have at least one non-coding functional loop. And when there is more than one functional domain, the functional domains can be same or different, e.g., two of the same or two different activators or repressors. In an aspect the invention provides a library of non-naturally occurring or engineered CRISPR-Cas complexes composition(s) comprising gRNAs of this invention and a Cpfl enzyme, wherein optionally the Cpfl enzyme comprises at least one mutation, such that the Cpfl enzyme has no more than 5% of the nuclease activity of the Cpfl enzyme not having the at least one mutation, and optionally one or more comprising at least one or more nuclear localization sequences. In an aspect the invention provides a 2023241400 09 Oct 2023 gRNA(s) or Cpfl CRISPR-Cas complex(es) of the invention including a non-naturally occurring or engineered composition comprising one or two or more adaptor proteins, wherein each protein is associated with one or more functional domains and wherein the adaptor protein binds to the distinct RNA sequence(s) inserted into the at least one loop of the gRNA.

[00265] In an aspect the invention provides a library of non-naturally occurring or engineered compositions, each comprising a Cpfl CRISPR guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, a Cpfl enzyme comprising at least one or more nuclear localization sequences, wherein the Cpfl enzyme comprises at least one mutation, such that the Cpfl enzyme has no more than 5% of the nuclease activity of the Cpfl enzyme not having the at least one mutation, wherein at least one loop of the gRNA is modified by the insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains, wherein the composition comprises one or more or two or more adaptor proteins, wherein the each protein is associated with one or more functional domains, and wherein the gRNAs comprise a genome wide library comprising a plurality of Cpfl guide RNAs (gRNAs) as detailed above. In particular embodimentsthe cell population of cells is a population of eukaryotic cells. In an aspect the invention provides a library as herein discussed, wherein the eukaryotic cell is a mammalian cell, a plant cell or a yeast cell. In an aspect the invention provides a library as herein discussed, wherein the mammalian cell is a human cell. In an aspect the invention provides a library as herein discussed, wherein the population of cells is a population of embryonic stem (ES) cells. In an aspect the invention provides a library as herein discussed, wherein the target sequence in the genomic locus is a non-coding sequence. In an aspect the invention provides a library as herein discussed, wherein gene function of one or more gene products is altered by said targeting; or wherein as to gene function there is gain of function; or wherein as to gene function there is change of function; or wherein as to gene function there is reduced function; or wherein the screen is for non-coding RNAs or potential regulatory regions (e.g. enhancers, repressors). In an aspect the invention provides a library as herein discussed, wherein said targeting results in a knockout of gene function. In an aspect the invention provides a library as herein discussed, wherein the targeting is of about 100 or more sequences. In an aspect the 2023241400 09 Oct 2023 invention provides a library as herein discussed, wherein the targeting is of about 1000 or more sequences. In an aspect the invention provides a library as herein discussed, wherein the targeting is of about 20,000 or more sequences. In an aspect the invention provides a library as herein discussed, wherein the targeting is of the entire genome. In an aspect the invention provides a library as herein discussed, wherein the targeting is of a panel of target sequences focused on a relevant or desirable pathway. In an aspect the invention provides a library as herein discussed, wherein the pathway is an immune pathway. In an aspect the invention provides a library as herein discussed, wherein the pathway is a cell division pathway. In an aspect the invention provides a library as herein discussed, wherein the alteration of gene function comprises: introducing into each cell in the population of cells a vector system of one or more vectors comprising an engineered, non-naturally occurring Cpfl CRISPR-Cas system comprising I. a Cpfl protein, and II. one or more type Cpfl guide RNAs, wherein components I and II may be same or on different vectors of the system, integrating components I and II into each cell, wherein the guide sequence targets a unique gene in each cell, wherein the Cpfl protein is operably linked to a regulatory element, wherein when transcribed, the guide RNA comprising the guide sequence directs sequence-specific binding of a Cpfl CRISPR-Cas system to a target sequence in the genomic loci of the unique gene, inducing cleavage of the genomic loci by the Cpfl protein, and confirming different mutations in a plurality of unique genes in each cell of the population of cells thereby generating a mutant cell library. In an aspect the invention provides a library as herein discussed, wherein the one or more vectors are plasmid vectors. In an aspect the invention provides a library as herein discussed, wherein the regulatory element is an inducible promoter. In an aspect the invention provides a library as herein discussed, wherein the inducible promoter is a doxycycline inducible promoter. In an aspect the invention provides a library as herein discussed wherein the confirming of different mutations is by whole exome sequencing. In an aspect the invention provides a library as herein discussed, wherein the mutation is achieved in 100 or more unique genes. In an aspect the invention provides a library as herein discussed, wherein the mutation is achieved in 1000 or more unique genes. In an aspect the invention provides a library as herein discussed, wherein the mutation is achieved in 20,000 or more unique genes. In an aspect the invention provides a library as herein discussed, wherein the mutation is achieved in the entire genome. In an aspect the 2023241400 09 Oct 2023 invention provides a library as herein discussed, wherein the alteration of gene function is achieved in a plurality of unique genes which function in a particular physiological pathway or condition. In an aspect the invention provides a library as herein discussed, wherein the pathway or condition is an immune pathway or condition. In an aspect the invention provides a library as herein discussed, wherein the pathway or condition is a cell division pathway or condition. In an aspect the invention provides a library as herein discussed, wherein a first adaptor protein is associated with a p65 domain and a second adaptor protein is associated with a HSF1 domain. In an aspect the invention provides a library as herein discussed, wherein each Cpfl CRISPR-Cas complex has at least three functional domains, at least one of which is associated with the Cpfl enzyme and at least two of which are associated with gRNA. In an aspect the invention provides a library as herein discussed, wherein the alteration in gene function is a knockout mutation. [002661 In an aspect the invention provides a method for functional screening genes of a genome in a pool of cells ex vivo or in vivo comprising the administration or expression of a library comprising a plurality of Cpfl CRISPR-Cas system guide RNAs (gRNAs) and wherein the screening further comprises use of a Cpfl enzyme, wherein the CRISPR complex is modified to comprise a heterologous functional domain. In an aspect the invention provides a method for screening a genome comprising the administration to a host or expression in a host in vivo of a library. In an aspect the invention provides a method as herein discussed further comprising an activator administered to the host or expressed in the host. In an aspect the invention provides a method as herein discussed wherein the activator is attached to a Cpfl enzyme. In an aspect the invention provides a method as herein discussed wherein the activator is attached to the N terminus or the C terminus of the Cpfl enzyme. In an aspect the invention provides a method as herein discussed wherein the activator is attached to the Cpfl CRISPR gRNA direct repeat. In an aspect the invention provides a method as herein discussed further comprising a repressor administered to the host or expressed in the host. In an aspect the invention provides a method as herein discussed, wherein the screening comprises affecting and detecting gene activation, gene inhibition, or cleavage in the locus. In an aspect the invention provides a pair of Cpfl CRISPR-Cas complexes, each comprising a Cpfl guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, wherein said gRNA is modified by the 2023241400 09 Oct 2023 insertion of distinct RNA sequence(s) that bind to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains, wherein each gRNA of each Cpfl CRISPR-Cas comprises a functional domain having a DNA cleavage activity. In an aspect the invention provides a paired Cpfl CRISPR-Cas complexes as herein-discussed, wherein the DNA cleavage activity is due to a Fokl nuclease. [00267J In one aspect, the invention provides a method of generating a model eukaryotic cell comprising a gene with modified expression. In some embodiments, a disease gene is any gene associated an increase in the risk of having or developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors described herein above into a eukaryotic cell, and (b) allowing a CRISPR complex to bind to a target polynucleotide so as to modify a genetic locus, thereby generating a model eukaryotic cell comprising a modified genetic locus. [002681 In one aspect, the invention provides a method for developing a biologically active agent that modulates a cell signaling event associated with a disease gene. In some embodiments, a disease gene is any gene associated an increase in the risk of having or developing a disease. In some embodiments, the method comprises (a) contacting a test compound with a model cell of any one of the above-described embodiments; and (b) detecting a change in a readout that is indicative of a reduction or an augmentation of a cell signaling event associated with said mutation in said disease gene, thereby developing said biologically active agent that modulates said cell signaling event associated with said disease gene. [002691 The invention comprehends optimized functional CRISPR-Cas Cpfl enzyme systems, especially in combination with the present modified guides and also where the Cpfl enzyme is also associated with a functional domain. In particular the Cpfl enzyme comprises one or more mutations that converts it to a DNA binding protein to which functional domains exhibiting a function of interest may be recruited or appended or inserted or attached. In certain embodiments, the Cpfl enzyme comprises one or more mutations which include but are not limited to D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A and N1257 (based on the amino acid position numbering of a Francisella tularensis 1 Novicida Cpfl), D908A, E993A or AsD1263A (based on the amino acid position numbering of a Acidaminococcus sp. BV3L6 Cpfl) D832A, E925A, D947A or 2023241400 09 Oct 2023 Dl 180A (based on the amino acid position numbering of a Lachnospiraceae bacterium Cpfl) and / or one or more mutations is in a RuvCl domain of the Cpfl enzyme or is a mutation as otherwise as discussed herein. In some embodiments, the Cpfl enzyme has one or more mutations in a catalytic domain, wherein when transcribed, the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the enzyme further comprises a functional domain. In some embodiments, a mutation at El006 according to FnCpfl protein is preferred.

