Novel crispr DNA targeting enzymes and systems

Abstract

To provide novel CRISPR DNA targeting enzymes and systems.SOLUTION: The disclosure describes novel systems, methods, and compositions for manipulation of nucleic acids in a targeted fashion. The disclosure describes non-naturally occurring, engineered CRISPR systems, components, and methods for targeted modification of nucleic acids such as DNA. Each system includes one or more protein components and one or more nucleic acid components that together target nucleic acids.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 62 / 642,919, filed March 14, 2018; U.S. patent application Ser. No. 62 / 666,397, filed May 3, 2018; U.S. patent application Ser. No. 62 / 672,489, filed May 16, 2018; U.S. patent application Ser. No. 62 / 679,628, filed June 1, 2018; U.S. patent application Ser. No. 62 / 679,628, filed July 26, 2018; No. 62 / 746,528, filed October 16, 2018; U.S. Patent Application No. 62 / 772,038, filed November 27, 2018; and U.S. Patent Application No. 62 / 775,885, filed December 5, 2018. The contents of each of the foregoing applications are hereby incorporated by reference in their entirety.

[0002] The present disclosure relates to systems, methods, and compositions used for gene expression control involving sequence targeting and nucleic acid editing, using vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its components. [Background technology]

[0003] In recent years, advances in genome sequencing technology and analysis have been applied to provide important insights into the genetic basis of biological activities in a wide variety of natural domains, ranging from prokaryotic biosynthetic pathways to human pathologies. To fully understand and evaluate the vast amount of information generated by gene sequencing technologies, corresponding improvements in the scale, efficacy, and ease of genome and epigenome engineering techniques are required. These novel genome and epigenome engineering technologies will accelerate the development of novel applications in numerous areas, including biotechnology, agriculture, and human therapeutics.

[0004] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR / Cas systems, are currently understood to confer immunity to phage infection in bacteria and archaea. The CRISPR-Cas system of prokaryotic adaptive immunity is an extremely diverse group of protein effectors, non-coding elements, and locus organizations, some examples of which have been engineered and adapted to generate important biotechnological applications.

[0005] Components of this system involved in host defense include one or more effector proteins capable of modifying DNA or RNA and an RNA guide element responsible for targeting the activity of these proteins to specific sequences on phage DNA or RNA. The RNA guide is composed of a CRISPR RNA (crRNA), which may require an additional trans-activating RNA (tracrRNA) to enable manipulation of target nucleic acids by one or more effector proteins. The crRNA consists of direct repeats responsible for protein binding to the crRNA and a spacer sequence complementary to the desired nucleic acid target sequence. The CRISPR system can be reprogrammed to target alternative DNA or RNA targets by modifying the spacer sequence of the crRNA.

[0006] CRISPR-Cas systems can be broadly divided into two classes: Class 1 systems consist of multiple effector proteins that together form a complex around the crRNA, whereas Class 2 systems consist of a single effector protein that complexes with an RNA guide to target DNA or RNA substrates. The single-subunit effector composition of Class 2 systems provides a more convenient set of components for engineering and translation, and thus has been an important source of programmable effectors to date. Thus, the discovery, engineering, and optimization of novel Class 2 systems may lead to widespread and powerful programmable technologies for genome engineering and beyond. Summary of the Invention [Problem to be solved by the invention]

[0007] The CRISPR-Cas system is an adaptive immune system in archaea and bacteria that defends species from foreign genetic elements. Class 2, exemplified by CRISPR-Cas9, Although the characterization and engineering of CRISPR-Cas systems has paved the way for a diverse array of biotechnological applications in genome editing and elsewhere, there remains a need for additional programmable effectors and systems for modifying nucleic acids and polynucleotides (i.e., DNA, RNA, or any hybrid, derivative, or modified form) that, due to their unique properties, enable novel applications beyond current CRISPR-Cas systems.

[0008] 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. [Means for solving the problem]

[0009] This disclosure provides non-naturally occurring engineered systems and compositions for novel single-effector Class 2 CRISPR-Cas systems, along with methods for computational identification from genome databases, development from natural loci into engineered systems, and experimental validation and application translation. These novel effectors differ in sequence from existing Class 2 CRISPR effector orthologs and homologs and also possess unique domain organization. They offer additional features, including, but not limited to: 1) novel DNA / RNA editing properties and mechanisms of control; 2) smaller size relative to greater versatility in delivery strategies; 3) genotype-driven cellular processes such as cell death; and 4) programmable RNA-guided DNA insertion, excision, and mobilization. The novel DNA targeting systems described herein add to the toolbox of genome and epigenome engineering techniques, enabling broad applications for specific, programmed perturbations.

[0010] In general, the present disclosure relates to novel CRISPR-Cas systems that include newly discovered enzymes and other components used to create minimal systems that can be used in non-native environments, such as bacteria other than the bacteria in which the system was originally discovered.

[0011] In one aspect, the present disclosure provides an engineered, non-naturally occurring CRISPR-Cas system comprising: (i) one or more type VI (CLUST.029130) RNA guides or one or more nucleic acids encoding one or more type VI RNA guides, the type VI RNA guides comprising or consisting of a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; and (ii) a type VI (CLUST.029130) CRISPR-Cas effector protein or a nucleic acid encoding a type VI CRISPR-Cas effector protein, the type VI CRISPR-Cas effector protein capable of binding to the type VI RNA guide and targeting a target nucleic acid sequence complementary to the spacer sequence, wherein the target nucleic acid is DNA. As used herein, a type VI (CLUST.029130) CRISPR-Cas effector protein is also referred to as a Cas12i effector protein, and these two terms are used interchangeably in this disclosure.

[0012] In some embodiments of any of the systems described herein, the Type VI CRISPR-Cas effector protein is about 1100 amino acids or less in length (excluding any amino acid signal sequence or peptide tag fused thereto) and comprises at least one RuvC domain. In some embodiments, the RuvC domains are none, one, or more catalytically inactive. In some embodiments, the Type VI CRISPR-Cas effector protein comprises or consists of the amino acid sequence X1SHX4DX6X7 (SEQ ID NO: 200), where X1 is S or T, X4 is Q or L, X6 is P or S, and X7 is F or L.

[0013] In some embodiments, the Type VI CRISPR-Cas effector protein has the amino acid sequence X1XDXNX6X7XXXX 11 (SEQ ID NO: 201) (wherein X1 is A or G or S, X is any amino acid, X6 is Q or I, X7 is T or S or V, and X 10is T or A. In some embodiments, the Type VI CRISPR-Cas effector protein comprises or consists of the amino acid sequence X1X2X3E (SEQ ID NO: 210), where X1 is C or F or I or L or M or P or V or W or Y, X2 is C or F or I or L or M or P or R or V or W or Y, and X3 is C or F or G or I or L or M or P or V or W or Y.

[0014] In some embodiments, the Type VI CRISPR-Cas effector protein comprises two or more sequences from the set SEQ ID NO:200, SEQ ID NO:201, and SEQ ID NO:210. In some embodiments, the Type VI CRISPR-Cas effector protein comprises or consists of an amino acid sequence at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to an amino acid sequence provided in Table 4 (e.g., SEQ ID NOs:1-5, and 11-18).

[0015] In some embodiments of any of the systems described herein, the Type VI CRISPR-Cas effector protein comprises or consists of an amino acid sequence at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of Cas12i1 (SEQ ID NO: 3) or Cas12i2 (SEQ ID NO: 5). In some embodiments, the Type VI CRISPR-Cas effector protein is Cas12i1 (SEQ ID NO: 3) or Cas12i2 (SEQ ID NO: 5).

[0016] In some embodiments, the Type VI CRISPR-Cas effector protein has the ability to recognize a protospacer adjacent motif (PAM), and the target nucleic acid comprises or consists of a PAM comprising or consisting of the nucleic acid sequence 5'-TTN-3' or 5'-TTH-3' or 5'-TTY-3' or 5'-TTC-3'.

[0017] In some embodiments of any of the systems described herein, the Type VI CRISPR-Cas effector protein comprises one or more amino acid substitutions within at least one of the RuvC domains. In some embodiments, the one or more amino acid substitutions include a substitution, e.g., an alanine substitution, at an amino acid residue corresponding to D647, E894, or D948 of SEQ ID NO: 3. In some embodiments, the one or more amino acid substitutions include an alanine substitution at an amino acid residue corresponding to D599, E833, or D886 of SEQ ID NO: 5. In some embodiments, the one or more amino acid substitutions result in a decrease in the nuclease activity of the Type VI CRISPR-Cas effector protein compared to the nuclease activity of the Type VI CRISPR-Cas effector protein without the one or more amino acid substitutions.

[0018] In some embodiments of any of the systems described herein, the type VI RNA guide comprises a direct repeat sequence comprising a stem-loop structure proximal to the 3' end (immediately adjacent to the spacer sequence). In some embodiments, the type VI RNA guide direct repeat comprises a stem-loop proximal to the 3' end, wherein the stem is 5 nucleotides long. In some embodiments, the type VI RNA guide direct repeat comprises a stem-loop proximal to the 3' end, wherein the stem is 5 nucleotides long and the loop is 7 nucleotides long. In some embodiments, the type VI RNA guide direct repeat comprises a stem-loop proximal to the 3' end, wherein the stem is 5 nucleotides long and the loop is 6, 7, or 8 nucleotides long.

[0019] In some embodiments, the type VI RNA guided direct repeat comprises the sequence 5'-CCGUCNNNNNNUGACGG-3' (SEQ ID NO: 202) (where N represents any nucleobase) proximal to the 3' end. In some embodiments, the type VI RNA guided direct repeat comprises the sequence 5'-GUGCCNNNNNNUGGCAC-3' (SEQ ID NO: 203) (where N represents any nucleobase) proximal to the 3' end.

[0020] In some embodiments, the type VI RNA guide direct repeat has the sequence 5'-GUGUCN proximal to the 3' end. 5-6 UGACAX1-3' (SEQ ID NO: 204) (wherein N 5-6 refers to any 5 or 6 consecutive nucleobase sequence, and X1 refers to C, T, or U. In some embodiments, a type VI RNA-guided direct repeat comprises the sequence 5'-UCX3UX5X6X7UUGACGG-3' (SEQ ID NO: 205) proximal to the 3' end, where X3 refers to C, T, or U, X5 refers to A, T, or U, X6 refers to A, C, or G, and X7 refers to A or G. In some embodiments, a type VI RNA-guided direct repeat comprises the sequence 5'-CCX3X4X5CX7UUGGCAC-3' (SEQ ID NO: 206) proximal to the 3' end, where X3 refers to C, T, or U, X4 refers to A, T, or U, X5 refers to C, T, or U, and X7 refers to A or G.

[0021] In some embodiments, the Type VI RNA guide comprises a direct repeat sequence that comprises or consists of a nucleotide sequence that is at least 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a nucleotide sequence provided in Table 5A (e.g., SEQ ID NOs: 6-19, and 19-24).

[0022] In some embodiments, a type VI RNA guide comprises or consists of a nucleotide sequence provided in Table 5B or a subsequence thereof (e.g., SEQ ID NOS: 150-163). In some embodiments, a type VI RNA guide comprises or consists of a direct repeat, a spacer, a nucleotide sequence constructed by concatenating the direct repeat sequence, where the direct repeat sequence is provided in Table 5A and the spacer length is provided in the Spacer Length 1 column of Table 5B. In some embodiments, a type VI RNA guide comprises or consists of a nucleotide sequence constructed by concatenating the direct repeat sequence, where the direct repeat sequence is provided in Table 5A and the spacer length is provided in the Spacer Length 2 column of Table 5B. In some embodiments, the Type VI RNA guide comprises or consists of a direct repeat, a spacer, and a nucleotide sequence constructed by concatenation of the direct repeat sequence, where the direct repeat sequence is provided in Table 5A and the spacer length is provided in the Spacer Length 3 column of Table 5B.

[0023] In some embodiments of any of the systems described herein, the spacer sequence of the Type VI RNA guide comprises or consists of about 15 to about 34 nucleotides (e.g., 16, 17, 18, 19, 20, 21, or 22 nucleotides). In some embodiments of any of the systems described herein, the spacer is 17 to 31 nucleotides in length.

[0024] In some embodiments of any of the systems provided herein, the target nucleic acid is DNA. In some embodiments of any of the systems described herein, the target nucleic acid comprises a protospacer adjacent motif (PAM), e.g., a PAM comprising or consisting of the nucleic acid sequence 5'-TTN-3', or 5'-TTH-3', or 5'-TTY-3', or 5'-TTC-3'.

[0025] In certain embodiments of any of the systems provided herein, targeting of the target nucleic acid by the Type VI CRISPR-Cas effector protein and the RNA guide results in a modification (e.g., a single- or double-strand break event) in the target nucleic acid. In some embodiments, the modification is a deletion event. In some embodiments, the modification is an insertion event. In some embodiments, the modification results in cytotoxicity and / or cell death.

[0026] In some embodiments, the Type VI CRISPR-Cas effector protein has non-specific (i.e., "collateral") nuclease (e.g., DNase) activity. In certain embodiments of any of the systems provided herein, the system further comprises a donor template nucleic acid (e.g., DNA or RNA).

[0027] In some embodiments of any of the systems provided herein, the system is in a cell (eg, a eukaryotic cell (eg, a mammalian cell) or a prokaryotic cell (eg, a bacterial cell)).

[0028] In another aspect, the present disclosure provides methods for targeting and editing a target nucleic acid, the methods comprising contacting the target nucleic acid with any of the systems described herein. This can be done by ex vivo or in vitro methods. In some embodiments, the methods described herein do not modify the identity of a human germline gene.

[0029] In other aspects, the present disclosure provides methods for targeting insertion of a payload nucleic acid at a site in a target nucleic acid, the method comprising contacting the target nucleic acid with any of the systems described herein.

[0030] In yet another aspect, the present disclosure provides a method for targeted excision of a payload nucleic acid from a site in a target nucleic acid, the method comprising contacting the target nucleic acid with any of the systems described herein.

[0031] In another aspect, the present disclosure provides a method for targeting and nicking a non-target strand (non-spacer complementary strand) of a double-stranded target DNA in response to recognition of a target strand (spacer complementary strand) of the double-stranded target DNA, the method comprising contacting the double-stranded target DNA with any of the systems described herein.

[0032] In yet another aspect, the present disclosure provides a method for targeting and cleaving double-stranded target DNA, the method comprising contacting the double-stranded target DNA with any of the systems described herein.

[0033] In some embodiments of the methods for targeting and cleaving double-stranded target DNA, the non-target strand (non-spacer complementary strand) of the double-stranded target DNA is nicked before nicking the target strand (spacer complementary strand) of the double-stranded target nucleic acid.

[0034] In yet another aspect, the disclosure provides a method for specifically editing a double-stranded nucleic acid, the method comprising contacting (a) a type VI effector protein and one other enzyme having sequence-specific nicking activity; (b) a type VI RNA guide that directs the type VI effector protein to nick the opposite strand relative to the activity of the other sequence-specific nickase; and (c) a double-stranded nucleic acid, wherein the method results in a reduced likelihood of off-target modification.

[0035] In some embodiments, the Type VI effector protein further comprises a linker sequence. In some embodiments, the Type VI effector protein comprises one or more mutations or amino acid substitutions that result in the CRISPR-associated protein being unable to cleave DNA.

[0036] In yet another aspect, the present disclosure provides a method for base editing a double-stranded nucleic acid, the method comprising contacting (a) a fusion protein comprising a type VI effector protein and a protein domain having DNA-modifying activity (e.g., cytidine deamination); (b) a type VI RNA guide that targets the double-stranded nucleic acid; and (c) the double-stranded nucleic acid. The type VI effector of the fusion protein may be modified to nick the non-target strand of the double-stranded nucleic acid. In some embodiments, the type VI effector of the fusion protein may be modified to be nuclease-deficient.

[0037] In another aspect, the present disclosure provides a method of modifying a DNA molecule, the method comprising contacting the DNA molecule with a system described herein.

[0038] In some embodiments of any of the methods (and compositions for use in such methods) described herein, the cell is a eukaryotic cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is a cancer cell (e.g., a tumor cell). In some embodiments, the cell is an infectious pathogen cell or a cell infected with an infectious pathogen. In some embodiments, the cell is a bacterial cell, a virus-infected cell, a prion-infected cell, a fungal cell, a protozoan, or a parasitic cell.

[0039] In another aspect, the present disclosure provides methods of treating a condition or disease in a subject in need thereof, and compositions for use in such methods, the method comprising administering to the subject the system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid associated with the condition or disease, wherein a Type VI CRISPR-Cas effector protein associates with an RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence complementary to at least 15 nucleotides of the spacer sequence, and wherein upon binding of the complex to the target nucleic acid sequence, the Type VI CRISPR-Cas effector protein cleaves or silences the target nucleic acid, thereby treating the condition or disease in the subject.

[0040] In some embodiments of the methods (and compositions for use in such methods) described herein, the condition or disease is cancer or an infectious disease. In some embodiments, the condition or disease is cancer, wherein the cancer is selected from the group consisting of Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and bladder cancer.

[0041] In some embodiments, a Type VI effector protein comprises or consists of at least one (e.g., 2, 3, 4, 5, 6, or more) nuclear localization signal (NLS). In some embodiments, a Type VI effector protein comprises or consists of at least one (e.g., 2, 3, 4, 5, 6, or more) nuclear export signal (NES). In some embodiments, a Type VI effector protein comprises at least one (e.g., 2, 3, 4, 5, 6, or more) NLS and at least one (e.g., 2, 3, 4, 5, 6, or more) NES.

[0042] In some embodiments, the systems described herein comprise a nucleic acid encoding one or more RNA guides, hi some embodiments, the nucleic acid encoding the one or more RNA guides is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter).

[0043] In some embodiments, the systems described herein include a nucleic acid encoding a target nucleic acid (e.g., a target DNA). In some embodiments, the nucleic acid encoding the target nucleic acid is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter).

[0044] In some embodiments, the systems described herein comprise a nucleic acid encoding a Type VI CRISPR-Cas effector protein in a vector, hi some embodiments, the systems further comprise one or more nucleic acids encoding RNA guides present in the vector.

[0045] In some embodiments, the vectors included in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors). In some embodiments, the vectors included in the system are phage vectors.

[0046] In some embodiments, the systems provided herein are in a delivery system, hi some embodiments, the delivery system is a nanoparticle, a liposome, an exosome, a microvesicle, or a gene gun.

[0047] The present disclosure also provides cells (e.g., eukaryotic or prokaryotic cells (e.g., bacterial cells)) comprising the systems described herein. In some embodiments, the eukaryotic cells are mammalian cells (e.g., human cells) or plant cells. The present disclosure also provides animal models (e.g., rodent, rabbit, dog, monkey, or ape models) and plant models comprising the cells. In some embodiments, the methods are used to treat a subject, e.g., a mammal, such as a human patient. The mammalian subject may also be a domesticated mammal, such as a dog, cat, horse, monkey, rabbit, rat, mouse, cow, goat, or sheep.

[0048] In yet another aspect, the present disclosure provides a method for detecting a target nucleic acid (e.g., DNA or RNA) in a sample, the method comprising: (a) contacting the sample with a system provided herein and a labeled reporter nucleic acid, wherein hybridization of the crRNA to the target nucleic acid results in cleavage of the labeled reporter nucleic acid; and (b) measuring a detectable signal generated by cleavage of the labeled reporter nucleic acid, thereby detecting the presence of the target nucleic acid in the sample.

[0049] In some embodiments, the method for detecting a target nucleic acid can also include comparing the level of the detectable signal to a reference signal level, and determining the amount of the target nucleic acid in the sample based on the level of the detectable signal.

[0050] In some embodiments, the measurement is performed using gold nanoparticle detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection, or semiconductor-based sensing.

[0051] In some embodiments, the labeled reporter nucleic acid can comprise a fluorescent dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher / fluorophore pair, where cleavage of the labeled reporter nucleic acid by the effector protein results in an increase or decrease in the amount of signal produced by the labeled reporter nucleic acid.

[0052] Viewed in another aspect, the disclosure includes a method of modifying a target DNA, the method comprising contacting the target DNA with a complex comprising a Cas12i effector protein and an engineered Type VI RNA guide designed to hybridize to (e.g., be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to) a target sequence of the target DNA, and the system is distinguished by (a) the absence of tracrRNA in the system, and (b) the Cas12i effector protein and the Type VI RNA guide form a complex that associates with the target DNA, thereby modifying the target DNA.

[0053] In certain embodiments, modifying the target DNA comprises cleaving at least one strand of the target DNA (e.g., creating a single-strand break or "nick" or creating a double-strand break). Alternatively or additionally, modifying the target DNA comprises either (i) binding to the target DNA, thereby preventing the target DNA from associating with another biomolecule or complex, or (ii) unwinding a portion of the target DNA. In some examples, the target DNA comprises a protospacer adjacent motif (PAM) sequence recognized by a Cas12i effector protein, such as 5'-TTN-3', 5'-TTH-3', 5'-TTY-3', or 5'-TTC-3'. The Cas12 effector protein, in certain embodiments, is a Cas12i1 effector protein or a Cas12i2 effector protein.

[0054] Continuing with this aspect of the disclosure, in certain embodiments, contacting the complex with the target DNA occurs intracellularly, for example, by (a) contacting the complex with a cell, whereby the complex forms in vitro, or (b) contacting the cell with one or more nucleic acids encoding a Cas12i effector protein and a Type VI RNA guide, whereby they are then expressed by the cell and form a complex within the cell. In some cases, the cell is a prokaryotic cell; in other cases, it is a eukaryotic cell.

[0055] In another aspect, the disclosure relates to a method of modifying target DNA, the method comprising contacting target DNA in a cell with a genome editing system comprising a Cas12i protein and a Type VI RNA guide (e.g., a crRNA, a guide RNA, or a similar structure, optionally comprising one or more nucleotide, nucleobase, or backbone modifications) comprising a 15-24 nucleotide spacer sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a sequence in the target DNA, but not a tracrRNA. In various embodiments, the Cas12i protein comprises or consists of an amino acid sequence having at least 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:3, and the type VI RNA guide comprises a direct repeat sequence having at least 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs:7 or 24; or the Cas12i protein comprises or consists of an amino acid sequence having at least 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:5, and the type VI RNA guide comprises a direct repeat sequence having at least 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs:9 or 10. The target DNA is optionally cellular DNA, and the contacting is optionally carried out in a cell, such as a prokaryotic or eukaryotic cell (eg, a mammalian cell, a plant cell, or a human cell).

[0056] In some embodiments, the Type VI CRISPR-Cas effector protein comprises an amino acid sequence having at least 90%, or at least 95%, sequence identity to one of SEQ ID NOs: 1-5 or 11-18. According to particular embodiments, the Type VI CRISPR-Cas effector protein comprises the amino acid sequence given by SEQ ID NO: 3 or the amino acid sequence given by SEQ ID NO: 5. The total length of the CRISPR-Cas effector protein of certain embodiments is less than 1100 amino acids, excluding any amino acid signal sequence or peptide tag fused thereto. In some cases, the CRISPR-Cas effector protein includes an amino acid substitution, for example, a substitution at an amino acid residue corresponding to D647, E894, or D948 of SEQ ID NO: 3, or a substitution at an amino acid residue corresponding to D599, E833, or D886 of SEQ ID NO: 5. The substitution is optionally alanine.

[0057] In yet another aspect, the disclosure relates to an engineered, non-naturally occurring CRISPR-Cas system comprising or consisting of a Cas12i effector protein and an engineered type VI RNA guide (e.g., a crRNA, guide RNA, or similar structure, optionally including one or more nucleotide, nucleobase, or backbone modifications) having a 15-34 nucleotide spacer sequence that is at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a target sequence. The system does not include a tracrRNA, and the Cas12i effector protein and type VI RNA guide form a complex that associates with the target sequence. In some examples, the complex between the Cas12i effector protein and type VI RNA guide causes cleavage of at least one strand of DNA containing the target sequence. The target sequence can include a protospacer adjacent motif (PAM) sequence recognized by the Cas12i effector protein, which is optionally 5'-TTN-3', 5'-TTY-3', 5'-TTH-3', or 5'-TTC-3'. The Type VI RNA guide can include a direct repeat sequence having at least 95%, e.g., 96%, 97%, 98%, 99%, or 100% sequence identity to one of SEQ ID NOs: 7, 9, 10, 24, 100, or 101.

[0058] In certain embodiments, the Cas12i effector protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3, and the direct repeat sequence has at least 95% sequence identity to SEQ ID NO: 100, or the Cas12i effector protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 5, and the direct repeat sequence has at least 95% sequence identity to SEQ ID NO: 101. Alternatively, or in addition, the Cas12i effector protein comprises an amino acid substitution (optionally an alanine substitution) selected from the group consisting of: (a) a substitution at an amino acid residue corresponding to D647, E894, or D948 of SEQ ID NO: 3; and (b) a substitution at an amino acid residue corresponding to D599, E833, or D886 of SEQ ID NO: 5.

[0059] In yet another aspect, the present disclosure relates to a composition comprising one or more nucleic acids encoding a CRISPR-Cas system (or genome editing system) according to one of the aspects of the present disclosure, and in another aspect, the present disclosure relates to a viral vector encoding a CRISPR-Cas system (or genome editing system) according to one of the aspects of the present disclosure.

[0060] The present disclosure also includes a method for targeting and nicking a non-spacer complementary strand of a double-stranded target DNA in response to recognition of a spacer complementary strand of the double-stranded target DNA, the method comprising contacting the double-stranded target DNA with any of the systems described herein.

[0061] In another aspect, the present disclosure includes methods for targeting and cleaving double-stranded target DNA, the methods comprising contacting the double-stranded target DNA with a system as described herein, wherein the non-spacer complementary strand of the double-stranded target DNA is nicked prior to nicking the spacer complementary strand of the double-stranded target nucleic acid.

[0062] In other embodiments, the present disclosure includes a method for detecting a target nucleic acid in a sample, the method comprising: (a) contacting a system as described herein and a labeled reporter nucleic acid with the sample, wherein hybridization of the crRNA to the target nucleic acid results in cleavage of the labeled reporter nucleic acid; and (b) measuring a detectable signal generated by cleavage of the labeled reporter nucleic acid, thereby detecting the presence of the target nucleic acid in the sample. These methods may further comprise comparing the level of the detectable signal with a reference signal level and determining the amount of the target nucleic acid in the sample based on the level of the detectable signal. In some embodiments, the measurement is performed using gold nanoparticle detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection, or semiconductor-based sensing. In some embodiments, the labeled reporter nucleic acid comprises a fluorescent dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher / fluorophore pair, wherein cleavage of the labeled reporter nucleic acid by an effector protein results in an increase or decrease in the amount of signal generated by the labeled reporter nucleic acid.

[0063] In another aspect, a method herein comprises specifically editing a double-stranded nucleic acid, the method comprising: (a) a Type VI CRISPR-Cas effector and one other enzyme having sequence-specific nicking activity, and a crRNA that directs the Type VI CRISPR-Cas effector to nick the opposite strand relative to the activity of the other sequence-specific nickase; and (b) contacting the double-stranded nucleic acid under sufficient conditions and for a sufficient length of time; wherein the method results in the formation of a double-stranded break.

[0064] Another aspect includes a method for editing double-stranded nucleic acid, the method comprising: (a) a fusion protein comprising a Type VI CRISPR-Cas effector, a protein domain having DNA-modifying activity, and an RNA guide that targets the double-stranded nucleic acid; and (b) contacting the double-stranded nucleic acid under sufficient conditions and for a sufficient length of time; wherein the Type VI CRISPR-Cas effector of the fusion protein is modified to nick the non-target strand of the double-stranded nucleic acid.

[0065] Another aspect includes a method for inducing genotype- or transcriptional state-specific cell death or dormancy in a cell, the method comprising contacting any of the systems disclosed herein with a cell, e.g., a prokaryotic or eukaryotic cell, wherein collateral DNase activity-mediated cell death or dormancy occurs upon hybridization of the RNA guide to the target DNA. For example, the cell may be a mammalian cell, e.g., a cancer cell. The cell may be an infectious cell or a cell infected with an infectious pathogen, e.g., a virus-infected cell, a prion-infected cell, a fungal cell, a protozoan, or a parasitic cell.

[0066] In another aspect, the present disclosure provides a method of treating a condition or disease in a subject in need thereof, the method comprising administering to the subject any of the systems described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid associated with the condition or disease; and wherein a Type VI CRISPR-Cas effector protein associates with the RNA guide to form a complex; wherein the complex binds to a target nucleic acid sequence that is complementary to at least 15 nucleotides of the spacer sequence; and wherein, in response to binding of the complex to the target nucleic acid sequence, the Type VI CRISPR-Cas effector protein cleaves the target nucleic acid, thereby treating a condition or disease in the subject. For example, the condition or disease can be cancer or an infectious disease. For example, the condition or disease may be cancer, wherein the cancer is selected from the group consisting of Wilms' tumor, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and bladder cancer.

[0067] The present disclosure also includes a system or cell as described herein for use as a medicament, or for use in the treatment or prevention of cancer or an infectious disease, for example, where the cancer is selected from the group consisting of Wilms' tumor, Ewing's sarcoma, neuroendocrine tumors, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and bladder cancer.

[0068] The present disclosure also provides in vitro or ex vivo a) Methods for targeting and editing target nucleic acids; b) a method for non-specific degradation of single-stranded DNA upon recognition of a DNA target nucleic acid; c) a method for targeting and nicking a non-spacer complementary strand of a double-stranded target DNA in response to recognition of a spacer complementary strand of the double-stranded target DNA; d) Methods for targeting and cleaving double-stranded target DNA; e) a method for detecting a target nucleic acid in a sample; f) A method for specific editing of double-stranded nucleic acids; g) A method for base editing of double-stranded nucleic acids; h) methods for inducing genotype-specific or transcriptional state-specific cell death or dormancy in cells; i) a method for creating indels in double-stranded target DNA; j) a method for inserting a sequence into double-stranded target DNA, or k) Methods for Deleting or Inverting Sequences in Double-Stranded Target DNA Also provided is the use of a system or cell as described herein in

[0069] In another aspect, the present disclosure provides: a) Methods for targeting and editing target nucleic acids; b) a method for non-specific degradation of single-stranded DNA upon recognition of a DNA target nucleic acid; c) a method for targeting and nicking a non-spacer complementary strand of a double-stranded target DNA in response to recognition of a spacer complementary strand of the double-stranded target DNA; d) Methods for targeting and cleaving double-stranded target DNA; e) a method for detecting a target nucleic acid in a sample; f) A method for specific editing of double-stranded nucleic acids; g) A method for base editing of double-stranded nucleic acids; h) methods for inducing genotype-specific or transcriptional state-specific cell death or dormancy in cells; i) methods for creating indels in double-stranded target DNA; j) a method for inserting a sequence into double-stranded target DNA, or k) Methods for Deleting or Inverting Sequences in Double-Stranded Target DNA Use of the system or cell described herein in The methods herein do not include processes that modify the identity of a human's germline genes, and do not include methods of treating a human or animal organism.

[0070] In the methods described herein, cleavage of the target DNA or target nucleic acid results in the formation of an indel, or cleavage of the target DNA or target nucleic acid results in the insertion of a nucleic acid sequence, or cleavage of the target DNA or target nucleic acid comprises cleavage of the target DNA or target nucleic acid at two sites, resulting in a deletion or inversion of the sequence between the two sites.

[0071] Various systems described herein may lack a tracrRNA. In some embodiments, a Type VI CRISPR-Cas effector protein and a Type VI RNA guide form a complex that associates with and thereby modifies a target nucleic acid.

[0072] In some embodiments of the systems described herein, the spacer sequence is 15 to 47 nucleotides in length, eg, 20 to 40 nucleotides in length, or 24 to 38 nucleotides in length.

[0073] In another aspect, the present disclosure provides a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell, comprising a modified target locus of interest, wherein the target locus of interest has been modified by a method or use of a composition thereof of any one of the preceding claims. For example, modification of the target locus of interest can be achieved by: (i) a eukaryotic cell containing an altered expression of at least one gene product; (ii) a eukaryotic cell containing an altered expression of at least one gene product, wherein the expression of at least one gene product is increased; (iii) a eukaryotic cell containing an altered expression of at least one gene product, wherein the expression of at least one gene product is decreased; or (iv) a eukaryotic cell containing the edited genome can be generated.

[0074] In another aspect, the present disclosure provides a eukaryotic cell line of or comprising a eukaryotic cell described herein, or a progeny thereof, or a multicellular organism comprising one or more eukaryotic cells described herein.

[0075] The present disclosure also provides a plant or animal model comprising one or more cells as described herein.

[0076] In another aspect, the present disclosure provides a method for producing a plant having a modified trait of interest encoded by a gene of interest, the method comprising contacting a plant cell with any of the systems described herein, thereby either modifying or introducing said gene of interest, and regenerating a plant from the plant cell.

[0077] The present disclosure also provides a method for identifying a trait of interest in a plant, wherein the trait of interest is encoded by a gene of interest, the method comprising contacting a plant cell with any of the systems described herein, thereby identifying the gene of interest. For example, the method may further comprise introducing the identified gene of interest into a plant cell, or a plant cell line, or plant germplasm, and generating a plant therefrom, whereby the plant contains the gene of interest. The method may also include causing the plant to exhibit the trait of interest.

[0078] The present disclosure also includes a method of targeting and cleaving single-stranded target DNA, the method comprising contacting the target nucleic acid with any of the systems described herein. The method may include the condition or disease being infectious, wherein the infectious pathogen is selected from the group consisting of human immunodeficiency virus (HIV), herpes simplex virus type 1 (HSV1), and herpes simplex virus type 2 (HSV2).

[0079] In some of the methods described herein, both strands of the target DNA may be cleaved at different sites, resulting in a sticky-end break. In other embodiments, both strands of the target DNA are cleaved at the same site, resulting in a blunt-end double-strand break (DSB).

[0080] In some of the treatment methods described herein, the condition or disease is selected from the group consisting of cystic fibrosis, Duchenne muscular dystrophy, Becker muscular dystrophy, alpha 1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, inherited chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber's congenital amaurosis, sickle cell disease, and beta thalassemia.

[0081] As used herein, term " cleavage event " refers to the DNA cleavage in target nucleic acid that is produced by the nuclease of the CRISPR system described herein.In some embodiments, cleavage event is double-stranded DNA cleavage.In some embodiments, cleavage event is single-stranded DNA cleavage.

[0082] The terms "CRISPR-Cas system," "Type VI CRISPR-Cas system," or "Type VI system," as used herein, refer to a Type VI CRISPR-Cas effector protein (i.e., a Cas12i effector protein) and one or more Type VI RNA guides, and / or a nucleic acid encoding a Type VI CRISPR-Cas effector protein or one or more Type VI RNA guides, and optionally a promoter operably linked to expression of the CRISPR effector or RNA guides or both.

[0083] The term "CRISPR array" as used herein refers to a nucleic acid (e.g., DNA) segment that includes CRISPR repeats and spacers, starting from the first nucleotide of the first CRISPR repeat and ending with the last nucleotide of the last (terminal) CRISPR repeat. Typically, each spacer in a CRISPR array is located between two repeats. The term "CRISPR repeat" or "CRISPR direct repeat" or "direct repeat" as used herein refers to multiple short, directional repeat sequences, which show little or no sequence variation within a CRISPR array. Preferably, type VI direct repeats can form a stem-loop structure.