[00270] The structural information provided herein allows for interrogation of guide RNA interaction with the target DNA and the Cpfl enzyme permitting engineering or alteration of guide RNA structure to optimize functionality of the entire Cpfl CRISPR-Cas system. For example, loops of the guide RNA may be extended, without colliding with the Cpfl protein by the insertion of adaptor proteins that can bind to RNA. These adaptor proteins can further recruit effector proteins or fusions which comprise one or more functional domains.

[00271] In general, the guide RNA are modified in a manner that provides specific binding sites (e.g. aptamers) for adapter proteins comprising one or more functional domains (e.g. via fusion protein) to bind to. The modified guide RNA are modified such that once the guide RNA forms a CRISPR complex (i.e. Cpfl enzyme binding to guide RNA and target) the adapter proteins bind and, the functional domain on the adapter protein is positioned in a spatial orientation which is advantageous for the attributed function to be effective. For example, if the functional domain is a transcription activator (e.g. VP64 or p65), the transcription activator is placed in a spatial orientation which allows it to affect the transcription of the target. Likewise, a transcription repressor will be advantageously positioned to affect the transcription of the target and a nuclease (e.g. Fokl) will be advantageously positioned to cleave or partially cleave the target.

[00272] The skilled person will understand that modifications to the guide RNA which allow for binding of the adapter + functional domain but not proper positioning of the adapter + functional domain (e.g. due to steric hindrance within the three dimensial structure of the CRISPR complex) are modifications which are not intended. The one or more modified guide RNA may be modified, by introduction of a distinct RNA sequence(s) 5’ of the direct repeat, within the direct repeat, or 3’ of the guide sequence. 2023241400 09 Oct 2023

[00273] As explained herein the functional domains may be, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g. light inducible). In some cases it is advantageous that additionally at least one NLS is provided. In some instances, it is advantageous to position the NLS at the N terminus. When more than one functional domain is included, the functional domains may be the same or different.

[00274] The guide RNA may be designed to include multiple binding recognition sites (e.g. aptamers) specific to the same or different adapter protein. The guide RNA of a Cpfl enzyme is characterized in that it typically is 37-43 nucleotides and in that it contains only one stem loop. The guide RNA may be designed to bind to the promoter region -1000 -+1 nucleic acids upstream of the transcription start site (i.e. TSS), preferably -200 nucleic acids. This positioning improves functional domains which affect gene activation (e.g. transcription activators) or gene inhibition (e.g. transcription repressors). The modified guide RNA may be one or more modified guide RNAs targeted to one or more target loci (e.g. at least 1 guide RNA, at least 2 guide RNA, at least 5 guide RNA, at least 10 guide RNA, at least 20 guide RNA, at least 30 guide RNA, at least 50 guide RNA) comprised in a composition.

[00275] Further, the Cpfl enzyme with diminished nuclease activity is most effective when the nuclease activity is inactivated (e.g. nuclease inactivation of at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% as compared with the wild type enzyme; or to put in another way, Cpfl enzyme having advantageously about 0% of the nuclease activity of the non-mutated or wild type Cpfl enzyme, or no more than about 3% or about 5% or about 10% of the nuclease activity of the non-mutated or wild type Cpfl enzyme). This is possible by introducing mutations into the RuvC nuclease domains of the FnCpfl or an ortholog thereof. For example utilizing mutations in a residue selected from the group consisting of D917A, E1006A, E1028A, D1227A, D1255A or N1257 as in FnCpfl and more preferably introducing one or more of the mutations selected from the group consisting of locations D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A and N1257 of FnCpfl or a corresponding ortholog. In particular embodiments, the mutations are D917A with E1006A in FnCpfl. Alternatively it can be a residue selected from 2023241400 09 Oct 2023 the group consisting of AsD908A, AsE993A, AsD1263A of AsCpfl or a corresponding ortholog or LbD832A, E925A, D947A or DI 180A of LbCpfl or a corresponding ortholog. [002761 The inactivated Cpfl enzyme may have associated (e.g. via fusion protein) one or more functional domains, like for example as described herein for the modified guide RNA adaptor proteins, including for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g. light inducible). Preferred domains are Fokl, VP64, P65, HSF1, MyoDl. In the event that Fokl is provided, it is advantageous that multiple Fokl functional domains are provided to allow for a functional dimer and that guide RNAs are designed to provide proper spacing for functional use (Fokl) as specifically described in Tsai et al. Nature Biotechnology, Vol. 32, Number 6, June 2014). The adaptor protein may utilize known linkers to attach such functional domains. In some cases it is advantageous that additionally at least one NLS is provided. In some instances, it is advantageous to position the NLS at the N terminus. When more than one functional domain is included, the functional domains may be the same or different.

[00277] In general, the positioning of the one or more functional domain on the inactivated Cpfl enzyme is one which allows for correct spatial orientation for the functional domain to affect the target with the attributed functional effect. For example, if the functional domain is a transcription activator (e.g. VP64 or p65), the transcription activator is placed in a spatial orientation which allows it to affect the transcription of the target. Likewise, a transcription repressor will be advantageously positioned to affect the transcription of the target, and a nuclease (e.g. Fokl) will be advantageously positioned to cleave or partially cleave the target. This may include positions other than the N- / C- terminus of the Cpfl enzyme.

[00278] The adaptor protein may be any number of proteins that binds to an aptamer or recognition site introduced into the modified guide RNA and which allows proper positioning of one or more functional domains, once the guide RNA has been incorporated into the CRISPR complex, to affect the target with the attributed function. As explained in detail in this application such may be coat proteins, preferably bacteriophage coat proteins. The functional domains associated with such adaptor proteins (e.g. in the form of fusion protein) 2023241400 09 Oct 2023 may include, for example, one or more domains from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g. light inducible). Preferred domains are Fokl, VP64, P65, HSF1, MyoDl. In the event that the functional domain is a transcription activator or transcription repressor it is advantageous that additionally at least an NLS is provided and preferably at the N terminus. When more than one functional domain is included, the functional domains may be the same or different. The adaptor protein may utilize known linkers to attach such functional domains. Enzyme mutations reducing off-target effects

[00279] In one aspect, the invention provides a non-naturally occurring or engineered CRISPR enzyme, preferably a class 2 CRISPR enzyme, preferably a Type V CRISPR enzyme as described herein, such as preferably, but without limitation Cpfl as described herein elsewhere, having one or more mutations resulting in reduced off-target effects, i.e. improved CRISPR enzymes for use in effecting modifications to target loci but which reduce or eliminate activity towards off-targets, such as when complexed to guide RNAs, as well as improved improved CRISPR enzymes for increasing the activity of CRISPR enzymes, such as when complexed with guide RNAs. It is to be understood that mutated enzymes as described herein below may be used in any of the methods according to the invention as described herein elsewhere. Any of the methods, products, compositions and uses as described herein elsewhere are equally applicable with the mutated CRISPR enzymes as further detailed below. It is to be understood, that in the aspects and embodiments as described herein, when referring to or reading on Cpfl as the CRISPR enzyme, reconstitution of a functional CRISPR-Cas system preferably does not require or is not dependent on a tracr sequence and / or direct repeat is 5’ (upstream) of the guide (target or spacer) sequence.

[00280] By means of further guidance, the following particular aspects and embodiments are provided.

[00281] The inventors have surprisingly determined that modifications may be made to CRISPR enzymes which confer reduced off-target activity compared to unmodified CRISPR enzymes and / or increased target activity compared to unmodified CRISPR enzymes. Thus, in certain aspects of the invention provided herein are improved CRISPR enzymes which may 2023241400 09 Oct 2023 have utility in a wide range of gene modifying applications. Also provided herein are CRISPR complexes, compositions and systems, as well as methods and uses, all comprising the herein disclosed modified CRISPR enzymes.

[00282] In the context of this aspect of the invention, a Cpfl or CRISPR enzyme is mutated or modified, “whereby the enzyme in the CRISPR complex has reduced capability of modifying one or more off-target loci as compared to an unmodified enzyme” (or like expressions); and, when reading this specification, the terms “Cpfl” or “Cas” or “CRISPR enzyme and the like are meant to include mutated or modified Cpfl or Cas or CRISPR enzyme in accordance with the invention, i.e., “whereby the enzyme in the CRISPR complex has reduced capability of modifying one or more off-target loci as compared to an unmodified enzyme” (or like expressions).

[00283] In an aspect, the altered activity of the engineered CRISPR protein comprises an altered binding property as to the nucleic acid molecule comprising RNA or the target polynucleotide loci, altered binding kinetics as to the nucleic acid molecule comprising RNA or the target polynucleotide loci, or altered binding specificity as to the nucleic acid molecule comprising RNA or the target polynucleotide loci compared to off-target polynucleotide loci.

[00284] In some embodiments, a Cpfl is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of the non-mutated form of the enzyme; an example can be when the DNA cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form. Thus, the Cpfl may comprise one or more mutations and may be used as a generic DNA binding protein with or without fusion to a functional domain. The mutations may be artificially introduced mutations or gain- or loss-of-function mutations. The instant invention modification(s) or mutation(s) “whereby the enzyme in the CRISPR complex has reduced capability of modifying one or more off-target loci as compared to an unmodified enzyme and / or whereby the enzyme in the CRISPR complex has increased capability of modifying the one or more target loci as compared to an unmodified enzyme” (or like expressions) can be combined with mutations that result in the enzyme being a nickase or dead. Such a dead enzyme can be an enhanced nucleic acid molecule binder. And such a nickase can be an enhanced nickase. For instance, changing neutral amino acid(s) in and / or near the groove and / or other charged residues in other 2023241400 09 Oct 2023 locations in Cas that are in close proximity to a nucleic acid (e.g., DNA, cDNA, RNA, gRNA to positive charged amino acid(s) may result in “whereby the enzyme in the CRISPR complex has reduced capability of modifying one or more off-target loci as compared to an unmodified enzyme and / or whereby the enzyme in the CRISPR complex has increased capability of modifying the one or more target loci as compared to an unmodified enzyme”, e.g., more cutting. As this can be both enhanced on- and off-target cutting (a super cutting Cpfl), using such with what is known in the art as a tru-guide or tru-sgRNAs (see, e.g., Fu et al., “Improving CRISPR-Cas nuclease specificity using truncated guide RNAs,” Nature Biotechnology 32, 279-284 (2014) doi:10.1038 / nbt.2808 Received 17 November 2013 Accepted 06 January 2014 Published online 26 January 2014 Corrected online 29 January 2014) to have enhanced on target activity without higher off target cutting or for making super cutting nickases, or for combination with a mutation that renders the Cas dead for a super binder.

[00285] In certain embodiments, the altered activity of the engineered Cpfl protein comprises increased targeting efficiency or decreased off-target binding. In certain embodiments, the altered activity of the engineered Cpfl protein comprises modified cleavage activity.

[00286] In certain embodiments, the altered activity comprises altered binding property as to the nucleic acid molecule comprising RNA or the target polynucleotide loci, altered binding kinetics as to the nucleic acid molecule comprising RNA or the target polynucleotide loci, or altered binding specificity as to the nucleic acid molecule comprising RNA or the target polynucleotide loci compared to off-target polynucleotide loci.

[00287] In certain embodiments, the altered activity comprises increased targeting efficiency or decreased off-target binding. In certain embodiments, the altered activity comprises modified cleavage activity. In certain embodiments, the altered activity comprises increased cleavage activity as to the target polynucleotide loci. In certain embodiments, the altered activity comprises decreased cleavage activity as to the target polynucleotide loci. In certain embodiments, the altered activity comprises decreased cleavage activity as to off-target polynucleotide loci. In certain embodiments, the altered activity comprises increased cleavage activity as to off-target polynucleotide loci. 2023241400 09 Oct 2023

[00288] In certain embodiments, the altered activity comprises increased cleavage activity as to the target polynucleotide loci. In certain embodiments, the altered activity comprises decreased cleavage activity as to the target polynucleotide loci. In certain embodiments, the altered activity comprises decreased cleavage activity as to off-target polynucleotide loci. In certain embodiments, the altered activity comprises increased cleavage activity as to off-target polynucleotide loci. Accordingly, in certain embodiments, there is increased specificity for target polynucleotide loci as compared to off-target polynucleotide loci. In other embodiments, there is reduced specificity for target polynucleotide loci as compared to off-target polynucleotide loci.

[00289] In an aspect of the invention, the altered activity of the engineered Cpfl protein comprises altered helicase kinetics.

[00290] In an aspect of the invention, the engineered Cpfl protein comprises a modification that alters association of the protein with the nucleic acid molecule comprising RNA, or a strand of the target polynucleotide loci, or a strand of off-target polynucleotide loci. In an aspect of the invention, the engineered Cpfl protein comprises a modification that alters formation of the CRISPR complex.

[00291] In certain embodiments, the modified Cpfl protein comprises a modification that alters targeting of the nucleic acid molecule to the polynucleotide loci. In certain embodiments, the modification comprises a mutation in a region of the protein that associates with the nucleic acid molecule. In certain embodiments, the modification comprises a mutation in a region of the protein that associates with a strand of the target polynucleotide loci. In certain embodiments, the modification comprises a mutation in a region of the protein that associates with a strand of the off-target polynucleotide loci. In certain embodiments, the modification or mutation comprises decreased positive charge in a region of the protein that associates with the nucleic acid molecule comprising RNA, or a strand of the target polynucleotide loci, or a strand of off-target polynucleotide loci. In certain embodiments, the modification or mutation comprises decreased negative charge in a region of the protein that associates with the nucleic acid molecule comprising RNA, or a strand of the target polynucleotide loci, or a strand of off-target polynucleotide loci. In certain embodiments, the modification or mutation comprises increased positive charge in a region of the protein that associates with the nucleic acid molecule comprising RNA, or a strand of the target 2023241400 09 Oct 2023 polynucleotide loci, or a strand of off-target polynucleotide loci. In certain embodiments, the modification or mutation comprises increased negative charge in a region of the protein that associates with the nucleic acid molecule comprising RNA, or a strand of the target polynucleotide loci, or a strand of off-target polynucleotide loci. In certain embodiments, the modification or mutation increases steric hindrance between the protein and the nucleic acid molecule comprising RNA, or a strand of the target polynucleotide loci, or a strand of off-target polynucleotide loci. In certain embodiments, the modification or mutation comprises a substitution of Lys, His, Arg, Glu, Asp, Ser, Gly, or Thr. In certain embodiments, the modification or mutation comprises a substitution with Gly, Ala, He, Glu, or Asp. In certain embodiments, the modification or mutation comprises an amino acid substitution in a binding groove.

[00292] In some embodiments, the CRISPR enzyme, such as preferably Cpfl enzyme is derived Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW201 l_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma tennitum, Eubacterium eligens, Moraxella bovoculi 237, Moraxella bovoculi AAX08 00205, Moraxella bovoculi AAX11 00205, Butyrivibrio sp. NC3005, Thiomicrospira sp. XS5, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae Cpfl(e.g., a Cpfl of one of these organisms modified as described herein), and may include further mutations or alterations or be a chimeric Cpfl.

[00293] In certain embodiments, the enzyme is modified by or comprises modification, e.g., comprises, consists essentially of or consists of modification by mutation of any one of the residues listed herein or a corresponding residue in the respective orthologue; or the enzyme comprises, consists essentially of or consists of modification in any one (single), two (double), three (triple), four (quadruple) or more position(s) in accordance with the disclosure throughout this application, or a corresponding residue or position in the CRISPR enzyme orthologue, e.g., an enzyme comprising, consisting essentially of or consisting of modification in any one of the Cpfl residues recited herein, or a corresponding residue or position in the 2023241400 09 Oct 2023 CRISPR enzyme orthologue. In such an enzyme, each residue may be modified by substitution with an alanine residue.

[00294] Applicants recently described a method for the generation of Cas9 orthologues with enhanced specificity (Slaymaker et al. 2015 “Rationally engineered Cas9 nucleases with improved specificity”). This strategy can be used to enhance the specificity of Cpfl orthologues. Primary residues for mutagenesis are preferably all positive charges residues within the RuvC domain. Additional residues are positive charged residues that are conserved between different orthologues.

[00295] In certain embodiments, specificity of Cpfl may be improved by mutating residues that stabilize the non-targeted DNA strand.

[00296] In certain of the above-described non-naturally-occurring Cpfl enzymes, the enzyme is modified by mutation of one or more residues (in the RuvC domain) including but not limited positions R909, R912, R930, R947, K949, R951, R955, K965, K968, K1000, K1002, R1003, K1009, K1017, K1022, K1029, K1035, K1054, K1072, K1086, R1094, K1095, KI 109, KI 118, KI 142, KI 150, KI 158, KI 159, R1220, R1226, R1242, and / or R1252 with reference to amino acid position numbering of AsCpfl (Acidaminococcus sp. BV3L6).

[00297] In certain of the above-described non-naturally-occurring Cpfl enzymes, the enzyme is modified by mutation of one or more residues (in the RAD50) domain including but not limited positions K324, K335, K337, R331, K369, K370, R386, R392, R393, K400, K404, K406, K408, K414, K429, K436, K438, K459, K460, K464, R670, K675, R681, K686, K689, R699, K705, R725, K729, K739, K748, and / or K752 with reference to amino acid position numbering of AsCpfl (Acidaminococcus sp. BV3L6).

[00298] In certain of the above-described non-naturally-occurring Cpfl enzymes, the enzyme is modified by mutation of one or more residues including but not limited positions R912, T923, R947, K949, R951, R955, K965, K968, K1000, R1003, K1009, K1017, K1022, K1029, K1072, K1086, Fl 103, R1226, and / or R1252 with reference to amino acid position numbering of AsCpfl (Acidaminococcus sp. BV3L6).