[0084] A "stem-loop structure" refers to a nucleic acid having a secondary structure that includes a nucleotide region (stem portion) known or predicted to form a double strand connected at one end by a predominantly single-stranded nucleotide region (loop portion). The terms "hairpin" and "foldback" structures are also used herein to refer to stem-loop structures. Such structures are well known in the art, and these terms are used consistent with their known meanings in the art. As known in the art, stem-loop structures do not require exact base pairing. Thus, the stem may contain one or more base mismatches. Alternatively, the base pairing may be exact, i.e., may not contain any mismatches. The predicted stem-loop structures of some type VI direct repeats are shown in Figure 3. The stems of type VI direct repeats contained in RNA guides are composed of complementary five nucleobases that hybridize with each other, and the loops are 6, 7, or 9 nucleotides long.

[0085] The term "CRISPR RNA" or "crRNA," as used herein, refers to an RNA molecule containing a guide sequence used by a CRISPR effector to target a specific nucleic acid sequence. Typically, a crRNA contains a spacer sequence that mediates target recognition and a direct repeat sequence (referred to herein as a direct repeat or "DR" sequence) that forms a complex with a CRISPR-Cas effector protein.

[0086] The term "donor template nucleic acid" as used herein refers to a nucleic acid molecule that can be used to modify the structure of a target nucleic acid by one or more cellular proteins after the CRISPR enzyme described herein has modified the target nucleic acid. In some embodiments, the donor template nucleic acid is a double-stranded nucleic acid. In some embodiments, the donor template nucleic acid is a single-stranded nucleic acid. In some embodiments, the donor template nucleic acid is linear. In some embodiments, the donor template nucleic acid is circular (e.g., a plasmid). In some embodiments, the donor template nucleic acid is an exogenous nucleic acid molecule. In some embodiments, the donor template nucleic acid is an endogenous nucleic acid molecule (e.g., a chromosome).

[0087] The terms "CRISPR-Cas effector," "CRISPR effector," "effector," "CRISPR-associated protein," or "CRISPR enzyme," "Type VI CRISPR-Cas effector protein," "Type VI CRISPR-Cas effector," "Type VI effector," or "Cas12i effector protein," as used herein, refer to a protein that performs an enzymatic activity or binds to a target site on a nucleic acid specified by an RNA guide. The associated CRISPR-Cas Type VI effector protein in a Type VI CRISPR-Cas system may also be referred to herein as "Cas12i" or "Cas12i enzyme." Cas12i enzymes can recognize a related short motif located near the target DNA, called a protospacer adjacent motif (PAM). Preferably, the Cas12i enzymes of the present disclosure can recognize a PAM that includes or consists of TTN (where N represents any nucleotide). For example, the PAM may be TTN, TTH, TTY, or TTC.

[0088] In some embodiments, the Type VI CRISPR-Cas effector protein has endonuclease activity, nickase activity, and / or exonuclease activity.

[0089] The terms "CRISPR effector complex," "effector complex," "binary complex," or "surveillance complex," as used herein, refer to a complex comprising a Type VI CRISPR-Cas effector protein and a Type VI RNA guide.

[0090] The term "RNA guide," as used herein, refers to any RNA molecule that facilitates targeting of a protein described herein to a target nucleic acid. Exemplary "RNA guides" include, but are not limited to, crRNA, pre-crRNA (e.g., DR-spacer-DR), and mature crRNA (e.g., mature_DR-spacer, mature DR-spacer-mature_DR).

[0091] As used herein, the term "targeting" refers to the ability of a complex comprising a CRISPR-associated protein and an RNA guide, such as a crRNA, to preferentially or specifically bind to, e.g., hybridize to, a particular target nucleic acid relative to other nucleic acids that do not have the same or similar sequence as the target nucleic acid.

[0092] As used herein, the term "target nucleic acid" refers to a specific nucleic acid substrate that includes a nucleic acid sequence that is complementary to all or part of a spacer in an RNA guide. In some embodiments, the target nucleic acid includes a gene or a sequence within a gene. In some embodiments, the target nucleic acid includes a non-coding region (e.g., a promoter). In some embodiments, the target nucleic acid is single-stranded. In some embodiments, the target nucleic acid is double-stranded.

[0093] The terms "activated CRISPR complex," "activation complex," or "ternary complex," as used herein, refer to a CRISPR effector complex after binding to or modifying a target nucleic acid.

[0094] The terms "collateral RNA" or "collateral DNA," as used herein, refer to nucleic acid substrates that are non-specifically cleaved by activated CRISPR complexes.

[0095] The term "collateral DNase activity," as used herein in reference to a CRISPR enzyme, refers to the non-specific DNase activity of an activated CRISPR complex.

[0096] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0097] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. In certain embodiments, for example, the following are provided: (Item 1) A type VI (CLUST.029130) RNA guide or a nucleic acid encoding said type VI RNA guide, said RNA guide comprising a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; and A type VI (CLUST.029130) CRISPR-Cas effector protein or a nucleic acid encoding said effector protein, said effector protein capable of binding to said RNA guide and targeting said target nucleic acid sequence complementary to said spacer sequence. 1. An engineered, non-naturally occurring clustered regularly interspaced short palindromic repeats (CRISPR) associated (Cas) system, comprising: (Item 2) 2. The system of item 1, comprising two or more RNA guides. (Item 3) Item 4. The system of item 1 or 2, wherein the Type VI CRISPR-Cas effector protein is less than about 1100 amino acids in length and comprises at least one RuvC domain. 3. The system of claim 1, wherein the type VI RNA guide comprises a direct repeat sequence, the spacer sequence, and a second direct repeat arranged in order within the type VI RNA guide. (Item 5) the type VI CRISPR-Cas effector protein a RuvC domain comprising the amino acid sequence X1SHX4DX6X7 (SEQ ID NO: 200), where X1 is S or T, X4 is Q or L, X6 is P or S, and X7 is F or L; Amino acid sequence X1XDXNX6X7XXXX 11 (SEQ ID NO: 201) wherein X1 is A, G, or S, X is any amino acid, X6 is Q or I, X7 is T, S, or V, and X 10 is T or A); and a RuvC domain comprising the amino acid sequence X1X2X3E (SEQ ID NO: 210), where X1 is C, F, I, L, M, P, V, W, or Y, X2 is C, F, I, L, M, P, R, V, W, or Y, and X3 is C, F, G, I, L, M, P, V, W, or Y. The system according to any one of items 1 to 4, comprising one or more of the following: (Item 6) 5. The system of any one of paragraphs 1 to 4, wherein the Type VI CRISPR-Cas effector protein comprises an amino acid sequence at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to an amino acid sequence in Table 4. (Item 7) 7. The system of claim 6, wherein the Type VI CRISPR-Cas effector protein comprises an amino acid sequence having at least 80% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5. (Item 8) The type VI RNA guide comprises a direct repeat sequence comprising a stem-loop structure proximal to the 3' end of the direct repeat sequence, wherein the stem-loop structure First stem nucleotide strand 5 nucleotides long; a second stem nucleotide strand 5 nucleotides in length, the first and second stem nucleotide strands being capable of hybridizing to each other; and a loop nucleotide strand disposed between the first and second stem nucleotide strands, the loop nucleotide strand comprising 6, 7, or 8 nucleotides; 8. The system according to any one of items 1 to 7, comprising: (Item 9) The direct repeat sequence is 5'-CCGUCNNNNNNUGACGG-3' (SEQ ID NO: 202) proximal to the 3' end, where N is any nucleobase; 5'-GUGCCNNNNNNUGGCAC-3' (SEQ ID NO: 203) proximal to the 3' end, where N is any nucleobase; 5'-GUGUCN proximal to the 3' end 5-6 UGACAX1-3' (SEQ ID NO: 204) (wherein N 5-6 is any consecutive sequence of 5 or 6 nucleobases, and X1 is C, T, or U; 5'-UCX3UX5X6X7UUGACGG-3' (SEQ ID NO: 205) proximal to the 3' end (wherein X3 is C, T, or U, X5 is A, T, or U, X6 is A, C, or G, and X7 is A or G); 9. The system of any one of items 1 to 8, comprising any one of 5'-CCX3X4X5CX7UUGGCAC-3' (SEQ ID NO: 206) (wherein X3 is C, T, or U, X4 is A, T, or U, X5 is C, T, or U, and X7 is A or G) located proximal to the 3' end. (Item 10) 10. The system of any one of items 1 to 9, wherein the direct repeat sequence comprises a nucleotide sequence at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a nucleotide sequence provided in Table 5. (Item 11) 11. The system of any one of items 1 to 10, wherein the RNA guide comprises an ordered direct repeat sequence, the spacer sequence, and a second direct repeat, wherein the RNA guide sequence comprises a nucleotide sequence at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a nucleotide sequence provided in Table 5B or a fragment thereof, and wherein the variable N region refers to the spacer and can have a length as specified in Table 5B. (Item 12) 12. The system of any one of items 1 to 11, wherein the Type VI CRISPR-Cas effector protein has the ability to recognize a protospacer adjacent motif (PAM), and the target nucleic acid comprises a PAM comprising the nucleic acid sequence 5'-TTN-3' (wherein N is any nucleotide), 5'-TTH-3', 5'-TTY-3', or 5'-TTC-3'. (Item 13) 13. The system according to any one of items 1 to 12, wherein the target nucleic acid is DNA. (Item 14) 14. The system of any one of items 1 to 13, wherein targeting of the target nucleic acid by the Type VI CRISPR-Cas effector protein and RNA guide results in modification of the target nucleic acid. (Item 15) Item 15. The system of item 14, wherein the modification of the target nucleic acid is a cleavage event. (Item 16) Item 15. The system of item 14, wherein the modification of the target nucleic acid is a nicking event. (Item 17) The system according to any one of Items 13 to 16, wherein the modification causes cytotoxicity. (Item 18) 18. The system of any one of items 3 to 17, wherein the Type VI CRISPR-Cas effector protein comprises one or more amino acid substitutions in the RuvC domain that result in reduced nuclease or nickase activity of the Type VI CRISPR-Cas effector protein compared to the nuclease or nickase activity of the Type VI CRISPR-Cas effector protein without the one or more amino acid substitutions. (Item 19) 19. The system of item 18, wherein the one or more amino acid substitutions comprise an alanine substitution at an amino acid residue corresponding to D647, E894, or D948 of SEQ ID NO: 3; or D599, E833, or D886 of SEQ ID NO: 5. (Item 20) 20. The system of claim 18 or 19, wherein the Type VI CRISPR-Cas effector protein is fused to a base editing domain. (Item 21) 20. The system of claim 18 or 19, wherein the Type VI CRISPR-Cas effector protein is fused to a DNA methylation domain, a histone residue modification domain, a localization factor, a transcriptional modifier, a photogating factor, a chemical inducible factor, or a chromatin visualization factor. (Item 22) 22. The system of any one of items 1 to 21, wherein the Type VI CRISPR-Cas effector protein comprises at least one nuclear localization signal (NLS), at least one nuclear export signal (NES), or both. (Item 23) 23. The system of any one of items 1 to 22, comprising the nucleic acid encoding the Type VI CRISPR-Cas effector protein operably linked to a promoter. (Item 24) 24. The system of item 23, wherein the promoter is a constitutive promoter. (Item 25) 24. The system of claim 23, wherein the nucleic acid encoding the Type VI CRISPR-Cas effector protein is codon-optimized for expression in a cell. (Item 26) 24. The system of claim 23, wherein the nucleic acid encoding the Type VI CRISPR-Cas effector protein operably linked to a promoter is in a vector. (Item 27) 27. The system of item 26, wherein the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, and a herpes simplex vector. (Item 28) 27. The system according to any one of items 1 to 26, wherein the system is present in a delivery system selected from the group consisting of nanoparticles, liposomes, exosomes, microvesicles, and gene guns. (Item 29) 29. The system according to any one of items 1 to 28, further comprising a target DNA or a nucleic acid encoding the target DNA, wherein the target DNA comprises a sequence capable of hybridizing to the spacer sequence of the RNA guide. (Item 30) 30. The system of any one of items 1 to 29, further comprising a donor template nucleic acid. (Item 31) 31. The system of item 30, wherein the donor template nucleic acid is DNA or RNA. (Item 32) A cell comprising the system according to any one of items 1 to 31. (Item 33) 33. The cell of item 32, wherein the cell is a eukaryotic cell. (Item 34) 33. The cell of item 32, wherein the cell is a prokaryotic cell. (Item 35) 32. A method for targeting and editing a target nucleic acid, comprising contacting the system according to any one of items 1 to 31 with the target nucleic acid. (Item 36) 37. A method for non-specifically degrading single-stranded DNA in response to recognition of a target DNA, the method comprising contacting the system according to any one of items 1 to 36 with the target nucleic acid. (Item 37) 37. A method for targeting and nicking a non-spacer complementary strand of a double-stranded target DNA in response to recognition of a spacer complementary strand of the double-stranded target DNA, the method comprising contacting the double-stranded target DNA with the system of any one of Items 1 to 36. (Item 38) 32. A method for targeting and cleaving double-stranded target DNA, comprising contacting the double-stranded target DNA with the system according to any one of items 1 to 31. (Item 39) 41. The method of claim 40, wherein the non-spacer complementary strand of the double-stranded target DNA is nicked prior to nicking the spacer complementary strand of the double-stranded target nucleic acid. (Item 40) 1. A method for detecting a target nucleic acid in a sample, comprising: (a) contacting the sample with the system of any one of items 1 to 31 and a labeled reporter nucleic acid, wherein hybridization of the crRNA to the target nucleic acid causes cleavage of the labeled reporter nucleic acid; and (b) measuring a detectable signal produced by cleavage of the labeled reporter nucleic acid, thereby detecting the presence of the target nucleic acid in the sample. (Item 41) 41. The method of claim 40, further comprising comparing the level of the detectable signal to a reference signal level, and determining the amount of target nucleic acid in the sample based on the level of the detectable signal. (Item 42) 42. The method of claim 41, wherein the measurement is performed using gold nanoparticle detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection, or semiconductor-based sensing. (Item 43) 43. The method of claim 42, wherein the labeled reporter nucleic acid comprises a fluorescent dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher / fluorophore pair, and cleavage of the labeled reporter nucleic acid by the effector protein results in an increase or decrease in the amount of signal produced by the labeled reporter nucleic acid. (Item 44) A method for specifically editing a double-stranded nucleic acid, comprising: (a) a type VI CRISPR-Cas effector and one other enzyme having sequence-specific nicking activity, and a crRNA that directs the type VI CRISPR-Cas effector to nick the opposite strand compared to the activity of the other sequence-specific nickase; and (b) the double-stranded nucleic acid contacting the A method wherein the method results in the formation of a double stranded break. (Item 45) A method for editing double-stranded nucleic acid, comprising: (a) a fusion protein comprising the type VI CRISPR-Cas effector, a protein domain having DNA-modifying activity, and an RNA guide that targets the double-stranded nucleic acid; and (b) the double-stranded nucleic acid contacting the wherein the Type VI CRISPR-Cas effector of the fusion protein is modified to nick a non-target strand of the double-stranded nucleic acid. (Item 46) 32. A method for inducing genotype-specific or transcriptional state-specific cell death or dormancy in a cell, the method comprising contacting a cell with the system according to any one of items 1 to 31, wherein collateral DNase activity-mediated cell death or dormancy occurs upon hybridization of the RNA guide to the target DNA. (Item 47) 48. The method of item 47, wherein the cell is a prokaryotic cell. (Item 48) 48. The method of item 47, wherein the cell is a eukaryotic cell. (Item 49) 49. The method of claim 48, wherein the cell is a mammalian cell. (Item 50) 50. The method of claim 49, wherein the cells are cancer cells. (Item 51) 48. The method of item 47, wherein the cell is an infectious cell or a cell infected with an infectious pathogen. (Item 52) 52. The method of claim 51, wherein the cell is a virus-infected cell, a prion-infected cell, a fungal cell, a protozoan, or a parasitic cell. (Item 53) A method for treating a condition or disease in a subject in need thereof, comprising administering to the subject the system according to any one of items 1 to 31, the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid associated with the condition or disease; the Type VI CRISPR-Cas effector protein associates with the RNA guide to form a complex; the complex binds to a target nucleic acid sequence complementary to the at least 15 nucleotides of the spacer sequence; and When the complex binds to the target nucleic acid sequence, the Type VI CRISPR-Cas effector protein cleaves the target nucleic acid, thereby treating the condition or disease in the subject. (Item 54) 54. The method of item 53, wherein the pathological condition or disease is cancer or an infectious disease. (Item 55) 55. The method of item 54, wherein the pathological condition or disease is cancer, and the cancer is selected from the group consisting of Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and bladder cancer. (Item 56) 35. The system or cell according to any one of items 1 to 34 for use as a medicament. (Item 57) 36. The system or cell according to any one of items 1 to 35 for use in the treatment or prevention of cancer or an infectious disease. (Item 58) 58. The system or cell for use according to Item 57, wherein the cancer is selected from the group consisting of Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and bladder cancer. (Item 59) In vitro or ex vivo a) Methods for targeting and editing target nucleic acids; b) a method for non-specific degradation of single-stranded DNA upon recognition of a DNA target nucleic acid; c) targeting and nicking of a non-spacer complementary strand of a double-stranded target DNA in response to recognition of a spacer complementary strand of said double-stranded target DNA; d) Methods for targeting and cleaving double-stranded target DNA; e) a method for detecting a target nucleic acid in a sample; f) A method for specific editing of double-stranded nucleic acids; g) A method for base editing of double-stranded nucleic acids; h) Methods for inducing genotype-specific or transcriptional state-specific cell death or dormancy in cells i) methods for creating indels in double-stranded target DNA; j) a method for inserting a sequence into double-stranded target DNA, or k) Methods for Deleting or Inverting Sequences in Double-Stranded Target DNA Use of the system or cell according to any one of items 1 to 35 in a method for treating a disease. (Item 60) a) Methods for targeting and editing target nucleic acids; b) a method for non-specific degradation of single-stranded DNA upon recognition of a DNA target nucleic acid; c) targeting and nicking of a non-spacer complementary strand of a double-stranded target DNA in response to recognition of a spacer complementary strand of said double-stranded target DNA; d) Methods for targeting and cleaving double-stranded target DNA; e) a method for detecting a target nucleic acid in a sample; f) A method for specific editing of double-stranded nucleic acids; g) A method for base editing of double-stranded nucleic acids; h) methods for inducing genotype-specific or transcriptional state-specific cell death or dormancy in cells; i) methods for creating indels in double-stranded target DNA; j) a method for inserting a sequence into double-stranded target DNA, or k) Methods for Deleting or Inverting Sequences in Double-Stranded Target DNA Use of the system or cell according to any one of items 1 to 35 in The use, wherein said method does not include a process that modifies the identity of a human's germline genes and does not include a method of treatment of said human or animal organism. (Item 61) 54. The method of claim 38 or 53, wherein cleavage of the target DNA or target nucleic acid results in the formation of indels. (Item 62) 54. The method of claim 38 or 53, wherein cleavage of the target DNA or target nucleic acid results in insertion of a nucleic acid sequence. (Item 63) 54. The method of claim 38 or 53, wherein cleavage of the target DNA or target nucleic acid comprises cleavage of the target DNA or target nucleic acid at two sites, resulting in a deletion or inversion of the sequence between the two sites. (Item 64) 32. The system of any one of items 1 to 31, wherein the system lacks tracrRNA. (Item 65) 32. The system of any one of items 1 to 31, wherein the Type VI CRISPR-Cas effector protein and the Type VI RNA guide form a complex that modifies the target nucleic acid by associating with the target nucleic acid. (Item 66) 32. The system according to any one of items 1 to 31, wherein the spacer sequence is 15 to 47 nucleotides in length, for example, 20 to 40 nucleotides in length, or 24 to 38 nucleotides in length. (Item 67) 67. A eukaryotic cell comprising a modified target locus of interest, wherein said target locus of interest has been modified by the method or by use of a composition according to any one of items 1 to 66. (Item 68) said modification of said target locus of interest comprising: (i) said eukaryotic cell comprising an altered expression of at least one gene product; (ii) said eukaryotic cell comprising an altered expression of at least one gene product, wherein said expression of said at least one gene product is increased; (iii) said eukaryotic cell comprising an altered expression of at least one gene product, wherein said expression of said at least one gene product is decreased; or (iv) the eukaryotic cell comprising an edited genome. 68. The eukaryotic cell according to item 67, which produces (Item 69) 69. The eukaryotic cell of item 67 or 68, wherein the eukaryotic cell comprises a mammalian cell. (Item 70) 70. The eukaryotic cell of item 69, wherein the mammalian cell comprises a human cell. (Item 71) 69. A eukaryotic cell according to any one of items 67 to 69, or a eukaryotic cell line comprising the same, or a progeny thereof. (Item 72) 70. A multicellular organism comprising one or more cells according to any one of items 67 to 69. (Item 73) 70. A plant or animal model comprising one or more cells according to any one of items 67 to 69. (Item 74) 32. A method for producing a plant having a modified trait of interest encoded by a gene of interest, the method comprising contacting a plant cell with the system according to any one of items 1 to 31, thereby either modifying or introducing the gene of interest, and regenerating a plant from the plant cell. (Item 75) 35. A method for identifying a trait of interest in a plant, wherein the trait of interest is encoded by a gene of interest, the method comprising contacting a plant cell with the system according to any one of items 1 to 34, thereby identifying the gene of interest. (Item 76) 76. The method of claim 75, further comprising introducing the identified gene of interest into a plant cell or plant cell line or plant germplasm and generating a plant therefrom, whereby the plant contains the gene of interest. (Item 77) 77. The method according to item 76, wherein the plant exhibits the trait of interest. (Item 78) 32. A method for targeting and cleaving single-stranded target DNA, comprising contacting the system according to any one of items 1 to 31 with the target nucleic acid. (Item 79) 70. The method of claim 69, wherein the condition or disease is infectious and the infectious pathogen is selected from the group consisting of human immunodeficiency virus (HIV), herpes simplex virus type 1 (HSV1), and herpes simplex virus type 2 (HSV2). (Item 80) 39. The method of claim 38, wherein both strands of the target DNA are cleaved at different sites, resulting in a sticky-end type cleavage. (Item 81) 39. The method of claim 38, wherein both strands of the target DNA are cleaved at the same site, resulting in a blunt-ended double-strand break (DSB). (Item 82) 54. The method of item 53, wherein the pathological condition or disease is selected from the group consisting of cystic fibrosis, Duchenne muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber's congenital amaurosis, sickle cell disease, and beta thalassemia.