[00299] In certain embodiments, the enzyme is modified by mutation of one or more residues including but not limited positions R833, R836, K847, K879, K881, R883, R887, K897, K900, K932, R935, K940, K948, K953, K960, K984, K1003, K1017, R1033, RI 138, 2023241400 09 Oct 2023 R1165, and / or R1252 with reference to amino acid position numbering of LbCpfl (Lachnospiraceae bacterium ND2006).

[00300] In certain embodiments, the Cpfl enzyme is modified by mutation of one or more residues including but not limited positions KI5, RI8, K26, Q34, R43, K48, K51, R56, R84, K85, K87, N93, R103, N104, T118, K123, K134, R176, K177, R192, K200, K226, K273, K275, T291, R301, K307, K369, S404, V409, K414, K436, K438, K468, D482, K516, R518, K524, K530, K532, K548, K559, K570, R574, K592, D596, K603, K607, K613, C647, R681, K686, H720, K739, K748, K757, T766, K780, R790, P791, K796, K809, K815, T816, K860, R862, R863, K868, K897, R909, R912, T923, R947, K949, R951, R955, K965, K968, K1000, R1003, K1009, K1017, K1022, K1029, A1053, K1072, K1086, Fl 103, S1209, R1226, R1252, K1273, K1282, and / or K1288 with reference to amino acid position numbering of AsCpfl (Acidaminococcus sp. BV3L6).

[00301] In certain embodiments, the Cpfl enzyme is modified by mutation of one or more residues including but not limited positions K15, R18, K26, R34, R43, K48, K51, K56, K87, K88, D90, K96, K106, K107, K120, Q125, K143, R186, K187, R202, K210, K235, K296, K298, K314, K320, K326, K397, K444, K449, E454, A483, E491, K527, K541, K581, R583, K589, K595, K597, K613, K624, K635, K639, K656, K660, K667, K671, K677, K719, K725, K730, K763, K782, K791, R800, K809, K823, R833, K834, K839, K852, K858, K859, K869, K871, R872, K877, K905, R918, R921, K932, I960, K962, R964, R968, K978, K981, K1013, R1016, K1021, K1029, K1034, K1041, K1065, K1084, and / or K1098 with reference to amino acid position numbering of FnCpfl (Francisella novicida UI 12).

[00302] In certain embodiments, the Cpfl enzyme is modified by mutation of one or more residues including but not limited positions K15, R18, K26, K34, R43, K48, K51, R56, K83, K84, R86, K92, R102, K103, KI 16, K121, R158, E159, R174, R182, K206, K251, K253, K269, K271, K278, P342, K380, R385, K390, K415, K421, K457, K471, A506, R508, K514, K520, K522, K538, Y548, K560, K564, K580, K584, K591, K595, K601, K634, K640, R645, K679, K689, K707, T716, K725, R737, R747, R748, K753, K768, K774, K775, K785, K787, R788, Q793, K821, R833, R836, K847, K879, K881, R883, R887, K897, K900, K932, R935, K940, K948, K953, K960, K984, K1003, K1017, R1033, KI 121, RI 138, RI 165, KI 190, KI 199, and / or K1208 with reference to amino acid position numbering of LbCpfl (Lachnospiraceae bacterium ND2006). 2023241400 09 Oct 2023

[00303] In certain embodiments, the enzyme is modified by mutation of one or more residues including but not limited positions K14, RI7, R25, K33, M42, Q47, K50, D55, K85, N86, K88, K94, R104, K105, KI 18, K123, K131, R174, K175, R190, R198, 1221, K267, Q269, K285, K291, K297, K357, K403, K409, K414, K448, K460, K501, K515, K550, R552, K558, K564, K566, K582, K593, K604, K608, K623, K627, K633, K637, E643, K780, Y787, K792, K830, Q846, K858, K867, K876, K890, R900, K901, M906, K921, K927, K928, K937, K939, R940, K945, Q975, R987, R990, K1001, R1034, 11036, R1038, R1042, K1052, K1055, K1087, R1090, K1095, N1103, K1108, K1115, K1139, K1158, R1172, KI 188, K1276, R1293, A1319, K1340, K1349, and / or K1356 with reference to amino acid position numbering of MbCpfl (Moraxella bovoculi 237).

[00304] In any of the non-naturally-occurring CRISPR enzymes: a single mismatch may exist between the target and a corresponding sequence of the one or more off-target loci; and / or two, three or four or more mismatches may exist between the target and a corresponding sequence of the one or more off-target loci, and / or wherein in (ii) said two, three or four or more mismatches are contiguous.

[00305] In an aspect, the invention provides CRISPR nucleases as defined herein, such as Cpf 1, that comprise an improved equilibrium towards conformations associated with cleavage activity when involved in on-target interactions and / or improved equilibrium away from conformations associated with cleavage activity when involved in off-target interactions. In one aspect, the invention provides Cas (e.g. Cpfl) nucleases with improved proof-reading function, i.e. a Cas (e.g. Cpfl) nuclease which adopts a conformation comprising nuclease activity at an on-target site, and which conformation has increased unfavorability at an off-target site. Sternberg et al., Nature 527(7576):110-3, doi: 10.1038 / naturel5544, published online 28 October 2015. Epub 2015 Oct 28, used Forster resonance energy transfer FRET) experiments to detect relative orientations of the Cas (e.g. Cpfl) catalytic domains when associated with on- and off-target DNA, and which may be extrapolated to the CRISPR enzymes of the present invention (e.g. Cpfl).

[00306] The invention further provides methods and mutations for modulating nuclease activity and / or specificity using modified guide RNAs. As discussed, on-target nuclease activity can be increased or decreased. Also, off-target nuclease activity can be increased or 2023241400 09 Oct 2023 decreased. Further, there can be increased or decreased specificity as to on-target activity vs. off-target activity. Modified guide RNAs include, without limitation, truncated guide RNAs, dead guide RNAs, chemically modified guide RNAs, guide RNAs associated with functional domains, modified guide RNAs comprising functional domains, modified guide RNAs comprising aptamers, modified guide RNAs comprising adapter proteins, and guide RNAs comprising added or modified loops. In some embodiments, one or more functional domains are associated with an dead gRNA (dRNA). In some embodiments, a dRNA complex with the CRISPR enzyme directs gene regulation by a functional domain at on gene locus while an gRNA directs DNA cleavage by the CRISPR enzyme at another locus. In some embodiments, dRNAs are selected to maximize selectivity of regulation for a gene locus of interest compared to off-target regulation. In some embodiments, dRNAs are selected to maximize target gene regulation and minimize target cleavage.

[00307] In an aspect, the invention also provides methods and mutations for modulating Cas (e.g. Cpfl) binding activity and / or binding specificity. In certain embodiments Cas (e.g. Cpfl) proteins lacking nuclease activity are used. In certain embodiments, modified guide RNAs are employed that promote binding but not nuclease activity of a Cas (e.g. Cpfl) nuclease. In such embodiments, on-target binding can be increased or decreased. Also, in such embodiments off-target binding can be increased or decreased. Moreover, there can be increased or decreased specificity as to on-target binding vs. off-target binding.

[00308] The methods and mutations which can be employed in various combinations to increase or decrease activity and / or specificity of on-target vs. off-target activity, or increase or decrease binding and / or specificity of on-target vs. off-target binding, can be used to compensate or enhance mutations or modifications made to promote other effects. Such mutations or modifications made to promote other effects include mutations or modification to the Cas (e.g. Cpfl) and / or design / mutation / modification made to a guide. In particular, whereas naturally occurring CRISPR / Cas systems involve guides consisting of ribonucleotides (i.e., guide RNAs), guides of engineered systems of the invention can comprise deoxyribonucleotides, non-naturally occurring nucleotides and / or nucleotide analogs as well as ribonucleotides. Further, guides of the invention can comprise base substitutions / additions / deletions. 2023241400 09 Oct 2023

[00309] In certain embodiments, the methods and Cpfl proteins are used with a guide comprising non-naturally occurring nucleic acids and / or non-naturally occurring nucleotides and / or nucleotide analogs, or the guide is a chemically modified guide RNA. Non-naturally occurring nucleic acids include, for example, mixtures of nucleotides. Non-naturally occurring nucleotides and / or nucleotide analogs may be modified at the ribose, phosphate, and / or base moiety. In an embodiment of the invention, a guide nucleic acid comprises ribonucleotides and non-ribonucleotides. In one such embodiment, a guide comprises one or more ribonucleotides and one or more deoxyribonucleotides. In an embodiment of the invention, the guide comprises one or more non-naturally occurring nucleotide or nucleotide analog such as a nucleotide with phosphorothioate linkage, a locked nucleic acid (LNA) nucleotides comprising a methylene bridge between the 2' and 4' carbons of the ribose ring, or bridged nucleic acids (BNA). Other examples of modified nucleotides include 2’-O-methyl analogs, 2’-deoxy analogs, or 2'-fluoro analogs. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. Examples of guide RNA chemical modifications include, without limitation, incorporation of 2'-O-methyl (M), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE (MSP) at one or more terminal nucleotides. Such chemically modified guide can comprise increased stability and increased activity as compared to unmodified guides, though on-target vs. off-target specificity is not predictable. (See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038 / nbt.3290, published online 29 June 2015). In certain embodients, a guide comprises ribonucleotides in a region that binds to a target DNA and one or more deoxyribonucletides and / or nucleotide analogs in a region that binds to Cpfl. In an embodiment of the invention, deoxyribonucleotides and / or nucleotide analogs are incorporated in engineered guide structures, such as, without limitation, stem-loop regions. The methods and mutations of the invention are used to modulate Cas (e.g. Cpfl) nuclease activity and / or dCpfl target binding activity and / or Cpfl binding with chemically modified guide RNAs.