[0098] These figures include a series of schematic diagrams and nucleic acid and amino acid sequences representing the results of locus analysis of various protein clusters. [Brief explanation of the drawings]

[0099] [Figure 1A] Figures 1A-1B show a classification tree of type V effectors (Cas12 proteins). For each branch, the corresponding CRISPR-Cas locus configuration is shown, where tracrRNA is required, indicated by a white rectangle adjacent to the CRISPR array. The CLUST.029130 (type VI) system is designated as Cas12i. [Figure 1B] Figures 1A-1B show a classification tree of type V effectors (Cas12 proteins). For each branch, the corresponding CRISPR-Cas locus configuration is shown, where tracrRNA is required, indicated by a white rectangle adjacent to the CRISPR array. The CLUST.029130 (type VI) system is designated as Cas12i. [Figure 2A-2B]Figure 2A is a schematic representation of the functional domains of the CLUST.029130 (Type VI) effector, designated Cas12i. The gray solid shading indicates the position of the C-terminal RuvC domain, where catalytic residues in three conserved sequence motifs (I, II, and III) are indicated and drawn to scale. The position of the bridging helix domain is indicated by a superscript h. Figure 2B is a schematic representation of a multiple sequence alignment of the Cas12i effector protein, displaying the relative positions of the conserved catalytic residues of the RuvC domain as RuvC I / II / III. [Figure 3] FIG. 3 is a set of schematic diagrams showing the predicted secondary structure of the RNA transcripts of each example of a type VI direct repeat sequence. [Figure 4A] Figure 4A is a schematic representation of the design of the in vivo screen effector and non-coding plasmids. A CRISPR array library was designed containing unique spacers flanked by two DRs and uniformly sampled from both strands of pACYC184 or E. coli essential genes expressed by J23119. [Figure 4B] Figure 4B is a schematic representation of the negative selection screening workflow: 1) the CRISPR array library was cloned into the effector plasmid; 2) the effector plasmid, and, if present, the non-coding plasmid, were transformed into E. coli and subsequently grown for negative selection of CRISPR arrays that confer interference with pACYC184 or DNA or RNA transcripts from essential E. coli genes; 3) targeted sequencing of the effector plasmid was used to identify depleted CRISPR arrays, and small RNA sequencing was used to identify mature crRNAs and tracrRNAs. [Figure 5A]Figures 5A-5B and 5C-5D are graphical representations showing the density of depleted and non-depleted targets for Cas12i1 and Cas12i2, respectively. Spacers targeting both pACYC184 and essential E. coli genes that were strongly depleted are shown in separate plots. Targets on the top and bottom strands are shown separately with respect to the annotated gene orientation. [Figure 5B] Figures 5A-5B and 5C-5D are graphical representations showing the density of depleted and non-depleted targets for Cas12i1 and Cas12i2, respectively. Spacers targeting both pACYC184 and essential E. coli genes that were strongly depleted are shown in separate plots. Targets on the top and bottom strands are shown separately with respect to the annotated gene orientation. [Figure 5C] Figures 5A-5B and 5C-5D are graphical representations showing the density of depleted and non-depleted targets for Cas12i1 and Cas12i2, respectively. Spacers targeting both pACYC184 and essential E. coli genes that were strongly depleted are shown in separate plots. Targets on the top and bottom strands are shown separately with respect to the annotated gene orientation. [Figure 5D] Figures 5A-5B and 5C-5D are graphical representations showing the density of depleted and non-depleted targets for Cas12i1 and Cas12i2, respectively. Spacers targeting both pACYC184 and essential E. coli genes that were strongly depleted are shown in separate plots. Targets on the top and bottom strands are shown separately with respect to the annotated gene orientation. [Figure 6A] Figures 6A and 6B are scatter plots showing the effect of mutating the RuvC-I catalytic residue aspartate (at position 647 for Cas12i1 and position 599 for Cas12i2) to alanine. Each point corresponds to a spacer, and values ​​indicate fold depletion under the conditions designated on the axis (wild type vs. mutant). Higher values ​​indicate stronger depletion (i.e., fewer surviving colonies). [Figure 6B] Figures 6A and 6B are scatter plots showing the effect of mutating the RuvC-I catalytic residue aspartate (at position 647 for Cas12i1 and position 599 for Cas12i2) to alanine. Each point corresponds to a spacer, and values ​​indicate fold depletion under the conditions designated on the axis (wild type vs. mutant). Higher values ​​indicate stronger depletion (i.e., fewer surviving colonies). [Figure 7A] Figures 7A and 7B are scatter plots showing the effect of adding or removing non-coding sequences from the Type VI CRISPR-Cas systems being screened. Each point corresponds to a spacer, and values ​​indicate fold depletion under the conditions designated on the axis (wild type vs. mutant). Higher values ​​indicate stronger depletion (i.e., fewer surviving colonies). [Figure 7B] Figures 7A and 7B are scatter plots showing the effect of adding or removing non-coding sequences from the Type VI CRISPR-Cas systems being screened. Each point corresponds to a spacer, and values ​​indicate fold depletion under the conditions designated on the axis (wild type vs. mutant). Higher values ​​indicate stronger depletion (i.e., fewer surviving colonies). [Figure 8A] Figures 8A and 8B show heat maps of the aggregated screening results for Cas12i1 and Cas12i2, respectively. The heat maps decompose dependent variables such as the orientation of the direct repeats, the need for noncoding sequences, and the requirement for an intact RuvC domain (here, dCas12i refers to a point mutation in a catalytically active residue in the RuvC-I domain). The Y-axis decomposes library targets by their compositional characteristics: whether they target pACYC184, an essential E. coli gene (E. coli EG), or the type of targeting strand (S, sense; AS, antisense). Cas12i1 and Cas12i2 in vivo screens were performed in Endura Stbl3 and E. cloni® competent cell lines, respectively. CRISPR arrays that were strongly depleted in negative controls without Cas12i1 or Cas12i2 effectors are subtracted from each analysis. [Figure 8B] Figures 8A and 8B show heat maps of the aggregated screening results for Cas12i1 and Cas12i2, respectively. The heat maps decompose dependent variables such as the orientation of the direct repeats, the need for noncoding sequences, and the requirement for an intact RuvC domain (here, dCas12i refers to a point mutation in a catalytically active residue in the RuvC-I domain). The Y-axis decomposes library targets by their compositional characteristics: whether they target pACYC184, an essential E. coli gene (E. coli EG), or the type of targeting strand (S, sense; AS, antisense). Cas12i1 and Cas12i2 in vivo screens were performed in Endura Stbl3 and E. cloni® competent cell lines, respectively. CRISPR arrays that were strongly depleted in negative controls without Cas12i1 or Cas12i2 effectors are subtracted from each analysis. [Figure 9A] Figures 9A and 9B are blogs of 5' PAM motifs identified from target-flanking sequences for strongly depleted spacers from the Cas12i1 and Cas12i2 in vivo screens, respectively. [Figure 9B] Figures 9A and 9B are blogs of 5' PAM motifs identified from target-flanking sequences for strongly depleted spacers from the Cas12i1 and Cas12i2 in vivo screens, respectively. [Figure 10A] Figures 10A and 10B are violin plots of the bit scores for all possible permutations of the target and flanking nucleotides, confirming that Cas12i1 and Cas12i2 have a preference for only a single 2-nt PAM motif at the second and third positions 5' to the spacer target, respectively. [Figure 10B] Figures 10A and 10B are violin plots of the bit scores for all possible permutations of the target and flanking nucleotides, confirming that Cas12i1 and Cas12i2 have a preference for only a single 2-nt PAM motif at the second and third positions 5' to the spacer target, respectively. [Figure 11A] Figure 11A and Figure 11B show small RNA sequencing read mapping of in vivo screening samples of minimal Cas12i systems, revealing mature crRNAs of the Cas12i1 and Cas12i2 systems, respectively. [Figure 11B] Figure 11A and Figure 11B show small RNA sequencing read mapping of in vivo screening samples of minimal Cas12i systems, revealing mature crRNAs of the Cas12i1 and Cas12i2 systems, respectively. [Figure 12] Figure 12 is a denaturing gel showing pre-crRNA processing by the Cas12i1 effector protein. Magnesium-independent processing by Cas12i1 of pre-crRNA expressed from a minimal CRISPR array (repeat-spacer-repeat-spacer-repeat) containing a 24-nt repeat and a 28-nt spacer. Pre-crRNA was incubated with Cas12i1 for 30 min at 37°C and analyzed on a 15% TBE-urea gel. [Figure 13] Figure 13 is a gel representation showing increasing doses of Cas12i1 binary complexes operating on target (left) or non-target (right) ssDNA labeled with IR800 dye. Samples were analyzed by 15% TBE-urea denaturing gel electrophoresis. [Figure 14] Figure 14 is a gel representation showing the operation of increasing doses of Cas12i1 binary complexes on collateral ssDNA (without sequence similarity to the target) labeled with IR800 dye in the presence of unlabeled target (left) or non-target (right) ssDNA. Samples were analyzed by 15% TBE-urea denaturing gel electrophoresis. [Figure 15] Figure 15 is a gel representation showing the operation of increasing doses of Cas12i1 binary complexes on target (left) or non-target (right) dsDNA labeled with IR800 dye. Samples were analyzed by 15% TBE-urea denaturing gel electrophoresis. [Figure 16]Figure 16 is a gel representation showing the operation of increasing doses of Cas12i1 binary complex on target dsDNA labeled with IR800 dye and either directly quenched (left) or treated with S1 nuclease before quenching (right). Samples were analyzed by 4-20% TBE native gel electrophoresis. [Figure 17A] Figures 17A and 17B are representations of gels showing the asymmetric cleavage efficiency of dsDNA target strands (spacer complementary; "SC") versus non-target strands (non-spacer complementary; "NSC"). Figure 17A is a denaturing gel imaged with IR800 (labeled DNA only), while Figure 17B is a denaturing gel imaged with SYBR staining (total DNA). Each gel shows the cleavage or nicking activity of increasing concentrations of Cas12i1 binary complexes against dsDNA containing the 5'IR800-labeled NSC strand (left) or the 5'IR800-labeled SC strand (right). Cas12i1 binary complexes were formed by pre-incubating Cas12i1 with pre-crRNA for 10 minutes at 37°C, followed by incubation with the substrate for 1 hour at 37°C. [Figure 17B] Figures 17A and 17B are representations of gels showing the asymmetric cleavage efficiency of dsDNA target strands (spacer complementary; "SC") versus non-target strands (non-spacer complementary; "NSC"). Figure 17A is a denaturing gel imaged with IR800 (labeled DNA only), while Figure 17B is a denaturing gel imaged with SYBR staining (total DNA). Each gel shows the cleavage or nicking activity of increasing concentrations of Cas12i1 binary complexes against dsDNA containing the 5'IR800-labeled NSC strand (left) or the 5'IR800-labeled SC strand (right). Cas12i1 binary complexes were formed by pre-incubating Cas12i1 with pre-crRNA for 10 minutes at 37°C, followed by incubation with the substrate for 1 hour at 37°C. [Figure 18A]Figure 18A is a schematic diagram of the design of an in vitro assay to detect gene silencing. In a one-pot reaction (indicated by the outer border), a linear DNA template encoding a Cas12i effector, an RNA guide, and sigma factor 28 is combined with reconstituted IVTT (in vitro transcription and translation) reagents and Escherichia coli (E. coli) RNA polymerase core enzyme (labeled RNAPc). A DNA plasmid encoding GFP targeted by the RNA guide is included, as well as a non-targeted linear DNA template expressing RFP as an internal control. Both GFP and RFP are expressed from the sigma factor 28 promoter (fliC), and GFP and RFP fluorescence is measured every 5 minutes for up to 12 hours. [Figure 18B] Figure 18B is a schematic representation of the design of a GFP-encoding plasmid used as a substrate in an in vitro gene silencing assay. This plasmid encodes GFP under the sig28 promoter, and engineered RNA guides are designed to target both strands of the promoter region and the GFP gene (indicated by short block arrows pointing in both directions). [Figure 19A] Figures 19A and 19B are graphs showing the fold GFP fluorescence depletion (y-axis) over 12 hours (720 min, x-axis) by Type VI effectors as indicated in complexes containing guides containing sequences complementary to the template strand (Figure 19A) and coding strand (Figure 19B) of the substrate GFP coding region. The fold GFP fluorescence depletion is calculated as the ratio of normalized GFP fluorescence by Type VI effectors in complexes containing non-targeting RNA guides compared to the normalized GFP fluorescence of Type VI effectors in complexes containing RNA guides targeting GFP. Cas12i1 (solid line) shows higher depletion (gene silencing) compared to the activity of mutant Cas12i1 D647A, Cas12i1 E894A, or Cas12i1 D948A, respectively. [Figure 19B]Figures 19A and 19B are graphs showing the fold GFP fluorescence depletion (y-axis) over 12 hours (720 min, x-axis) by Type VI effectors as indicated in complexes containing guides containing sequences complementary to the template strand (Figure 19A) and coding strand (Figure 19B) of the substrate GFP coding region. The fold GFP fluorescence depletion is calculated as the ratio of normalized GFP fluorescence by Type VI effectors in complexes containing non-targeting RNA guides compared to the normalized GFP fluorescence of Type VI effectors in complexes containing RNA guides targeting GFP. Cas12i1 (solid line) shows higher depletion (gene silencing) compared to the activity of mutant Cas12i1 D647A, Cas12i1 E894A, or Cas12i1 D948A, respectively. [Figure 20] Figure 20 shows various forms of protein and / or RNA in in vitro reconstitution of the CRISPR-Cas system used in the in vitro pooled screen. The direction of transcription is indicated by the T7 promoter arrow. [Figure 21] Figure 21 shows one embodiment of ssDNA and dsDNA substrates for in vitro pooled screening. The target sequence is flanked on both the 5' and 3' sides by six degenerate bases ("N"), which flank a consensus region used as a fiducial mark for downstream data analysis after next-generation sequencing. For dsDNA substrates, second-strand synthesis is completed using DNA polymerase I fill-in after pre-annealing of a primer to the 3' fiducial mark. [Figure 22] FIG. 22 shows a schematic diagram of unidirectional sequencing library preparation of ssDNA fragments after incubation with the reconstituted CRISPR-Cas system. [Figure 23] Figure 23 shows a schematic diagram of bidirectional sequencing library preparation possible for dsDNA fragments after incubation with the reconstituted CRISPR-Cas system. Sequencing adapters can be ligated to both cleaved fragments, followed by selection using a primer combination common to the adapters and common to the dsDNA substrate. [Figure 24A]Figures 24A-B show the morphology of full-length and cleavage products captured by next-generation sequencing library preparation and readout using A) I5 / P5 ligation adapters and 3' standards for I7 / P7 targeted amplification and addition, or B) I7 / P7 ligation adapters and 5' standards for I5 / P5 targeted amplification and addition. [Figure 24B] Figures 24A-B show the morphology of full-length and cleavage products captured by next-generation sequencing library preparation and readout using A) I5 / P5 ligation adapters and 3' standards for I7 / P7 targeted amplification and addition, or B) I7 / P7 ligation adapters and 5' standards for I5 / P5 targeted amplification and addition. [Figure 25A] 25A-25B show schematic diagrams for A) ssDNA target length mapping and B) substrate length mapping, respectively. [Figure 25B] 25A-25B show schematic diagrams for A) ssDNA target length mapping and B) substrate length mapping, respectively. [Figure 26A] Figures 26A-B show the distribution of dsDNA substrate lengths for Cas12i1 by IVTT expression in complexes containing top-strand (active orientation) crRNA targeting dsDNA (red) versus the apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 26B]Figures 26A-B show the distribution of dsDNA substrate lengths for Cas12i1 by IVTT expression in complexes containing top-strand (active orientation) crRNA targeting dsDNA (red) versus the apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 27A] Figures 27A-27B show the distribution of dsDNA target lengths for Cas12i1 by IVTT expression in complexes containing top-strand (active orientation) crRNA targeting dsDNA (red) versus the apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of a full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of a full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 27B]Figures 27A-27B show the distribution of dsDNA target lengths for Cas12i1 by IVTT expression in complexes containing top-strand (active orientation) crRNA targeting dsDNA (red) versus the apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of a full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of a full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 28A] Figures 28A-B show the distribution of dsDNA substrate length (X) versus target length (Y) for Cas12i1 by IVTT expression in complexes containing the top-strand (active orientation) crRNA targeting dsDNA (red) versus the apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 28B]Figures 28A-B show the distribution of dsDNA substrate length (X) versus target length (Y) for Cas12i1 by IVTT expression in complexes containing the top-strand (active orientation) crRNA targeting dsDNA (red) versus the apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 29] Figure 29 shows a weblog showing the 5'TTN PAM motif (left of the target sequence) for Cas12i1 associated with non-target strand cleavage between +24 / +25 nucleotides relative to the PAM. No PAM sequence requirement is observed for the right side of the Cas12i1 target. [Figure 30] Figure 30 shows the 5 nt 3' overhang associated with double-stranded DNA cleavage by Cas12i1, with cleavage observed between +24 / +25 nucleotides relative to the PAM on the non-target strand and between +19 / +20 nucleotides relative to the PAM on the target strand. [Figure 31A]Figures 31A-31B show the distribution of dsDNA substrate lengths for Cas12i1 by IVTT expression in complexes containing non-targeting crRNA (red) versus the apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 31B] Figures 31A-31B show the distribution of dsDNA substrate lengths for Cas12i1 by IVTT expression in complexes containing non-targeting crRNA (red) versus the apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 32A]Figures 32A-B show the distribution of dsDNA substrate lengths for Cas12i1 by IVTT expression in complexes containing bottom-strand (inactive orientation) crRNA targeting dsDNA (red) versus the apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 32B] Figures 32A-B show the distribution of dsDNA substrate lengths for Cas12i1 by IVTT expression in complexes containing bottom-strand (inactive orientation) crRNA targeting dsDNA (red) versus the apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 33A]Figures 33A-B show the distribution of dsDNA substrate lengths for Cas12i2 by IVTT expression in complexes containing top-strand (active orientation) crRNA targeting dsDNA (red) versus an apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of a full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of a full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 33B] Figures 33A-B show the distribution of dsDNA substrate lengths for Cas12i2 by IVTT expression in complexes containing top-strand (active orientation) crRNA targeting dsDNA (red) versus an apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of a full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of a full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 34A]Figures 34A-B show the distribution of dsDNA target lengths for Cas12i2 by IVTT expression in complexes containing top-strand (active orientation) crRNA targeting dsDNA (red) versus apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of a full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of a full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 34B] Figures 34A-B show the distribution of dsDNA target lengths for Cas12i2 by IVTT expression in complexes containing top-strand (active orientation) crRNA targeting dsDNA (red) versus apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of a full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of a full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 35A]Figures 35A-35B show the distribution of dsDNA substrate length (X) versus target length (Y) for Cas12i2 by IVTT expression in complexes containing top-strand (active orientation) crRNA targeting dsDNA (red) versus apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of a full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of a full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 35B] Figures 35A-35B show the distribution of dsDNA substrate length (X) versus target length (Y) for Cas12i2 by IVTT expression in complexes containing top-strand (active orientation) crRNA targeting dsDNA (red) versus apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of a full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of a full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 36] Figure 36 shows a weblog showing the 5'TTN PAM motif (left of the target sequence) for Cas12i2 associated with non-target strand cleavage between +24 / +25 nucleotides relative to the PAM. No PAM sequence requirement is observed for the right side of the Cas12i2 target. [Figure 37]Figure 37 shows blunt-end cleavage associated with double-stranded DNA cleavage by Cas12i2, as indicated by cleavage observed between +24 / +25 nucleotides relative to the PAM on the non-target strand and between +24 / +25 nucleotides relative to the PAM on the target strand. [Figure 38A] Figures 38A-B show the distribution of dsDNA substrate lengths for Cas12i2 by IVTT expression in complexes containing non-targeting crRNA (red) versus apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 38B] Figures 38A-B show the distribution of dsDNA substrate lengths for Cas12i2 by IVTT expression in complexes containing non-targeting crRNA (red) versus apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 39A]Figures 39A-39B show the distribution of dsDNA substrate lengths for Cas12i2 by IVTT expression in complexes containing bottom-strand (inactive orientation) crRNA targeting dsDNA (red) versus an apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of a full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of a full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 39B] Figures 39A-39B show the distribution of dsDNA substrate lengths for Cas12i2 by IVTT expression in complexes containing bottom-strand (inactive orientation) crRNA targeting dsDNA (red) versus an apo (effector-only) control (blue). (A) Next-generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of a full-length or truncated substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence). (B) Next-generation sequencing libraries for readout were prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of a full-length or truncated substrate (and containing the I7 / P7 sequence). [Figure 40]Figure 40 is a schematic diagram of the construct used for mammalian validation of the Type VI CRISPR system as described herein. The effector is mammalian codon-optimized, and a nucleoplasmin nuclear localization sequence (npNLS) is added to the C-terminus of the protein. Mammalian expression from a plasmid uses the EF1α short promoter (EFS) and a polyA sequence derived from bGH (bGHpA). The RNA guide is expressed from a linear dsDNA fragment, driven by an RNA polymerase III promoter (U6). This schematic illustrates different embodiments, where the RNA guide is expressed as either a single-target-bearing pre-crRNA, a mature crRNA, or multiplexed with multiple targets, in the configurations shown. [Figure 41A] Figure 41A is a bar graph showing indel activity induced by Cas12i2 CRISPR effectors targeted to the VEGFA locus in 293T cell lines 72 hours after transient transfection of the effector and RNA guide constructs described in Figure 40. Various RNA guide designs were assayed, exhibiting varying degrees of efficacy. Error bars represent SEM from 3 replicates. [Figure 41B] Figure 41B is a representation of a representative indel from next generation sequencing. The TTC PAM sequence is labeled, with a representative indel occurring more than 20 bp downstream of the PAM. DETAILED DESCRIPTION OF THE INVENTION

[0100] The wide diversity of natural CRISPR-Cas defense systems contains a variety of active mechanisms and functional elements that can be exploited for programmable biotechnology. In natural systems, these mechanisms and parameters provide efficient defense against foreign DNA and viruses while also providing self-nonself discrimination and avoiding self-targeting. In engineered systems, these same mechanisms and parameters also define the boundaries of targeting space, providing a diverse toolbox for molecular engineering. For example, systems Ca9 and Ca13a possess canonical DNA and RNA endonuclease activities, whose targeting space is defined by a protospacer adjacent motif (PAM) on the target DNA and a protospacer adjacent site (PFS) on the target RNA, respectively.

[0101] Using the methods described herein, additional mechanisms and parameters within single-subunit class 2 effector systems have been discovered that can expand the capabilities of RNA-programmable nucleic acid manipulation.

[0102] In one aspect, the present disclosure relates to the use of computational methods and algorithms to explore and identify novel protein families that exhibit strong co-occurrence patterns with certain other features within naturally occurring genomic sequences. In certain embodiments, these computational methods relate to identifying protein families that co-occur in close proximity to CRISPR arrays. However, the methods disclosed herein are applicable to both non-coding and protein-coding sequences (e.g., fragments of phage sequences in non-coding regions of bacterial loci; or CRISPR sequences). These methods are useful in identifying naturally occurring proteins within close proximity of other features, such as Cas1 proteins. It is understood that the methods and calculations described herein may be performed on one or more computing devices.

[0103] In some embodiments, the set of genome sequences is obtained from a genome or metagenomic database. The database may include short reads, contig-level data, assembled scaffolds, or complete genome sequences of an organism. Similarly, the database may include genome sequence data from prokaryotes, eukaryotes, or data from metagenomic environmental samples. Examples of database repositories include the National Center for Biotechnology Information (NCBI) RefSeq, NCBI's GenBank, NCBI's Whole Genome Shotgun (WGS), and the Joint Genome Institute's (JGI) Integrated Microbial Genomes (IMG).

[0104] In some embodiments, the selection of genome sequence data of a specified minimum length imposes a minimum size requirement, in certain exemplary embodiments, the minimum contig length may be 100 nucleotides, 500 nt, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 40 kb, or 50 kb.

[0105] In some embodiments, known or predicted proteins are extracted from a complete or selected set of genome sequence data. In some embodiments, known or predicted proteins are taken from extracting coding sequence (CDS) annotations provided by a source database. In some embodiments, predicted proteins are determined by applying computational methods to identify proteins from nucleotide sequences. In some embodiments, proteins are predicted from genome sequences using the GeneMark suite. In some embodiments, proteins are predicted from genome sequences using Prodigal. In some embodiments, multiple protein prediction algorithms may be used on the same set of sequence data, and redundancies may be removed from the resulting set of proteins.

[0106] In some embodiments, CRISPR arrays are identified from genome sequence data. In some embodiments, CRISPR arrays are identified using PILER-CR. In some embodiments, CRISPR arrays are identified using CRISPR Recognition Tool (CRT). In some embodiments, CRISPR arrays are identified by a heuristic method that identifies a nucleotide motif that repeats a minimum number of times (for example, 2, 3, or 4 times), and the interval between consecutive occurrences of the repeated motif does not exceed a specified length (for example, 50, 100, or 150 nucleotides). In some embodiments, multiple CRISPR array identification tools can be used on the same set of sequence data, and duplicates can be removed from the resulting set of CRISPR arrays.

[0107] In some embodiments, proteins that are close to the CRISPR array are identified. In some embodiments, proximity is defined as a nucleotide distance, and may be within 20 kb, 15 kb, or 5 kb. In some embodiments, proximity is defined as the number of open reading frames (ORFs) between the protein and the CRISPR array, and certain exemplary distances may be 10, 5, 4, 3, 2, 1, or 0 ORFs. The proteins identified to be close to the CRISPR array are then grouped into homologous protein clusters. In some embodiments, protein clusters are formed using blastclust. In certain other embodiments, protein clusters are formed using mmseqs2.

[0108] To establish strong co-occurrence patterns between members of a protein cluster with a CRISPR array, a BLAST search of each member of a protein family against a complete, pre-organized set of known and predicted proteins may be performed. In some embodiments, UBLAST or mmseqs2 may be used to search for similar proteins. In some embodiments, a search may be performed on only a representative subset of proteins within the family.

[0109] In some embodiments, the clusters of proteins that are in close proximity to the CRISPR array are ranked or filtered by a metric to determine co-occurrence.One exemplary metric is the ratio of the number of elements in a protein cluster to the number of BLAST matches up to a certain E-value threshold.In some embodiments, a fixed E-value threshold may be used.In other embodiments, the E-value threshold may be determined by the most distant member of the protein cluster.In some embodiments, a global set of proteins is clustered, and the co-occurrence metric is the ratio of the number of elements in the CRISPR-associated cluster to the number of elements in one or more global clusters that it contains.

[0110] In some embodiments, a manual review process is used to evaluate the potential functionality and minimal set of components of an engineered system based on the naturally occurring locus structure of proteins in the cluster. In some embodiments, the manual review can be aided by a graphical representation of the protein cluster, which can include information such as pairwise sequence similarity, phylogenetic tree, source organism / environment, predicted functional domains, and a graphical representation of the locus structure. In some embodiments, the graphical representation of the locus structure can filter for neighboring protein families with high representation. In some embodiments, representation can be calculated by the ratio of the number of related neighboring proteins to one or more sizes of the containing global cluster(s). In certain exemplary embodiments, the graphical representation of the protein cluster can include a representation of the CRISPR array structure of the naturally occurring locus. In some embodiments, the graphical representation of the protein cluster can include a representation of the number of conserved direct repeats relative to the length of the estimated CRISPR array, or the number of unique spacer sequences relative to the length of the estimated CRISPR array. In some embodiments, the graphical representation of the protein clusters may include depictions of various co-occurrence metrics of putative effectors with CRISPR arrays to predict novel CRISPR-Cas systems and identify their components.

[0111] Pooled Screening To efficiently validate the activity of novel engineered CRISPR-Cas systems and simultaneously evaluate various mechanistic and functional parameters in an unbiased manner, we employ a novel pooled screening approach in Escherichia coli (E. coli). First, we computationally identify conserved proteins and non-coding elements of novel CRISPR-Cas systems, then assemble the individual components into a single artificial expression vector (based in one embodiment on the pET-28a+ backbone) using DNA synthesis and molecular cloning. In a second embodiment, the effector and non-coding elements are transcribed into a single mRNA transcript, and individual effectors are translated using distinct ribosome binding sites.

[0112] Second, the natural crRNA and targeting spacer are replaced with a library of unprocessed crRNAs containing a non-natural spacer that targets a second plasmid, pACYC184. This crRNA library is cloned into a vector backbone (e.g., pET-28a+) containing protein effectors and non-coding elements, and then this library is subsequently transformed into E. coli along with the pACYC184 plasmid target. As a result, each resulting E. coli cell contains only one targeting spacer. In an alternative embodiment, a library of unprocessed crRNAs containing non-natural spacers further targets essential E. coli genes drawn from sources such as those described in Baba et al. (2006) Mol. Syst. Biol. 2:2006.0008; and Gerdes et al. (2003) J. Bacteriol. 185(19):5673-84, the entire contents of each of which are incorporated herein by reference. In this embodiment, positive targeted activation of the novel CRISPR-Cas system to disrupt essential gene function results in cell death or growth arrest. In some embodiments, essential gene targeting spacers can be combined with the pACYC184 target to add another dimension to the assay. In another embodiment, non-coding sequences flanking the CRISPR array, putative effector or accessory open reading frames, and predicted anti-repeats indicative of tracrRNA elements were concatemerized together and cloned into pACYC184 for expression by lac and IPTG-inducible T7 promoters.

[0113] Third, E. coli is grown under antibiotic selection. In one embodiment, triple antibiotic selection is used: kanamycin to confirm successful transformation of the pET-28a+ vector containing the engineered CRISPR-Cas effector system, and chloramphenicol and tetracycline to confirm successful co-transformation of the pACYC184 targeting vector. Because pACYC184 normally confers resistance to chloramphenicol and tetracycline, under antibiotic selection, positive activity of the novel CRISPR-Cas system targeting this plasmid will eliminate cells actively expressing the effector, non-coding elements, and specific active elements of the crRNA library. Examination of surviving cell populations at later time points compared to earlier time points typically results in a depletion of signal compared to inactive crRNA. In some embodiments, double antibiotic selection is used. For example, removing selection pressure by withdrawing either chloramphenicol or tetracycline can provide new information about targeting substrates, sequence specificity, and potency. In some embodiments, successful transformation of pET-28a+ vectors containing engineered CRISPR-Cas effector systems is confirmed using only kanamycin. This embodiment is suitable for libraries containing spacers targeting essential E. coli genes, as no further selection beyond kanamycin is required to observe growth changes. In this embodiment, chloramphenicol and tetracycline dependence is eliminated, and their targets (if present) in the library provide an additional source of negative or positive information regarding targeting substrate, sequence specificity, and potency.