[00310] The use of Cas (e.g. Cpfl) as an RNA-guided binding protein is not limited to nuclease-null Cas (e.g. Cpfl). Cas (e.g. Cpfl) enzymes comprising nuclease activity can also function as RNA-guided binding proteins when used with certain guide RNAs. For example short guide RNAs and guide RNAs comprising nucleotides mismatched to the target can 2023241400 09 Oct 2023 promote RNA directed Cas (e.g. Cpfl) binding to a target sequence with little or no target cleavage. (See, e.g., Dahlman, 2015, Nat Biotechnol. 33(11):1159-1161, doi: 10.1038 / nbt.3390, published online 05 October 2015). In an aspect, the invention provides methods and mutations for modulating binding of Cas (e.g. Cpfl) proteins that comprise nuclease activity. In certain embodiments, on-target binding is increased. In certain embodiments, off-target binding is decreased. In certain embodiments, on-target binding is decreased. In certain embodiments, off-target binding is increased. In certain embodiments, there is increased or decreased specificity of on-target binding vs. off-target binding. In certain embodiments, nuclease activity of guide RNA-Cas (e.g. Cpfl) enzyme is also modulated.

[00311] RNA-DNA heteroduplex formation is important for cleavage activity and specificity throughout the target region, not only the seed region sequence closest to the PAM. Thus, truncated guide RNAs show reduced cleavage activity and specificity. In an aspect, the invention provides method and mutations for increasing activity and specificity of cleavage using altered guide RNAs.

[00312] The invention also demonstrates that modifications of Cas (e.g. Cpfl) nuclease specificity can be made in concert with modifications to targeting range. Cas (e.g. Cpfl) mutants can be designed that have increased target specificity as well as accommodating modifications in PAM recognition, for example by choosing mutations that alter PAM specificity and combining those mutations with nt-groove mutations that increase (or if desired, decrease) specificity for on-target sequences vs. off-target sequences. In one such embodiment, a PI domain residue is mutated to accommodate recognition of a desired PAM sequence while one or more nt-groove amino acids is mutated to alter target specificity. The Cas (e.g. Cpfl) methods and modifications described herein can be used to counter loss of specificity resulting from alteration of PAM recognition, enhance gain of specificity resulting from alteration of PAM recognition, counter gain of specificity resulting from alteration of PAM recognition, or enhance loss of specificity resulting from alteration of PAM recognition.

[00313] The methods and mutations can be used with any Cas (e.g. Cpfl) enzyme with altered PAM recognition. Non-limiting examples of PAMs included are as described herein elsewhere. 2023241400 09 Oct 2023 [00314J In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzyme may comprise one or more heterologous functional domains as described elsewhere herein. [003151 In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzyme may comprise a CRISPR enzyme from an organism from a genus comprising Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW201 l_GWA2 33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11 _00205, Butyrivibrio sp. NC3005, Thiomicrospira sp. XS5, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae (e.g., a Cpfl of one of these organisms modified as described herein), and may include further mutations or alterations or be a chimeric Cas (e.g. Cpfl).

[00316] In any of the non-naturally-occurring CRISPR enzymes, the CRISPR enzyme may comprise a chimeric Cas (e.g. Cpfl) enzyme comprising a first fragment from a first Cas (e.g. Cpfl) ortho log and a second fragment from a second Cas (e.g. Cpfl) ortholog, and the first and second Cas (e.g. Cpfl) orthologs are different. At least one of the first and second Cas (e.g. Cpfl) orthologs may comprise a Cas (e.g. Cpfl) from an organism comprising Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW201 l_GWA2_33_10, Parcubacteria bacterium GW2011 _GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Moraxella bovoculi AAX08_00205, Moraxella bovoculi AAX11 _00205, Butyrivibrio sp. NC3005, Thiomicrospira sp. XS5, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae.

[00317] In certain embodiments, the methods as described herein may comprise providing a Cas (e.g. Cpfl) transgenic cell in which one or more nucleic acids encoding one or more guide RNAs are provided or introduced operably connected in the cell with a regulatory element comprising a promoter of one or more gene of interest. As used herein, the term “Cas 2023241400 09 Oct 2023 transgenic cell” refers to a cell, such as a eukaryotic cell, in which a Cas gene has been genomically integrated. The nature, type, or origin of the cell are not particularly limiting according to the present invention. Also the way how the Cas transgene is introduced in the cell is may vary and can be any method as is known in the art. In certain embodiments, the Cas transgenic cell is obtained by introducing the Cas transgene in an isolated cell. In certain other embodiments, the Cas transgenic cell is obtained by isolating cells from a Cas transgenic organism. By means of example, and without limitation, the Cas transgenic cell as referred to herein may be derived from a Cas transgenic eukaryote, such as a Cas knock-in eukaryote. Reference is made to WO 2014 / 093622 (PCT / US13 / 74667), incorporated herein by reference. Methods of US Patent Publication Nos. 20120017290 and 20110265198 assigned to Sangamo BioSciences, Inc. directed to targeting the Rosa locus may be modified to utilize the CRISPR Cas system of the present invention. Methods of US Patent Publication No. 20130236946 assigned to Cellectis directed to targeting the Rosa locus may also be modified to utilize the CRISPR Cas system of the present invention. By means of further example reference is made to Platt et. al. (Cell; 159(2):440-455 (2014)), describing a Cas9 knock-in mouse, which is incorporated herein by reference, and which can be extrapolated to the CRISPR enzymes of the present invention as defined herein. The Cas transgene can further comprise a Lox-Stop-polyA-Lox(LSL) cassette thereby rendering Cas expression inducible by Cre recombinase. Alternatively, the Cas transgenic cell may be obtained by introducing the Cas transgene in an isolated cell. Delivery systems for transgenes are well known in the art. By means of example, the Cas transgene may be delivered in for instance eukaryotic cell by means of vector (e.g., AAV, adenovirus, lentivirus) and / or particle and / or nanoparticle delivery, as also described herein elsewhere.

[00318] It will be understood by the skilled person that the cell, such as the Cas transgenic cell, as referred to herein may comprise further genomic alterations besides having an integrated Cas gene or the mutations arising from the sequence specific action of Cas when complexed with RNA capable of guiding Cas to a target locus, such as for instance one or more oncogenic mutations, as for instance and without limitation described in Platt et al. (2014), Chen et al., (2014) or Kumar et al.. (2009). 2023241400 09 Oct 2023

[00319] The invention also provides an engineered, non-naturally occurring Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) vector system comprising one or more vectors comprising: a) a first regulatory element operably linked to a nucleotide sequence encoding a non-naturally-occurring CRISPR enzyme of any one of the inventive constructs herein; and b) a second regulatory element operably linked to one or more nucleotide sequences encoding one or more of the guide RNAs, the guide RNA comprising a guide sequence, a direct repeat sequence, wherein: components (a) and (b) are located on same or different vectors, the CRISPR complex is formed; the guide RNA targets the target polynucleotide loci and the enzyme alters the polynucleotide loci, and the enzyme in the CRISPR complex has reduced capability of modifying one or more off-target loci as compared to an unmodified enzyme and / or whereby the enzyme in the CRISPR complex has increased capability of modifying the one or more target loci as compared to an unmodified enzyme.

[00320] In such a system, component (II) may comprise a first regulatory element operably linked to a polynucleotide sequence which comprises the guide sequence, the direct repeat sequence, and wherein component (II) may comprise a second regulatory element operably linked to a polynucleotide sequence encoding the CRISPR enzyme. In such a system, where applicable the guide RNA may comprise a chimeric RNA.

[00321] In such a system, component (I) may comprise a first regulatory element operably linked to the guide sequence and the direct repeat sequence, and wherein component (II) may comprise a second regulatory element operably linked to a polynucleotide sequence encoding the CRISPR enzyme. Such a system may comprise more than one guide RNA, and each guide RNA has a different target whereby there is multiplexing. Components (a) and (b) may be on the same vector.

[00322] The invention also provides a method of modifying a locus of interest in a cell comprising contacting the cell with any of the herein-described engineered CRISPR enzymes (e.g. engineered Cpfl), compositions or any of the herein-described systems or vector 2023241400 09 Oct 2023 systems, or wherein the cell comprises any of the herein-described CRISPR complexes present within the cell. In such methods the cell may be a prokaryotic or eukaryotic cell, preferably a eukaryotic cell. In such methods, an organism may comprise the cell. In such methods the organism may not be a human or other animal.

[00323] The invention also provides the use of any of the engineered CRISPR enzymes (e.g. engineered Cpfl), compositions, systems or CRISPR complexes described above for gene or genome editing.