[0114] Because the pACYC184 plasmid contains a diverse set of features and sequences that can affect the activity of CRISPR-Cas systems, mapping active crRNAs from pooled screens to pACYC184 provides a broad, non-hypothesis-bound activity pattern that may suggest various activity mechanisms and functional parameters. In this way, the features required for reconstitution of novel CRISPR-Cas systems in heterologous prokaryotic species can be more comprehensively tested and studied.

[0115] Certain important advantages of the in vivo pooled screens described herein include: (1) Versatility - the plasmid design allows for the expression of multiple effectors and / or non-coding elements; the library cloning strategy allows for the expression of both computationally predicted crRNA transcriptional directions; (2) By using a comprehensive examination of activity mechanisms and functional parameters, we can evaluate various interference mechanisms, including DNA or RNA cleavage; co-occurrence of features such as transcription and plasmid DNA replication; and examine flanking sequences for crRNA libraries to reliably determine 4N complexity-equivalent PAMs; (3) Sensitivity—pACYC184 is a low-copy plasmid, allowing high sensitivity for CRISPR-Cas activity because even small interference rates can eliminate the antibiotic resistance encoded by the plasmid; and (4) Efficiency—Pooled screening involves optimized molecular biology steps that allow for higher speed and throughput for RNA sequencing, and protein expression samples can be taken directly from surviving cells in the screen.

[0116] As discussed in more detail in the Examples below, this in vivo pooled screen was used to evaluate the novel CRISPR-Cas family described herein by assessing its operable elements, mechanisms and parameters, as well as its ability to be active and reprogrammed in an engineered system outside of its native cellular environment.

[0117] In vitro pooled screening In vitro pooled screening methods can also be used, which complement in vivo pooled screens. In vitro pooled screens allow for rapid biochemical characterization and reduction of CRISPR systems to the essential components required for system activity. In one embodiment, a cell-free in vitro transcription and translation (IVTT) system is used to directly synthesize RNA and proteins from DNA encoding the non-coding and effector proteins of the CRISPR system, thus providing a faster, higher-throughput method for evaluating a greater number of different, distinct CRISPR-Cas effector systems than traditional biochemical assays that rely on FPLC-purified proteins. In addition to achieving higher throughput and efficiency of biochemical reactions, in vitro screening offers several advantages that complement the in vivo pooled screening methods described above.

[0118] (1) Direct observation of both enrichment and depletion signals—In vitro pooled screens provide readouts for both cleavage enrichment, where the specific cleavage sites, cleavage patterns, and sequence motifs for active effector systems can be identified by directly capturing and sequencing cleavage products, and target depletion, where a negative signal from depletion of a specific target in the uncleaved population is used as a surrogate for activity. Because in vivo pooled screens utilize a target depletion readout, the enrichment mode provides further insight into effector activity.

[0119] (2) Greater control of reaction components and environment—The well-defined components and activities of proprietary IVTT, compared to the complex E. coli cellular environment of in vivo screens, allow for precise control of reaction components, enabling identification of the minimal components required for further active translation. Additionally, non-natural modifications to reaction components may be made to enhance activity or facilitate readout; for example, the addition of phosphorothioate linkages to ssDNA and dsDNA substrates reduces noise by limiting substrate degradation by exonucleases.

[0120] (3) Robustness against toxic / growth-inhibitory proteins—For proteins that may be toxic to E. coli cell growth, in vitro pooled screens allow for functional screening without subjecting live cells to growth inhibition, ultimately enabling greater versatility in protein selection and screening.

[0121] The novel CRISPR-Cas family described herein was evaluated using a combination of in vivo and in vitro pooled screens to assess its operable elements, mechanisms and parameters, as well as its ability to be active and reprogrammed in engineered systems outside of its native cellular environment.

[0122] Class 2 CRISPR-Cas effectors containing the RuvC domain In one aspect, the present disclosure provides a Class 2 CRISPR-Cas system, referred to herein as the CLUST.029130 (Type VI) CRISPR-Cas system, which includes an isolated CRISPR-associated protein having a RuvC domain and an isolated crRNA, also referred to as an RNA guide, guide RNA, or gRNA, that includes a spacer sequence complementary to a target nucleic acid sequence, such as a DNA sequence.

[0123] Suitably, the CRISPR-Cas effector protein having a RuvC domain comprises a RuvC III motif, X1SHX4DX6X7 (SEQ ID NO: 200) (wherein X1 is S or T, X4 is Q or L, X6 is P or S, and X7 is F or L); a RuvC I motif, X1XDXNX6X7XXXX 11 (SEQ ID NO: 201) (wherein X1 is A or G or S, X is any amino acid, X6 is Q or I, X7 is T or S or V, and X 11 is T or A); and the RuvC II motif, X1X2X3E (SEQ ID NO: 210) (wherein X1 is C or F or I or L or M or P or V or W or Y, X2 is C or F or I or L or M or P or R or V or W or Y, and X3 is C or F or G or I or L or M or P or V or W or Y).

[0124] Preferably, the type VI CRISPR-Cas system comprises a CRISPR-Cas effector having a RuvC domain and a type VI crRNA. Preferably, the Cas12i effector is about 1100 amino acids or less in length and comprises a functional PAM-interacting domain that recognizes a PAM in the target DNA. The type VI CRISPR-Cas effector protein is capable of binding to a type VI RNA guide to form a type VI CRISPR-Cas system, wherein the type VI RNA guide comprises a stem-loop structure of a 5-nucleotide stem and a 6-, 7-, or 8-nucleotide loop. The type VI CRISPR-Cas system is capable of targeting and binding to sequence-specific DNA even in the absence of tracrRNA.

[0125] In some embodiments, the Type VI CRISPR-Cas effector protein and the Type VI RNA guide form a binary complex, which may include other components. The binary complex is activated upon binding to a nucleic acid substrate (i.e., a sequence-specific substrate or target nucleic acid) complementary to the spacer sequence in the RNA guide. In some embodiments, the sequence-specific substrate is double-stranded DNA. In some embodiments, the sequence-specific substrate is single-stranded DNA. In some embodiments, sequence specificity requires a perfect match between the spacer sequence in the RNA guide (e.g., crRNA) and the target substrate. In other embodiments, sequence specificity requires a partial (contiguous or non-contiguous) match between the spacer sequence in the RNA guide (e.g., crRNA) and the target substrate. In certain embodiments, sequence specificity further requires a perfect match between the protospacer adjacent motif ("PAM") sequence adjacent to the spacer sequence and the canonical PAM sequence recognized by the CRISPR-associated protein. In some instances, a perfect PAM sequence match is not required; a partial match is sufficient for sequence-specific association of the binary complex with the DNA substrate.

[0126] In some embodiments, the target nucleic acid substrate is double-stranded DNA (dsDNA). In some embodiments, the target nucleic acid substrate is dsDNA and includes a PAM. In some embodiments, the binary complex modifies the target sequence-specific dsDNA substrate upon binding thereto. In some embodiments, the binary complex preferentially nicks the non-target strand of the target dsDNA substrate. In some embodiments, the binary complex cleaves both strands of the target dsDNA substrate. In some embodiments, the binary complex cleaves both strands of the target dsDNA substrate with sticky end cleavage. In some embodiments, the binary complex creates a blunt-ended double-strand break (DSB) in the target dsDNA substrate.

[0127] In some embodiments, the target nucleic acid substrate is single-stranded DNA (ssDNA). In some embodiments, the target nucleic acid substrate is ssDNA and does not contain PAM. In some embodiments, the binary complex modifies the target sequence-specific ssDNA substrate upon binding thereto. In some embodiments, the binary complex cleaves the target ssDNA substrate.

[0128] In some embodiments, the binary complex becomes activated upon binding to the target substrate. In some embodiments, the activated complex exhibits "multiple turnover" activity, such that upon acting on the target substrate (e.g., cleaving it), the activated complex remains in an activated state. In some embodiments, the binary complex exhibits "single turnover" activity, such that upon acting on the target substrate, the binary complex returns to an inactive state. In some embodiments, the activated complex exhibits nonspecific (i.e., "collateral") cleavage activity, such that the activated complex cleaves nucleic acids that have no sequence similarity to the target. In some embodiments, the collateral nucleic acid substrate is ssDNA.

[0129] CRISPR enzyme modification Nuclease-deficient CRISPR enzyme If the CRISPR enzyme described herein has nuclease activity, the CRISPR enzyme can be modified to reduce nuclease activity, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% nuclease inactivation compared to the wild-type CRISPR enzyme. Nuclease activity can be reduced by several methods, for example, by introducing mutations into the nuclease or PAM-interacting domain of the CRISPR enzyme. In some embodiments, catalytic residues for nuclease activity may be identified, and nuclease activity may be reduced by substituting these amino acid residues with different amino acid residues (e.g., glycine or alanine). Examples of such mutations for Cas12i1 include D647A, E894A, or D948A. Examples of such mutations for Cas12i2 include D599A, E833A, or D886A.

[0130] The inactivated CRISPR enzyme can comprise or be associated with one or more functional domains (e.g., via a fusion protein, a linker peptide, a Gly4Ser (GS) peptide linker, etc.) (e.g., through co-expression of multiple proteins). Such functional domains can have various activities, such as methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and switch activity (e.g., light-inducible). In some embodiments, the functional domains are Krüppel-associated box (KRAB), VP64, VP16, Fok1, P65, HSF1, MyoD1, and biotin-APEX.

[0131] By positioning one or more functional domains on the inactivated CRISPR enzyme, the functional domain can be oriented in the correct space to affect the target with the functional effect it ascribes. For example, if the functional domain is a transcriptional activator (e.g., VP16, VP64, or p65), the transcriptional activator is oriented in the space to allow it to affect the transcription of the target. Similarly, a transcriptional repressor is positioned to affect the transcription of the target, and a nuclease (e.g., Fok1) is positioned to cleave or partially cleave the target. In some embodiments, the functional domain is located at the N-terminus of the CRISPR enzyme. In some embodiments, the functional domain is located at the C-terminus of the CRISPR enzyme. In some embodiments, the inactivated CRISPR enzyme is modified to include a first functional domain at the N-terminus and a second functional domain at the C-terminus.

[0132] Split Enzyme The present disclosure also provides the split version of the CRISPR enzyme described herein.The split version of the CRISPR enzyme can be advantageous for delivery.In some embodiments, the CRISPR enzyme is split into two parts of the enzyme, and together they comprise substantially functional CRISPR enzyme.

[0133] The division can be performed in such a way that one or more catalytic domains are unaffected. The CRISPR enzyme may function as a nuclease, or may be an inactivated enzyme that is essentially an RNA-binding protein with little or no catalytic activity (e.g., due to one or more mutations in its catalytic domain).

[0134] In some embodiments, the nuclease lobe and the α-helical lobe are expressed as separate polypeptides. These lobes do not interact with each other, but the RNA guide recruits them into a complex that mimics the activity of a full-length CRISPR enzyme and catalyzes site-specific DNA cleavage. The use of modified RNA guides prevents dimerization, thereby abolishing split enzyme activity and allowing the development of an inducible dimerization system. Split enzymes are described, for example, in Wright, Addison V., et al., "Rational design of a split-Cas9 enzyme complex," Proc. Nat'l. Acad. Sci., 112.10 (2015): 2984-2989 (incorporated herein by reference in its entirety).

[0135] In some embodiments, the split enzyme can be fused to a dimerization partner, for example, by utilizing a rapamycin-sensitive dimerization domain. This allows the creation of a chemically inducible CRISPR enzyme for temporal control of CRISPR enzyme activity. By splitting the CRISPR enzyme into two fragments in this way, the CRISPR enzyme can be chemically inducible, and the rapamycin-sensitive dimerization domain can be used to control the reassembly of the CRISPR enzyme.

[0136] The split point is typically designed in silico and cloned into the construct. During this process, mutations may be introduced into the split enzyme and non-functional domains may be removed. In some embodiments, the two portions or fragments (i.e., N-terminal and C-terminal fragments) of the split CRISPR enzyme can form a complete CRISPR enzyme that contains, for example, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the sequence of the wild-type CRISPR enzyme.

[0137] Self-activating or inactivating enzymes The CRISPR enzymes described herein may be designed to be self-activating or self-inactivating. In some embodiments, the CRISPR enzyme is self-inactivating. For example, a target sequence can be introduced into a construct encoding the CRISPR enzyme. The CRISPR enzyme can then cleave the target sequence, thereby causing the construct encoding the enzyme to self-inactivate its expression. Methods for constructing self-inactivating CRISPR systems are described, for example, in Epstein, Benjamin E., and David V. Schaffer, "Engineering a Self-Inactivating CRISPR System for AAV Vectors," Mol. Ther., 24 (2016): S50 (incorporated herein by reference in its entirety).

[0138] In some other embodiments, an additional RNA guide expressed under the control of a weak promoter (e.g., a 7SK promoter) can target the nucleic acid sequence encoding the CRISPR enzyme and interfere with and / or prevent its expression (e.g., by preventing transcription and / or translation of the nucleic acid). Transfecting a cell with a vector expressing a CRISPR enzyme, an RNA guide, and an RNA guide targeting the nucleic acid encoding the CRISPR enzyme can lead to the efficient destruction of the nucleic acid encoding the CRISPR enzyme, reducing CRISPR enzyme levels and thus limiting genome editing activity.

[0139] In some embodiments, the genome editing activity of CRISPR enzymes can be regulated through endogenous RNA signatures (e.g., miRNAs) in mammalian cells. A CRISPR enzyme switch can be created by using miRNA-complementary sequences in the 5'-UTR of mRNA encoding the CRISPR enzyme. This switch selectively and efficiently responds to miRNAs in target cells. Thus, this switch can differentially control genome editing by sensing endogenous miRNA activity within heterogeneous cell populations. Therefore, this switch system may provide a framework for cell-type-selective genome editing and cell engineering based on intracellular miRNA information (Hirosawa, Moe et al., "Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch," Nucl. Acids Res., 2017 Jul. 27;45(13):e118).

[0140] Inducible CRISPR enzymes CRISPR enzymes can be inducible, for example, light-inducible or chemically inducible. This mechanism allows functional domains in CRISPR enzymes to be activated by known triggers. Light-inducibility can be achieved by various methods known in the art, for example, by designing a fusion complex in which the CRY2PHR / CIBN pair is used in a split CRISPR enzyme (see, for example, Konermann et al. "Optical control of mammalian endogenous transcription and epigenetic states," Nature, 500.7463 (2013): 472). Chemical inducibility can be achieved, for example, by designing a fusion complex in which the FKBP / FRB (FK506-binding protein / FKBP rapamycin-binding domain) pair is used in a split CRISPR enzyme. Rapamycin is required for the formation of the fusion complex, thus activating the CRISPR enzyme (see, e.g., Zetsche, Volz, and Zhang, “A split-Cas9 architecture for inducible genome editing and transcription modulation,” Nature Biotech., 33.2 (2015): 139-142).

[0141] Furthermore, expression of CRISPR enzymes can be regulated by inducible promoters, such as tetracycline- or doxycycline-controlled transcriptional activation (Tet-On and Tet-Off expression systems), hormone-inducible gene expression systems (e.g., ecdysone-inducible gene expression systems), and arabinose-inducible gene expression systems. When delivered as RNA, expression of RNA-targeting effector proteins may be regulated by riboswitches capable of sensing small molecules like tetracycline (see, e.g., Goldfless, Stephen J. et al., "Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction," Nucl. Acids Res., 40.9 (2012): e64-e64).

[0142] Various embodiments of inducible CRISPR enzymes and inducible CRISPR systems are described, for example, in U.S. Pat. No. 8,871,445, U.S. Patent Application Publication No. 20160208243, and WO 2016205764, each of which is herein incorporated by reference in its entirety.

[0143] Functional mutations Various mutations or modifications can be introduced into the CRISPR enzymes described herein to improve specificity and / or robustness. In some embodiments, amino acid residues that recognize a protospacer adjacent motif (PAM) are identified. The CRISPR enzymes described herein can be further modified to recognize different PAMs, for example, by substituting amino acid residues that recognize PAMs with other amino acid residues. In some embodiments, the CRISPR enzymes can recognize other PAMs, for example, as described herein.

[0144] In some embodiments, a CRISPR-associated protein comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nuclear localization signal (NLS) attached to the N-terminus or C-terminus of the protein. Non-limiting examples of NLSs include the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 300); an NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 301)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 302) or RQRRNELKRSP (SEQ ID NO: 303); the hRNPA1M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 304); the IBB domain from importin-α, sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 305); the sequences VSRKRPRP (SEQ ID NO: 306) and PPKKARED (SEQ ID NO: 307) of the fibroid T protein; the sequence PQPKKKPL (SEQ ID NO: 308) of human p53; and the mouse c-abl Examples of NLS sequences include the sequence SALIKKKKKMAP (SEQ ID NO: 309) of IV; the sequences DRLRR (SEQ ID NO: 310) and PKQKKRK (SEQ ID NO: 311) of influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 312) of hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 313) of mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 314) of human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 315) of human glucocorticoid receptor. In some embodiments, the CRISPR-associated protein comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nuclear export signal (NES) attached to the N- or C-terminus of the protein. In preferred embodiments, the C- and / or N-terminal NLS or NES is attached for optimal expression and nuclear targeting in eukaryotic cells, such as human cells.

[0145] In some embodiments, the CRISPR enzymes described herein have one or more functional activities altered by mutating one or more amino acid residues. For example, in some embodiments, the CRISPR enzyme has its helicase activity altered by mutating one or more amino acid residues. In some embodiments, the CRISPR enzyme has its nuclease activity (e.g., endonuclease activity or exonuclease activity) altered by mutating one or more amino acid residues. In some embodiments, the CRISPR enzyme has its ability to functionally associate with an RNA guide altered by mutating one or more amino acid residues. In some embodiments, the CRISPR enzyme has its ability to functionally associate with a target nucleic acid altered by mutating one or more amino acid residues.

[0146] In some embodiments, the CRISPR enzymes described herein are capable of cleaving a target nucleic acid molecule. In some embodiments, the CRISPR enzyme cleaves both strands of a target nucleic acid molecule. However, in some embodiments, the cleavage activity of a CRISPR enzyme is modified by mutating one or more amino acid residues. For example, in some embodiments, a CRISPR enzyme may contain one or more mutations that render the enzyme incapable of cleaving a target nucleic acid. In other embodiments, a CRISPR enzyme may contain one or more mutations that render the enzyme capable of cleaving a single strand of a target nucleic acid (i.e., nickase activity). In some embodiments, a CRISPR enzyme is capable of cleaving a strand of a target nucleic acid that is complementary to the strand to which an RNA guide is hybridized. In some embodiments, a CRISPR enzyme is capable of cleaving a strand of a target nucleic acid to which an RNA guide is hybridized.

[0147] In some embodiments, the CRISPR enzymes described herein may be engineered to contain deletions of one or more amino acid residues to reduce the size of the enzyme while retaining one or more desired functional activities (e.g., nuclease activity and the ability to functionally interact with an RNA guide). This truncated CRISPR enzyme may be advantageously used in combination with a payload-limited delivery system.

[0148] In one aspect, the present disclosure provides nucleic acid sequences that are at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic sequences described herein. In another aspect, the present disclosure also provides amino acid sequences that are at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences described herein.

[0149] In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is identical to a sequence described herein. In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that differs from a sequence described herein.

[0150] In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is identical to a sequence described herein. In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that differs from a sequence described herein.

[0151] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences to ensure optimal alignment, and non-homologous sequences may be ignored for comparison purposes). Generally, the length of the reference sequence aligned for comparison purposes should be at least 80% of the length of the reference sequence, and in some embodiments, at least 90%, 95%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of gaps that need to be introduced for optimal alignment of the two sequences and the number of identical positions shared by the sequences, taking into account the length of each gap. For purposes of this disclosure, comparison of sequences and determination of percent identity between two sequences may be accomplished using a Blosum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0152] In addition to the biochemical and diagnostic applications described herein, the programmable Type VI CRISPR-Cas systems described herein have important applications in eukaryotic cells, including therapeutic modification of the genome, including, but not limited to, genotype correction, gene knockout, gene sequence insertion / deletion (by homology-directed repair or other methods), single nucleotide modification, or gene regulation. These gene modification modalities can utilize the nuclease activity of Cas12i, double nicking, or programmable DNA binding of catalytically inactive Cas12i fused to additional effector domains.

[0153] In some embodiments, the CRISPR-associated proteins and accessory proteins described herein can be fused to one or more peptide tags, including His tags, GST tags, FLAG tags, or myc tags. In some embodiments, the CRISPR-associated proteins or accessory proteins described herein can be fused to a detectable moiety, such as a fluorescent protein (e.g., green fluorescent protein or yellow fluorescent protein). And in some embodiments, the CRISPR-associated proteins or accessory proteins of the present disclosure are fused to a peptide or non-peptide moiety that allows the protein to enter or localize to a tissue, cell, or region of a cell. For example, the CRISPR-associated proteins or accessory proteins (such as Cas12i) of the present disclosure can include a nuclear localization sequence (NLS), such as the SV40 (simian virus 40) NLS, c-Myc NLS, or other suitable monopartite NLS. The NLS may be fused to the N-terminus and / or C-terminus of a CRISPR-associated protein or accessory protein, and may be fused singly (i.e., a single NLS) or concatemerized (e.g., a chain of 2, 3, 4, etc. NLSs).

[0154] In embodiments in which a tag is fused to a CRISPR-associated protein, such a tag can facilitate affinity- or charge-based purification of the CRISPR-associated protein, for example, by liquid chromatography or bead separation utilizing immobilized affinity or ion exchange reagents. As a non-limiting example, a recombinant CRISPR-associated protein of the present disclosure (such as Cas12i) can contain a polyhistidine (His) tag and be loaded onto a chromatography column containing an immobilized metal ion for purification (e.g., Zn chelated by a chelating ligand immobilized on the resin). 2+ , Ni 2+ , Cu 2+ ion, this resin is a custom formulated resin or a commercially available resin or GE Healthcare (The HisTrap FF column may be a pre-made column, such as the HisTrap FF column commercialized by Life Sciences, Marlborough, Massachusetts.) After the loading step, the column is optionally rinsed, for example, using one or more suitable buffer solutions, and the His-tagged protein is then eluted using a suitable elution buffer. Alternatively, or in addition, where a recombinant CRISPR-associated protein of the disclosure utilizes a FLAG tag, such protein may be purified using immunoprecipitation methods known in the art. Other suitable purification methods for tagged CRISPR-associated proteins or accessory proteins of the disclosure will be apparent to those of skill in the art.

[0155] The proteins described herein (e.g., CRISPR-associated proteins or accessory proteins) can be delivered or used as either nucleic acid molecules or polypeptides. When nucleic acid molecules are used, the nucleic acid molecules encoding the CRISPR-associated proteins can be codon-optimized, as discussed in more detail below. Nucleic acids can be codon-optimized for use in any organism of interest, particularly human cells or bacteria. For example, nucleic acids can be codon-optimized for any non-human eukaryote, including mouse, rat, rabbit, dog, livestock, or non-human primates. Codon usage tables are readily available, for example, in the "Codon Usage Database," available at www.kazusa.orjp / codon / , and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000) (incorporated herein by reference in its entirety). Computer algorithms for codon-optimizing specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen; Jacobus, PA).

[0156] In some examples, nucleic acids of the present disclosure encoding CRISPR-associated proteins or accessory proteins for expression in eukaryotic (e.g., human or other mammalian) cells include one or more introns, i.e., one or more non-coding sequences comprising a splice donor sequence at a first end (e.g., the 5' end) and a splice acceptor sequence at a second end (e.g., the 3' end). In various embodiments of the present disclosure, any suitable splice donor / splice acceptor can be used, including, without limitation, a Simian Virus 40 (SV40) intron, a β-globin intron, and a synthetic intron. Alternatively or additionally, nucleic acids of the present disclosure encoding CRISPR-associated proteins or accessory proteins can include a transcription termination signal, such as a polyadenylation (polyA) signal, at the 3' end of the DNA coding sequence. In some examples, the polyA signal is located in close proximity to or adjacent to an intron, such as an SV40 intron.

[0157] RNA guide In some embodiments, the CRISPR systems described herein comprise at least one type VI RNA guide. Many RNA guide configurations are known in the art (see, for example, International Publication Nos. 2014 / 093622 and 2015 / 070083, the entire contents of each of which are incorporated herein by reference). In some embodiments, the CRISPR systems described herein comprise multiple RNA guides (e.g., 2, 3, 4, 5, 6, 7, 8, or more RNA guides).

[0158] In some embodiments, the CRISPR systems described herein comprise at least one type VI RNA guide or a nucleic acid encoding at least one type VI RNA guide. In some embodiments, the RNA guide comprises a crRNA. Generally, the crRNA described herein comprises a direct repeat sequence and a spacer sequence. In certain embodiments, the crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or a spacer sequence. In some embodiments, the crRNA comprises a direct repeat sequence, a spacer sequence, and a direct repeat sequence (DR-spacer-DR), which is typical of precursor crRNA (pre-crRNA) configurations in other CRISPR systems. In some embodiments, the crRNA comprises a truncated direct repeat sequence and a spacer sequence, which is typical of processed or mature crRNA. In some embodiments, a CRISPR-Cas effector protein forms a complex with the RNA guide, and the spacer sequence directs the complex to sequence-specific binding to a target nucleic acid complementary to the spacer sequence.

[0159] Suitably, the CRISPR systems described herein comprise at least one type VI RNA guide or a nucleic acid encoding a type VI RNA guide, wherein the RNA guide comprises a direct repeat. Suitably, the type VI RNA guide may form a secondary structure, such as a stem-loop structure, for example as described herein.

[0160] A direct repeat can contain two nucleotide stretches that may be complementary to each other, separated by an intervening nucleotide; thus, when the direct repeat hybridizes to form a double-stranded RNA duplex (dsRNA duplex), a stem-loop structure can result, in which the two complementary nucleotide stretches form a stem and the intervening nucleotides form a loop or hairpin (Figure 3). For example, the intervening nucleotides that form the "loop" have a length of about 6 nucleotides to about 8 nucleotides, or about 7 nucleotides. In different embodiments, the stem can contain at least 2, at least 3, at least 4, or 5 base pairs.

[0161] Preferably, the direct repeat may comprise two complementary stretches of nucleotides that are about 5 nucleotides in length separated by an intervening interval of about 7 nucleotides.

[0162] Some exemplary direct repeats of a type VI system are illustrated in Figure 3, and preferably, one skilled in the art can mimic the structure of such direct repeats illustrated in Figure 3 while deviating from naturally occurring type VI direct repeats.

[0163] The direct repeat can comprise or consist of about 22 to 40 nucleotides, or about 23 to 38 nucleotides, or about 23 to 36 nucleotides.

[0164] In some embodiments, the CRISPR systems described herein include multiple RNA guides (e.g., 2, 3, 4, 5, 10, 15, or more) or multiple nucleic acids encoding multiple RNA guides.

[0165] In some embodiments, the CRISPR systems described herein include an RNA guide or a nucleic acid encoding the RNA guide. In some embodiments, the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid (e.g., hybridizing to it under appropriate conditions), where the direct repeat sequence comprises 5'-CCGUCNNNNNNNGACGG-3' (SEQ ID NO: 202) proximal to its 3' end and adjacent to the spacer sequence. In some embodiments, the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid (e.g., hybridizing to it under appropriate conditions), where the direct repeat sequence comprises 5'-GUGCCNNNNNNNGGCAC-3' (SEQ ID NO: 203) proximal to its 3' end and adjacent to the spacer sequence. In some embodiments, the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing to (e.g., hybridizing to) a target nucleic acid, wherein the direct repeat sequence has a 5'-GUGUCN sequence proximal to the 3' end and adjacent to the spacer sequence. 5-6 UGACAX1-3' (SEQ ID NO: 204) (wherein N 5-6 refers to any contiguous sequence of 5 or 6 nucleic acid bases, and X1 refers to C, T, or U).

[0166] Examples of pairs of RNA guide direct repeat sequences and effector proteins are provided in Table 5A. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid sequence listed in Table 5A (e.g., SEQ ID NOS: 6-10, 19-24). In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the first three 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the first four 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the first five 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the first six 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the first seven 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the first eight 5' nucleotides.

[0167] RNA guide multiplexing It has been demonstrated that the CLUST.029130 (Type VI) CRISPR-Cas effector utilizes two or more RNA guides, thus enabling these effectors, and systems and complexes comprising them, to target multiple different nucleic acid targets. In some embodiments, the CRISPR systems described herein comprise multiple RNA guides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or more RNA guides). In some embodiments, the CRISPR systems described herein comprise a single RNA strand or a nucleic acid encoding a single RNA strand, where the RNA guides are arranged in tandem. The single RNA strand can comprise multiple copies of the same RNA guide, multiple copies of different RNA guides, or a combination thereof.

[0168] In some embodiments, the CLUST.029130 (type VI) CRISPR-Cas effector protein is delivered in a complex with multiple RNA guides directed to different target nucleic acids. In some embodiments, the CLUST.029130 (type VI) CRISPR-Cas effector protein can be co-delivered with multiple RNA guides, each specific to a different target nucleic acid. Methods for multiplexing using CRISPR-associated proteins are described, for example, in U.S. Patent No. 9,790,490 and European Patent No. 3009511 (the entire contents of each of which are expressly incorporated herein by reference).

[0169] RNA-guided modifications Spacer Length The spacer length of the RNA guide may range from about 15 to 50 nucleotides. In some embodiments, the spacer length of the RNA guide is at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides. In some embodiments, the spacer length is 15 to 17 nucleotides, 15 to 23 nucleotides, 16 to 22 nucleotides, 17 to 20 nucleotides, 20 to 24 nucleotides (e.g., 20, 21, 22, 23, or 24 nucleotides), 23 to 25 nucleotides (e.g., 23, 24, or 25 nucleotides), 24 to 27 nucleotides, 27 to 30 nucleotides, 30 to 45 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 40, or 45 nucleotides), 30 or 35 to 40 nucleotides, 41 to 45 nucleotides, 45 to 50 nucleotides, or more. In some embodiments, the spacer length of the RNA guide is 31 nucleotides. In some embodiments, the direct repeat length of the RNA guide is at least 21 nucleotides, or is between 21 and 37 nucleotides (e.g., 23, 24, 25, 30, 35, or 36 nucleotides), hi some embodiments, the direct repeat length of the RNA guide is 23 nucleotides.

[0170] RNA guide sequences may be modified in a way that allows for successful CRISPR effector complex formation and target binding, but simultaneously disallows successful nuclease activity (i.e., no nuclease activity / no indels). Such modified guide sequences are referred to as "dead guides" or "dead guide sequences." Such dead guides or dead guide sequences may be catalytically inactive or conformationally inactive with respect to nuclease activity. Dead guide sequences are typically shorter than the respective guide sequences that result in active RNA cleavage. In some embodiments, dead guides are 5%, 10%, 20%, 30%, 40%, or 50% shorter than the respective RNA guides that have nuclease activity. The dead guide sequences of an RNA guide may be 13-15 nucleotides in length (e.g., 13, 14, or 15 nucleotides in length), 15-19 nucleotides in length, or 17-18 nucleotides in length (e.g., 17 nucleotides in length).

[0171] Accordingly, in one aspect, the present disclosure provides a non-naturally occurring or engineered CRISPR system comprising a functional CRISPR enzyme as described herein and an RNA guide (gRNA), wherein the gRNA comprises a dead guide sequence such that the gRNA is capable of hybridizing to a target sequence such that the CRISPR system is directed to a genomic locus of interest in a cell without detectable cleavage activity.

[0172] A detailed description of dead guides is provided, for example, in International Publication No. WO2016094872 (incorporated herein by reference in its entirety).

[0173] Navigational Guide The RNA guide can be made as a component of an inducible system. The inducible nature of this system allows for spatiotemporal control of gene editing or gene expression. In some embodiments, the stimulus for the inducible system can include, for example, electromagnetic radiation, acoustic energy, chemical energy, and / or thermal energy.

[0174] In some embodiments, transcription of the RNA guide can be regulated by an inducible promoter, such as tetracycline- or doxycycline-controlled transcriptional activation (Tet-On and Tet-Off expression systems), hormone-inducible gene expression systems (e.g., ecdysone-inducible gene expression systems), and arabinose-inducible gene expression systems. Other examples of inducible systems include, for example, small molecule two-hybrid transcription activation systems (FKBP, ABA, etc.), light-inducible systems (phytochrome, LOV domain, or cryptochrome), or light-inducible transcription effectors (LITEs). These inducible systems are described, for example, in WO2016205764 and U.S. Pat. No. 8,795,965, both of which are incorporated herein by reference in their entireties.

[0175] chemical modification Chemical modifications can be applied to the phosphate backbone, sugar, and / or base of the RNA guide. Backbone modifications such as phosphorothioates modify the charge on the phosphate backbone and aid in oligonucleotide delivery and nuclease resistance (see, e.g., Eckstein, "Phosphorothioates, essential components of therapeutic oligonucleotides," Nucl. Acid Ther., 24 (2014), pp. 374-387); sugar modifications such as 2'-O-methyl (2'-OMe), 2'-F, and locked nucleic acid (LNA) enhance both base pairing and nuclease resistance (see, e.g., Allerson et al., "Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA," J. Med. Chem., 48.4 (2005): 901-904). Chemically modified bases, such as 2-thiouridine or N6-methyladenosine, among others, can allow for either stronger or weaker base pairing (see, e.g., Bramsen et al., "Development of therapeutic-grade small interfering RNAs by chemical engineering," Front. Genet., 2012 Aug. 20;3:154). In addition, RNA is suitable for conjugation at both the 5' and 3' ends with various functional moieties, including fluorescent dyes, polyethylene glycol, or proteins.

[0176] A wide variety of modifications can be applied to chemically synthesized RNA guide molecules. For example, oligonucleotides can be modified with 2'-OMe to improve nuclease resistance and alter the binding energy of Watson-Crick base pairing. Furthermore, 2'-OMe modifications can affect how the oligonucleotide interacts with transfection reagents, proteins, or any other molecules in the cell. The effects of these modifications can be determined through empirical testing.

[0177] In some embodiments, the RNA guide comprises one or more phosphorothioate modifications, hi some embodiments, the RNA guide comprises one or more locked nucleic acids for the purposes of enhancing base pairing and / or increasing nuclease resistance.

[0178] For an overview of these chemical modifications, see, for example, Kelley et al., "Versatility of chemically synthesized guides." See, for example, “RNAs for CRISPR-Cas9 genome editing,” J. Biotechnol. 2016 Sep 10;233:74-83; International Publication No. WO 2016205764; and U.S. Patent No. 8,795,965 B2 (each of which is incorporated by reference in its entirety).