[00324] The invention also provides a method of altering the expression of a genomic locus of interest in a mammalian cell comprising contacting the cell with the engineered CRISPR enzymes (e.g. engineered Cpfl), compositions, systems or CRISPR complexes described herein and thereby delivering the CRISPR-Cas (vector) and allowing the CRISPR-Cas complex to form and bind to target, and determining if the expression of the genomic locus has been altered, such as increased or decreased expression, or modification of a gene product.

[00325] The invention also provides any of the engineered CRISPR enzymes (e.g. engineered Cpfl), compositions, systems or CRISPR complexes described above for use as a therapeutic. The therapeutic may be for gene or genome editing, or gene therapy. In particular embodiments, the target sequence in a genomic locus of interest, is in a HSC (hematopoietic stemm cell), wherein the genomic locus of interest is associated with a mutation associated with an aberrant protein expression or with a disease condition or state.

[00326] In one aspect, the invention provides a method of modifying an organism or a nonhuman organism by manipulation of a target sequence in a genomic locus of interest of for instance an HSC(hematopoietic stem cell), e.g., wherein the genomic locus of interest is associated with a mutation associated with an aberrant protein expression or with a disease condition or state, comprising: delivering to an HSC, e.g., via contacting an HSC with a particle containing, a non-naturally occurring or engineered composition comprising: I. a CRISPR-Cas system guide RNA (gRNA) polynucleotide sequence, comprising: (a) a guide sequence capable of hybridizing to a target sequence in a HSC, 2023241400 09 Oct 2023 (b) a direct repeat sequence, and II. a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences, wherein, the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence,; and the method may optionally include also delivering a HDR template, e.g., via the particle contacting the HSC containing or contacting the HSC with another particle containing, the HDR template wherein the HDR template provides expression of a normal or less aberrant form of the protein; wherein “normal” is as to wild type, and “aberrant” can be a protein expression that gives rise to a condition or disease state; and optionally the method may include isolating or obtaining HSC from the organism or non-human organism, optionally expanding the HSC population, performing contacting of the particle(s) with the HSC to obtain a modified HSC population, optionally expanding the population of modified HSCs, and optionally administering modified HSCs to the organism or non-human organism.

[00327] In one aspect, the invention provides a method of modifying an organism or a nonhuman organism by manipulation of a target sequence in a genomic locus of interest of for instance a HSC, e.g., wherein the genomic locus of interest is associated with a mutation associated with an aberrant protein expression or with a disease condition or state, comprising: delivering to an HSC, e.g., via contacting an HSC with a particle containing, a non-naturally occurring or engineered composition comprising: I. (a) a guide sequence capable of hybridizing to a target sequence in a HSC, and (b) at least one or more direct repeat sequences, and II. a CRISPR enzyme optionally having one or more NLSs,, and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with the guide sequence that is hybridized to the target sequence,; and the method may optionally include also delivering a HDR template, e.g., via the particle contacting the HSC containing or contacting the HSC with another particle containing, the HDR template wherein the HDR template provides expression of a normal or 2023241400 09 Oct 2023 less aberrant form of the protein; wherein “normal” is as to wild type, and “aberrant” can be a protein expression that gives rise to a condition or disease state; and optionally the method may include isolating or obtaining HSC from the organism or non-human organism, optionally expanding the HSC population, performing contacting of the particle(s) with the HSC to obtain a modified HSC population, optionally expanding the population of modified HSCs, and optionally administering modified HSCs to the organism or non-human organism. [003281 The delivery can be of one or more polynucleotides encoding any one or more or all of the CRISPR-complex, advantageously linked to one or more regulatory elements for in vivo expression, e.g. via particle(s), containing a vector containing the polynucleotide(s) operably linked to the regulatory element(s). Any or all of the polynucleotide sequence encoding a CRISPR enzyme, guide sequence, direct repeat sequence, may be RNA. It will be appreciated that where reference is made to a polynucleotide, which is RNA and is said to ‘comprise’ a feature such a direct repeat sequence, the RNA sequence includes the feature. Where the polynucleotide is DNA and is said to comprise a feature such a direct repeat sequence, the DNA sequence is or can be transcribed into the RNA including the feature at issue. Where the feature is a protein, such as the CRISPR enzyme, the DNA or RNA sequence referred to is, or can be, translated (and in the case of DNA transcribed first).

[00329] In certain embodiments the invention provides a method of modifying an organism, e.g., mammal including human or a non-human mammal or organism by manipulation of a target sequence in a genomic locus of interest of an HSC e.g., wherein the genomic locus of interest is associated with a mutation associated with an aberrant protein expression or with a disease condition or state, comprising delivering, e.g., via contacting of a non-naturally occurring or engineered composition with the HSC, wherein the composition comprises one or more particles comprising viral, plasmid or nucleic acid molecule vector(s) (e.g. RNA) operably encoding a composition for expression thereof, wherein the composition comprises: (A) I. a first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a direct repeat sequence and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences 2023241400 09 Oct 2023 (or optionally at least one or more nuclear localization sequences as some embodiments can involve no NLS), wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation, wherein components I and II are located on the same or different vectors of the system, wherein when transcribed and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with the guide sequence that is hybridized to the target sequence, or (B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) at least one or more direct repeat sequences, II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and optionally, where applicable, wherein components I, and II are located on the same or different vectors of the system, wherein when transcribed and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with the guide sequence that is hybridized to the target sequence; the method may optionally include also delivering a HDR template, e.g., via the particle contacting the HSC containing or contacting the HSC with another particle containing, the HDR template wherein the HDR template provides expression of a normal or less aberrant form of the protein; wherein “normal” is as to wild type, and “aberrant” can be a protein expression that gives rise to a condition or disease state; and optionally the method may include isolating or obtaining HSC from the organism or non-human organism, optionally expanding the HSC population, performing contacting of the particle(s) with the HSC to obtain a modified HSC population, optionally expanding the population of modified HSCs, and optionally administering modified HSCs to the organism or non-human organism. In some embodiments, components I, II and III are located on the same vector. In other embodiments, components I and II are located on the same vector, while component III is located on another vector. In other embodiments, components I and III are located on the same vector, while component II is located on another vector. In other embodiments, components II and III are located on the same vector, while component I is located on another vector. In other embodiments, each of components I, II and III is located on different vectors. The invention also provides a viral or plasmid vector system as described herein. 2023241400 09 Oct 2023

[00330] By manipulation of a target sequence, Applicants also mean the epigenetic manipulation of a target sequence. This may be f the chromatin state of a target sequence, such as by modification of the methylation state of the target sequence (i.e. addition or removal of methylation or methylation patterns or CpG islands), histone modification, increasing or reducing accessibility to the target sequence, or by promoting 3D folding. It will be appreciated that where reference is made to a method of modifying an organism or mammal including human or a non-human mammal or organism by manipulation of a target sequence in a genomic locus of interest, this may apply to the organism (or mammal) as a whole or just a single cell or population of cells from that organism (if the organism is multicellular). In the case of humans, for instance, Applicants envisage, inter alia, a single cell or a population of cells and these may preferably be modified ex vivo and then reintroduced. In this case, a biopsy or other tissue or biological fluid sample may be necessary. Stem cells are also particularly preferred in this regard. But, of course, in vivo embodiments are also envisaged. And the invention is especially advantageous as to HSCs.

[00331] The invention in some embodiments comprehends a method of modifying an organism or a non-human organism by manipulation of a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in a HSC e.g., wherein the genomic locus of interest is associated with a mutation associated with an aberrant protein expression or with a disease condition or state, comprising delivering, e.g., by contacting HSCs with particle(s) comprising a non-naturally occurring or engineered composition comprising : I. a first CRISPR-Cas (e.g. Cpfl) system RNA polynucleotide sequence, wherein the first polynucleotide sequence comprises: (a) a first guide sequence capable of hybridizing to the first target sequence, (b) a first direct repeat sequence, and II. a second CRISPR-Cas (e.g. Cpfl) system guide RNA polynucleotide sequence, wherein the second polynucleotide sequence comprises: (a) a second guide sequence capable of hybridizing to the second target sequence, (b) a second direct repeat sequence, and 2023241400 09 Oct 2023 III. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences and comprising one or more mutations, wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation; or IV. expression product(s) of one or more of I. to III., e.g., the the first and the second direct repeat sequence, the CRISPR enzyme; wherein when transcribed, the first and the second guide sequence directs sequencespecific binding of a first and a second CRISPR complex to the first and second target sequences respectively, wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized to the first target sequence, wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridized to the second target sequence, wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of the other strand near the second target sequence inducing a double strand break, thereby modifying the organism or the nonhuman organism; and the method may optionally include also delivering a HDR template, e.g., via the particle contacting the HSC containing or contacting the HSC with another particle containing, the HDR template wherein the HDR template provides expression of a normal or less aberrant form of the protein; wherein “normal” is as to wild type, and “aberrant” can be a protein expression that gives rise to a condition or disease state; and optionally the method may include isolating or obtaining HSC from the organism or nonhuman organism, optionally expanding the HSC population, performing contacting of the particle(s) with the HSC to obtain a modified HSC population, optionally expanding the population of modified HSCs, and optionally administering modified HSCs to the organism or non-human organism. In some methods of the invention any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second direct repeat sequence. In further embodiments of the invention the polynucleotides encoding the sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second direct repeat sequence, is / are RNA and are delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun; but, it is advantageous that the delivery is via a particle. In certain embodiments of the invention, the 2023241400 09 Oct 2023 first and second direct repeat sequence share 100% identity. In some embodiments, the polynucleotides may be comprised within a vector system comprising one or more vectors. In preferred embodiments, the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand nicking enzyme, and the second CRISPR enzyme has one or more mutations such that the enzyme is a non-complementary strand nicking enzyme. Alternatively the first enzyme may be a non-complementary strand nicking enzyme, and the second enzyme may be a complementary strand nicking enzyme. In preferred methods of the invention the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of the other strand near the second target sequence results in a 5’ overhang. In embodiments of the invention the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In embodiments of the invention the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs.

[00332] The invention in some embodiments comprehends a method of modifying an organism or a non-human organism by manipulation of a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in for instance a HSC e.g., wherein the genomic locus of interest is associated with a mutation associated with an aberrant protein expression or with a disease condition or state, comprising delivering, e.g., by contacting HSCs with particle(s) comprising a non-naturally occurring or engineered composition comprising : I. a first regulatory element operably linked to (a) a first guide sequence capable of hybridizing to the first target sequence, and (b) at least one or more direct repeat sequences, II. a second regulatory element operably linked to (a) a second guide sequence capable of hybridizing to the second target sequence, and (b) at least one or more direct repeat sequences, III. a third regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme (e.g. Cpfl), and 2023241400 09 Oct 2023 V. expression product(s) of one or more of I. to IV., e.g., the the first and the second direct repeat sequence, the CRISPR enzyme; wherein components I, II, III and IV are located on the same or different vectors of the system, when transcribed, and the first and the second guide sequence direct sequencespecific binding of a first and a second CRISPR complex to the first and second target sequences respectively, wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized to the first target sequence, wherein the second CRISPR complex comprises the CRISPR enzyme complexed with the second guide sequence that is hybridized to the second target sequence, wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of the other strand near the second target sequence inducing a double strand break, thereby modifying the organism or the nonhuman organism; and the method may optionally include also delivering a HDR template, e.g., via the particle contacting the HSC containing or contacting the HSC with another particle containing, the HDR template wherein the HDR template provides expression of a normal or less aberrant form of the protein; wherein “normal” is as to wild type, and “aberrant” can be a protein expression that gives rise to a condition or disease state; and optionally the method may include isolating or obtaining HSC from the organism or nonhuman organism, optionally expanding the HSC population, performing contacting of the particle(s) with the HSC to obtain a modified HSC population, optionally expanding the population of modified HSCs, and optionally administering modified HSCs to the organism or non-human organism.

[00333] The invention also provides a vector system as described herein. The system may comprise one, two, three or four different vectors. Components I, II, III and IV may thus be located on one, two, three or four different vectors, and all combinations for possible locations of the components are herein envisaged, for example: components I, II, III and IV can be located on the same vector; components I, II, III and IV can each be located on different vectors; components I, II, II I and IV may be located on a total of two or three different vectors, with all combinations of locations envisaged, etc. In some methods of the invention any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the 2023241400 09 Oct 2023 second guide sequence, the first and the second direct repeat sequence is / are RNA. In further embodiments of the invention the first and second direct repeat sequence share 100% identity. In preferred embodiments, the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand nicking enzyme, and the second CRISPR enzyme has one or more mutations such that the enzyme is a non-complementary strand nicking enzyme. Alternatively the first enzyme may be a non-complementary strand nicking enzyme, and the second enzyme may be a complementary strand nicking enzyme. In a further embodiment of the invention, one or more of the viral vectors are delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun; but, particle delivery is advantageous.

[00334] In preferred methods of the invention the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a 5’ overhang. In embodiments of the invention the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In embodiments of the invention the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs.

[00335] The invention in some embodiments comprehends a method of modifying a genomic locus of interest in for instance HSC e.g., wherein the genomic locus of interest is associated with a mutation associated with an aberrant protein expression or with a disease condition or state, by introducing into the HSC, e.g., by contacting HSCs with particle(s) comprising, a Cas protein having one or more mutations and two guide RNAs that target a first strand and a second strand of the DNA molecule respectively in the HSC, whereby the guide RNAs target the DNA molecule and the Cas protein nicks each of the first strand and the second strand of the DNA molecule, whereby a target in the HSC is altered; and, wherein the Cas protein and the two guide RNAs do not naturally occur together and the method may optionally include also delivering a HDR template, e.g., via the particle contacting the HSC containing or contacting the HSC with another particle containing, the HDR template wherein the HDR template provides expression of a normal or less aberrant form of the protein; wherein “normal” is as to wild type, and “aberrant” can be a protein expression that gives rise to a condition or disease state; and optionally the method may include isolating or obtaining HSC from the organism or non-human organism, optionally expanding the HSC population, 2023241400 09 Oct 2023 performing contacting of the particle(s) with the HSC to obtain a modified HSC population, optionally expanding the population of modified HSCs, and optionally administering modified HSCs to the organism or non-human organism. In preferred methods of the invention the Cas protein nicking each of the first strand and the second strand of the DNA molecule results in a 5’ overhang. In embodiments of the invention the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In embodiments of the invention the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs. In an aspect of the invention the Cas protein is codon optimized for expression in a eukaryotic cell, preferably a mammalian cell or a human cell. Aspects of the invention relate to the expression of a gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased. In an embodiment of the invention, the gene product is a protein. In an aspect, the invention provides cells which transiently comprise CRISPR systems, or components. For example, CRISPR proteins or enzymes and nucleic acids are transiently provided to a cell and a genetic locus is altered, followed by a decline in the amount of one or more components of the CRISPR system. Subsequently, the cells, progeny of the cells, and organisms which comprise the cells, having acquired a CRISPR mediated genetic alteration, comprise a diminished amount of one or more CRISPR system components, or no longer contain the one or more CRISPR system components. One non-limiting example is a selfinactivating CRISPR-Cas system such as further described herein. Thus, the invention provides cells, and organisms, and progeny of the cells and organisms which comprise one or more CRISPR-Cas system-altered genetic loci, but essentially lack one or more CRISPR system component. In certain embodiments, the CRISPR system components are substantially absent. Such cells, tissues and organisms advantageously comprise a desired or selected genetic alteration but have lost CRISPR-Cas components or remnants thereof that potentially might act non-specifically, lead to questions of safety, or hinder regulatory approval. As well, the invention provides products made by the cells, organisms, and progeny of the cells and organisms. 2023241400 09 Oct 2023 Inducible Cpfl CRISPR-Cas systems (“Split-Cpfl ”)

[00336] In an aspect the invention provides a non-naturally occurring or engineered inducible Cpfl CRISPR-Cas system, comprising: a first Cpfl fusion construct attached to a first half of an inducible dimer and a second Cpfl fusion construct attached to a second half of the inducible dimer, wherein the first Cpfl fusion construct is operably linked to one or more nuclear localization signals, wherein the second Cpfl fusion construct is operably linked to one or more nuclear export signals, wherein contact with an inducer energy source brings the first and second halves of the inducible dimer together, wherein bringing the first and second halves of the inducible dimer together allows the first and second Cpfl fusion constructs to constitute a functional Cpfl CRISPR-Cas system, wherein the Cpfl CRISPR-Cas system comprises a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, and wherein the functional Cpfl CRISPR-Cas system binds to the target sequence and, optionally, edits the genomic locus to alter gene expression.

[00337] In an aspect of the invention in the inducible Cpfl CRISPR-Cas system, the inducible dimer is or comprises or consists essentially of or consists of an inducible heterodimer. In an aspect, in inducible Cpfl CRISPR-Cas system, the first half or a first portion or a first fragment of the inducible heterodimer is or comprises or consists of or consists essentially of an FKBP, optionally FKBP12. In an aspect of the invention, in the inducible Cpfl CRISPR-Cas system, the second half or a second portion or a second fragment of the inducible heterodimer is or comprises or consists of or consists essentially of FRB. In an aspect of the invention, in the inducible Cpfl CRISPR-Cas system, the arrangement of the first Cpfl fusion construct is or comprises or consists of or consists essentially of N’ terminal Cpfl part-FRB-NES. In an aspect of the invention, in the inducible Cpfl CRISPR-Cas system, the arrangement of the first Cpfl fusion construct is or comprises or consists of or consists essentially of NES-N’ terminal Cpfl part-FRB-NES. In an aspect of the invention, in the inducible Cpfl CRISPR-Cas system, the arrangement of the second Cpfl fusion construct 2023241400 09 Oct 2023 is or comprises or consists essentially of or consists of C’ terminal Cpfl part-FKBP-NLS. In an aspect the invention provides in the inducible Cpfl CRISPR-Cas system, the arrangement of the second Cpfl fusion construct is or comprises or consists of or consists essentially of NLS-C’ terminal Cpfl part-FKBP-NLS. In an aspect, in inducible Cpfl CRISPR-Cas system there can be a linker that separates the Cpfl part from the half or portion or fragment of the inducible dimer. In an aspect, in the inducible Cpfl CRISPR-Cas system, the inducer energy source is or comprises or consists essentially of or consists of rapamycin. In an aspect, in inducible Cpfl CRISPR-Cas system, the inducible dimer is an inducible homodimer. In an aspect, in inducible Cpfl CRISPR-Cas system, the Cpfl is FnCpfl, AsCpfl or LbCpfl. In an aspect, in the inducible Cpfl CRISPR-Cas system, one or more functional domains are associated with one or both parts of the Cpfl, e.g., the functional domains optionally including a transcriptional activator, a transcriptional or a nuclease such as a Fokl nuclease. In an aspect, in the inducible Cpfl CRISPR-Cas system, the functional Cpfl CRISPR-Cas system binds to the target sequence and the enzyme is a dead-Cpfl, optionally having a diminished nuclease activity of at least 97%, or 100% (or no more than 3% and advantageously 0% nuclease activity) as compared with the Cpfl not having the at least one mutation. The invention further comprehends and an aspect of the invention provides, a polynucleotide encoding the inducible Cpfl CRISPR-Cas system as herein discussed. [00338J In an aspect, the invention provides a method of treating a subject in need thereof, comprising inducing gene editing by transforming the subject with the polynucleotide as herein discussed or any of the vectors herein discussed and administering an inducer energy source to the subject. The invention also provides a method of treating a subject in need thereof, comprising inducing transcriptional activation or repression by transforming the subject with the polynucleotide herein discussed or any of the vectors herein discussed, wherein said polynucleotide or vector encodes or comprises the catalytically inactive Cpfl and one or more associated functional domains as herein discussed; the method further comprising administering an inducer energy source to the subject. The invention also provides the polynucleotide herein discussed or any of the vectors herein discussed for use in a method of treating a subject in need thereof comprising inducing transcriptional activation or repression, wherein the method further comprises administering an inducer energy source to the subject. 2023241400 09 Oct 2023

[00339] In an aspect the invention involves a non-naturally occurring or engineered inducible Cpfl CRISPR-Cas system, comprising a first Cpfl fusion construct attached to a first half of an inducible heterodimer and a second Cpfl fusion construct attached to a second half of the inducible heterodimer, wherein the first CPfl fusion construct is operably linked to one or more nuclear localization signals, wherein the second CPfl fusion construct is operably linked to a nuclear export signal, wherein contact with an inducer energy source brings the first and second halves of the inducible heterodimer together, wherein bringing the first and second halves of the inducible heterodimer together allows the first and second Cpfl fusion constructs to constitute a functional Cpfl CRISPR-Cas system, wherein the Cpfl CRISPR-Cas system comprises a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence in a genomic locus of interest in a cell, and wherein the functional Cpfl CRISPR-Cas system edits the genomic locus to alter gene expression. In an embodiment of the invention the first half of the inducible heterodimer is FKBP12 and the second half of the inducible heterodimer is FRB. In another embodiment of the invention the inducer energy source is rapamycin.

[00340] An inducer energy source may be considered to be simply an inducer or a dimerizing agent. The term ‘inducer energy source’ is used herein throughout for consistency. The inducer energy source (or inducer) acts to reconstitute the Cpfl. In some embodiments, the inducer energy source brings the two parts of the Cpfl together through the action of the two halves of the inducible dimer. The two halves of the inducible dimer therefore are brought tougher in the presence of the inducer energy source. The two halves of the dimer will not form into the dimer (dimerize) without the inducer energy source.

[00341] Thus, the two halves of the inducible dimer cooperate with the inducer energy source to dimerize the dimer. This in turn reconstitutes the Cpfl by bringing the first and second parts of the Cpfl together.

[00342] The CRISPR enzyme fusion constructs each comprise one part of the split Cpfl. These are fused, preferably via a linker such as a GlySer linker described herein, to one of the two halves of the dimer. The two halves of the dimer may be substantially the same two monomers that together that form the homodimer, or they may be different monomers that together form the heterodimer. As such, the two monomers can be thought of as one half of the full dimer. 2023241400 09 Oct 2023

[00343] The Cpfl is split in the sense that the two parts of the Cpfl enzyme substantially comprise a functioning Cpfl. That Cpfl may function as a genome editing enzyme (when forming a complex with the target DNA and the guide), such as a nickase or a nuclease (cleaving both strands of the DNA), or it may be a dead-Cpfl which is essentially a DNA-binding protein with very little or no catalytic activity, due to typically mutation(s) in its catalytic domains.

[00344] The two parts of the split Cpfl can be thought of as the N’ terminal part and the C’ terminal part of the split Cpfl. The fusion is typically at the split point of the Cpfl. In other words, the C’ terminal of the N’ terminal part of the split Cpfl is fused to one of the dimer halves, whilst the N’ terminal of the C’ terminal part is fused to the other dimer half.

[00345] The Cpfl does not have to be split in the sense that the break is newly created. The split point is typically designed in silico and cloned into the constructs. Together, the two parts of the split Cpfl, the N’ terminal and C’ terminal parts, form a full Cpfl, comprising preferably at least 70% or more of t...

Claims

1. An adeno-associated virus (AAV) vector comprising (a) a first regulatory elementoperably linked to a nucleotide sequence encoding a Cpf1 effector protein, and (b) a second regulatory element operably linked to a nucleotide sequence encoding a guide RNA comprising a guide sequence linked to a direct repeat sequence,wherein the guide sequence is capable of hybridizing with a target sequence 3’ of a Protospacer Adjacent Motif (PAM),wherein the guide RNA is capable of forming a complex with the Cpf1 effector protein, andwherein the Cpf1 effector protein has at least 95% sequence identity with Moraxella bovoculi AAX08_00205 Cpf1 (Mb2Cpf1) or Moraxella bovoculi AAX11_00205 Cpf1 (Mb3Cpf1).

2. An adeno-associated virus (AAV) vector comprising (a) a first regulatory elementoperably linked to a nucleotide sequence encoding a Cpf1 effector protein, and (b) a second regulatory element operably linked to a plurality of nucleotide sequences encoding a plurality of guide RNAs each comprising a guide sequence linked to a direct repeat sequence,wherein the guide sequence is capable of hybridizing with a target sequence 3’ of a Protospacer Adjacent Motif (PAM),wherein each guide RNA is capable of forming a complex with the Cpf1 effector protein, wherein the Cpf1 effector protein has at least 95% sequence identity with Moraxellabovoculi AAX08_00205 Cpf1 (Mb2Cpf1) or Moraxella bovoculi AAX11_00205 Cpf1 (Mb3Cpf1),wherein the plurality of guide RNAs target different target sequences, andwherein the plurality of nucleotide sequences encoding the plurality of guide RNAs are operably linked to the second regulatory element in tandem.

3. The AAV vector of claim 1 or claim 2, wherein the nucleotide sequence encoding theCpf1 effector protein is codon optimized for expression in a eukaryotic cell.2023241400   28 Apr 20264.     The AAV vector of any one of claims 1-3, wherein the Cpf1 effector protein is fused to atleast one nuclear localization signal (NLS).

5. The AAV vector of any one of claims 1-3, wherein the Cpf1 effector protein is fused to atleast two NLSs.

6. The AAV vector of any one of claims 1 -5, wherein the Cpf1 effector protein is Mb2Cpf1or Mb3Cpf1.

7. The AAV vector of any one of claims 1-6, wherein the Cpf1 effector protein comprises atleast one mutation in a catalytic domain.

8. The AAV vector of any one of claims 1-7, wherein the Cpf1 effector protein is fused to atleast one heterologous functional domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, or deaminase activity.

9. The AAV vector of any one of claims 1-8, wherein the direct repeat sequence comprisesAAUUUCUACUAAGUGUAGAU, AAUUUCUACUGUUGUAGAU, AAUUUCUACUAUUGUAGAU, AAUUUCUACUUUUGUAGAU, AAUUUCUACUCUUGUAGAU, or AAUUUCUACUGUUUGUAGAU.

10. The AAV vector of any one of claims 1-9, wherein the first regulatory element is a constitutive promoter or an inducible promoter.

11. The AAV vector of any one of claims 1-9, wherein the first regulatory element is a tissue-specific promoter.

12. The AAV vector of any one of claims 1-11, wherein the second regulatory element is a constitutive promoter or an inducible promoter.

13. The AAV vector of any one of claims 1-11, wherein the second regulatory element is a tissue-specific promoter.2023241400   28 Apr 202614. The AAV vector of any one of claims 1-13, wherein the PAM comprises a 5’ T-rich motif.

15. The AAV vector of any one of claims 1-13, wherein the PAM is TTN, wherein N is A / C / G or T.

16. The AAV vector of any one of claims 1-13, wherein the PAM is TTTV, wherein V is A / C or G.

17. The AAV vector of any one of claims 1-16, wherein the target sequence is within a eukaryotic cell.

18. The AAV vector of claim 17, wherein the target sequence resides within the nucleus of a eukaryotic cell.

19. Use of the AAV vector of any one of claims 1-18 for treating a genetic disease or disorder.

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