[0179] Array Modification The sequences and lengths of the RNA guides and crRNAs described herein can be optimized. In some embodiments, the optimized length of the RNA guide may be determined by identifying the processed form of the crRNA or by empirical length studies of the RNA guide of the crRNA.

[0180] The RNA guide can also include one or more aptamer sequences. Aptamers are oligonucleotide or peptide molecules capable of binding to specific target molecules. Aptamers may be specific for gene effectors, gene activators, or gene repressors. In some embodiments, the aptamer may be specific for a protein that, in turn, is specific for and recruits / binds to a specific gene effector, gene activator, or gene repressor. The effector, activator, or repressor can exist in the form of a fusion protein. In some embodiments, the RNA guide has two or more aptamer sequences specific for the same adaptor protein. In some embodiments, the two or more aptamer sequences are specific for different adaptor proteins. Examples of adaptor proteins include MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1. Thus, in some embodiments, the aptamer is selected from binding proteins that specifically bind to any one of the adaptor proteins described herein. In some embodiments, the aptamer sequence is the MS2 loop. For a detailed description of aptamers, see, for example, Nowak et al., "Guide RNA engineering for versatile Cas9 functionality," Nucl. Acid. Res., 2016 Nov 16;44(20):9555-9564; and WO 2016205764 (which are incorporated by reference in their entireties).

[0181] Guide: Target sequence match requirements In classical CRISPR systems, the degree of complementarity between a guide sequence and its corresponding target sequence may be about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%. In some embodiments, the degree of complementarity is 100%. The RNA guide may be 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.

[0182] To reduce off-target interactions, e.g., to reduce guide interactions with target sequences with low complementarity, mutations can be introduced into the CRISPR system to enable the CRISPR system to distinguish between target sequences and off-target sequences with greater than 80%, 85%, 90%, or 95% complementarity. In some embodiments, the degree of complementarity is 80% to 95%, e.g., about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% (e.g., to distinguish between a target with 18 nucleotides and an 18 nucleotide off-target with 1, 2, or 3 mismatches). Thus, in some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9%, hi some embodiments, the degree of complementarity is 100%.

[0183] It is known in the art that perfect complementarity is not a requirement, as long as there is sufficient complementarity to be functional. The introduction of mismatches, for example, one or more mismatches, for example, one or two mismatches, between the spacer sequence and the target sequence, including the location of the mismatch along the spacer / target, can be used to adjust the cleavage efficiency. The more centrally located the mismatch, for example, a double mismatch (i.e., not at the 3' or 5' end), the greater the impact on cleavage efficiency. Thus, the selection of the mismatch location along the spacer sequence can adjust the cleavage efficiency. For example, if less than 100% cleavage of the target is desired (e.g., in a cell population), one or two mismatches between the spacer and the target sequence may be introduced into the spacer sequence.

[0184] Optimizing CRISPR systems for use in the organism of choice Codon optimization The present invention contemplates all possible variations of nucleic acids, such as cDNAs, that can be created by selecting combinations based on possible codon choices. These combinations are created according to the standard triplet genetic code as applied to polynucleotides encoding naturally occurring variants, and all such variations should be considered specifically disclosed. Nucleotide sequences encoding Type VI CRISPR-Cas-associated effector protein variants that are codon-optimized for expression in bacteria (e.g., E. coli) and human cells are disclosed herein. For example, sequences codon-optimized for human cells can be created by substituting codons that occur frequently in human cells for codons in the nucleotide sequence that occur less frequently in human cells. Codon frequency can be determined computationally using methods known in the art. Examples of such codon frequency calculations for various host cells (e.g., E. coli, yeast, insect, C. elegans, D. melanogaster, human, mouse, rat, pig, P. pastoris, A. thalian, corn, and tobacco) are published or made available through sources such as the GenScript® Codon Usage Table Tool (included below are exemplary codon usage tables for E. coli and human).

[0185] [Table 1]

[0186] [Table 2]

[0187] How to use the CRISPR system The CRISPR system described herein has a wide variety of utilities, including modification (e.g., deletion, insertion, rearrangement, inactivation, or activation) of target polynucleotides in numerous cell types. The CRISPR system has a wide range of applications, for example, in DNA / RNA detection (e.g., specific high-sensitivity enzymatic reporter unlocking (SHERLOCK)), nucleic acid tracking and labeling, enrichment assays (extraction of desired sequences from background), detection of circulating tumor DNA, next-generation library preparation, drug screening, disease diagnosis and prognosis, and treatment of various genetic disorders. Without wishing to be bound by any particular theory, CRISPR systems containing Cas12i proteins may exhibit increased activity or may be preferentially active when targeting in specific environments, such as DNA plasmids, supercoiled DNA, or transcriptionally active genomic loci.

[0188] Introduction to genome editing systems The term "genome editing system" refers to an engineered CRISPR system of the present disclosure that has RNA-guided DNA editing activity. The genome editing system of the present disclosure includes at least two components of the CRISPR system described above: an RNA guide and a cognate CRISPR effector protein. In certain embodiments of the present disclosure, the effector is a Cas12i protein, and the RNA guide is a cognate type VI RNA guide. As described above, these two components form a complex that associates with a specific nucleic acid sequence and has the ability to edit DNA by creating one or more of, for example, a single-strand break (SSB or nick), a double-strand break (DSB), a nucleobase modification, DNA methylation or demethylation, a chromatin modification, etc., within or around the nucleic acid sequence of interest.

[0189] In certain embodiments, the genome editing system is transiently active (e.g., incorporates an inducible CRISPR effector as discussed above), while in other embodiments, the system is constitutive (e.g., is encoded by a nucleic acid in which one or more strong promoters control the expression of the CRISPR system components).

[0190] The genome editing system of the present disclosure, upon introduction into a cell, may modify (a) endogenous genomic DNA (gDNA), including, without limitation, DNA encoding a gene target of interest, exon sequences of a gene, intron sequences of a gene, regulatory elements of a gene or genes, etc.; (b) DNA outside the endogenous genome, such as mitochondrial DNA (mtDNA); and / or (c) exogenous DNA, such as non-integrated viral genomes, plasmids, artificial chromosomes, etc. Throughout this disclosure, these DNA substrates are referred to as "target DNA."

[0191] In instances where genome editing works by generating SSBs or DSBs, the modifications made by the system can take the form of short DNA insertions or deletions, collectively referred to as "indels." These indels may form within or near predicted cleavage sites, typically adjacent to the PAM sequence and / or within the complementary region of the spacer sequence, although in some cases indels may appear outside of such predicted cleavage sites. Without wishing to be bound by any theory, indels are thought to often be the result of repair of SSBs or DSBs by "error-prone" DNA damage repair pathways, such as non-homologous end joining (NHEJ).

[0192] In some cases, genome editing is used to create two DSBs within 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, or 2000 base pairs of each other, which can have one or more consequences, including the formation of indels at one or both break sites, and deletion or inversion of DNA sequences located between the DSBs.

[0193] Alternatively, the genome editing system of the present disclosure may modify the target DNA through the integration of new sequences. These new sequences may differ from existing sequences in the target DNA (as a non-limiting example, integrated by NHEJ via blunt-end ligation) or may correspond to a DNA template that has one or more regions homologous to the targeted DNA. The integration of a homologous template sequence is also referred to as "homologous recombination repair" or "HDR." The template DNA for HDR may be endogenous to the cell, including, without limitation, in the form of a homologous sequence located on another copy of the same chromosome as the target DNA, a homologous sequence from the same gene cluster as the target DNA, etc. Alternatively, or in addition, the template DNA may be provided exogenously, including, without limitation, as free linear or circular DNA, as DNA bound (covalently or non-covalently) to one or more genome editing system components, or as part of a vector genome.

[0194] In some cases, editing occurs via the transcription factor cleavage pathway, as described by Matthew H. Larson et al., “CRISPR interference (CRISPRi) for sequence-specific control of gene expression,” Nature This includes transient or permanent silencing of genes by CRISPR-mediated interference, as described by J. Med. Proc. Natl. Acad. Sci. USA 8, 2180-2196 (2013), which is incorporated by reference in its entirety and for all purposes.

[0195] Genome editing systems may include other components, including, without limitation, one or more heterologous functional domains that mediate site-specific nucleobase modification, DNA methylation or demethylation, or chromatin modification. In some cases, the heterologous functional domain is covalently linked to a CRISPR-associated protein, such as Cas12i, for example, by a direct peptide bond or an intervening peptide linker. This type of fusion is described in more detail below. In some embodiments, the heterologous functional domain is covalently linked to the crRNA, for example, by chemical crosslinking. And in some embodiments, one or more functional groups may be non-covalently associated with the CRISPR-associated protein and / or crRNA. This is variously accomplished by aptamers appended to the crRNA and / or the heterologous functional group, peptide motifs fused to the CRISPR-associated protein and binding domains configured to bind such motifs fused to the heterologous functional domain, or vice versa.

[0196] Genome editing system design and genome editing results are described in more detail elsewhere herein.

[0197] DNA / RNA detection In one embodiment, the CRISPR-Cas system described herein can be used in DNA / RNA detection by DNA sensing.By reprogramming single-effector RNA-guided DNase with RNA guide, it can provide a platform for specific single-stranded DNA (ssDNA) sensing.When recognizing its DNA target, activated CRISPR type VI effector protein is involved in "collateral" cleavage of neighboring ssDNA that does not have sequence similarity with the target sequence.This collateral cleavage activity programmed by RNA allows the CRISPR system to detect the presence of specific DNA through non-specific degradation of labeled ssDNA.

[0198] In DNA detection applications, collateral ssDNase activity can be combined with a reporter, for example, a DNA Endonuclease-Targeted CRISPR trans reporter. These include methods known as "detector (DETECTR)" that, when combined with amplification, achieve attomolar DNA detection sensitivity (see, e.g., Chen et al., Science, 360(6387):436-439, 2018, which is incorporated herein by reference in its entirety). One application using the enzymes described herein is the degradation of non-target ssDNA in an in vitro environment. A "reporter" ssDNA molecule linked to a fluorophore and a quencher can also be added to this in vitro system along with an unknown DNA sample (either single-stranded or double-stranded). Upon recognition of the target sequence in the unknown piece of DNA, the surveillance complex, which contains a type VI effector, cleaves the reporter ssDNA, producing a fluorescent readout.

[0199] In another embodiment, the SHERLOCK method (Specific High Sensitivity Enzymatic Reporter Unlocking) also provides an in vitro nucleic acid detection platform with attomolar (or single molecule) sensitivity based on nucleic acid amplification and collateral cleavage of a reporter ssDNA, enabling real-time detection of targets. The use of CRISPR in SHERLOCK is described in detail, for example, in Gootenberg, et al. "Nucleic acid detection with CRISPR-Cas13a / C2c2," Science, 356(6336):438-442(2017), which is incorporated herein by reference in its entirety.

[0200] In some embodiments, the CRISPR systems described herein can be used in multiplexed error-robust fluorescence in situ hybridization (MERFISH). Such methods are described, for example, in Chen et al., "Spatially resolved, highly multiplexed RNA profiling in single cells,” Science, 2015 Apr 24;348(6233):aaa6090, which is incorporated herein by reference in its entirety.

[0201] In some embodiments, the CRISPR systems described herein can be used to detect target DNA in a sample (e.g., a clinical sample, cells, or cell lysate). The collateral DNase activity of the CLUST.029130 (Type VI) CRISPR-Cas effector protein described herein is activated when the effector protein binds to a target nucleic acid. Upon binding to the target DNA of interest, the effector protein cleaves the labeled detector ssDNA, generating or altering a signal (e.g., an increase or decrease in signal), thereby enabling qualitative and quantitative detection of target DNA in a sample. Specific detection and quantification of DNA in a sample enables numerous applications, including diagnostic methods.

[0202] In some embodiments, the method includes a) contacting a sample with (i) an RNA guide (e.g., crRNA) and / or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence capable of hybridizing to a target RNA; (ii) a CLUST.029130 (Type VI) CRISPR-Cas effector protein and / or a nucleic acid encoding the effector protein; and (iii) a labeled detector ssDNA; wherein the effector protein associates with the RNA guide to form a surveillance complex; the surveillance complex hybridizes to the target DNA; and upon binding of the surveillance complex to the target DNA, the effector protein exhibits collateral DNase activity and cleaves the labeled detector ssDNA; and b) measuring a detectable signal generated by cleavage of the labeled detector ssDNA, wherein said measuring provides for detection of the target DNA in the sample.

[0203] In some embodiments, the method further includes comparing the detectable signal with a reference signal and determining the amount of target DNA in the sample. In some embodiments, the measurement is performed using gold nanoparticle detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection, and semiconductor-based sensing. In some embodiments, the labeled detector ssDNA comprises a fluorescent dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher / fluorophore pair. In some embodiments, when the labeled detector ssDNA is cleaved by the effector protein, the amount of detectable signal generated by the labeled detector ssDNA decreases or increases. In some embodiments, the labeled detector ssDNA generates a first detectable signal before cleavage by the effector protein and a second detectable signal after cleavage by the effector protein.

[0204] In some embodiments, a detectable signal is generated when the labeled detector ssDNA is cleaved by the effector protein. In some embodiments, the labeled detector ssDNA comprises a modified nucleobase, a modified sugar moiety, a modified nucleobase linkage, or a combination thereof.

[0205] In some embodiments, the methods involve multichannel detection of multiple independent target DNAs (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or more target RNAs) in a sample by using multiple CLUST.029130 (Type VI) CRISPR-Cas systems, each containing a different orthologous effector protein and corresponding RNA guide, thereby enabling differentiation of the multiple target DNAs in the sample. In some embodiments, the methods involve multichannel detection of multiple independent target DNAs in a sample by using multiple instances of CLUST.029130 (Type VI) CRISPR-Cas systems, each containing orthologous effector proteins with distinct collateral ssDNase substrates. Methods for detecting DNA in a sample using CRISPR-associated proteins are described, for example, in U.S. Patent Application Publication No. 2017 / 0362644, the entire contents of which are incorporated herein by reference.

[0206] Nucleic Acid Tracking and Labeling Cellular processes depend on a network of molecular interactions between proteins, RNA, and DNA. Accurate detection of protein-DNA and protein-RNA interactions is key to understanding such processes. In vitro proximity labeling techniques use affinity tags combined with reporter groups, such as photoactivatable groups, to label polypeptides and DNA in vitro near a protein or DNA of interest. After ultraviolet irradiation, the photoactivatable groups react with proteins and other molecules in close proximity to the tagged molecule, thereby labeling them. The labeled interacting molecules can then be recovered and identified. This DNA-targeting effector protein can be used, for example, to target probes to selected DNA sequences. Such applications can also be applied to in vivo imaging of disease or difficult-to-culture cell types in animal models. Methods for tracking and labeling nucleic acids are described, for example, in U.S. Pat. No. 8,795,965; WO 2016205764; and WO 2017070605 (each of which is incorporated by reference in its entirety).

[0207] Genome editing using paired CRISPR nickases The CRISPR system described herein can be used in tandem to generate double-stranded breaks with overhangs using two Cas12i nicking enzymes, or one Cas12i enzyme and one other CRISPR Cas enzyme with nicking activity, targeted to opposite strands of a target locus by a pair of RNA guides. This method is expected to generate double-stranded breaks only at loci where both enzymes generate nicks, reducing the likelihood of off-target modifications and thus increasing the specificity of genome editing. This method is referred to as the "double nicking" or "paired nickase" strategy and is described, for example, in Ran et al., "Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity," Cell, 2013 Sep 12;154(6):1380-1389, and Mali et al., "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering," Nature Biotechnology, 2013 Aug 01;31:833-838 (both of which are incorporated herein by reference in their entireties).

[0208] The first application of paired nickases demonstrated the utility of this strategy in mammalian cell lines. The application of paired nickases has also been demonstrated in the model plant Arabidopsis thaliana (e.g., Fauser et al., "Both CRISPR / Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana," The Plant Journal 79(2):348-59 (2014) and Shiml et al., "The CRISPR / Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny,” The Plant Journal 80(6):1139-50(2014); in crops such as rice (e.g., Mikami et al., “Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice,”Plant and Cell Physiology 57(5):1058-68(2016) and wheat (e.g., Czermak et al., "A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants,”Plant Cell 29:1196-1217(2017); Genomes Using CRISPR-Nickases,” ACS Synthetic Biology 4(11):1217-25 (2015); and in primary human cells for therapeutic purposes (e.g., Dabrowska et al., “Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases,” Frontiers in Neuroscience 12:75 (2018), and Kocher et al., “Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR / Cas9 Nickases,” Molecular Therapy 25(11):2585-2598 (2017)), all of which are incorporated herein by reference in their entireties.

[0209] The CRISPR systems described herein can also be used as paired nickases to detect splice junctions, as described, for example, in Santo & Paik, “A splice junction-targeted CRISPR approach (spJCRISPR) reveals human FOXO3B to be a protein-coding gene,” Gene 673:95-101 (2018).

[0210] The CRISPR systems described herein may also be used in conjunction with other methods, such as those described in, for example, Wang et al., "Therapeutic Genome Editing for Myotonic The CRISPR system described herein can also be used as a paired nickase to insert DNA molecules into target loci, as described in, for example, Gao et al., "Single Cas9 nickase-induced generation of NRAMP1 knockin cattle with reduced expression," Molecular Therapy 26(11):2617-2630 (2018). It can also be used as a single nickase to insert genes, as described in “Off-target effects,” Genome Biology 18(1):13 (2017).

[0211] Enhanced base editing using CRISPR nickases The CRISPR system described herein can be used to enhance the efficiency of CRISPR base editing. In base editing, a protein domain with DNA nucleotide modifying activity (e.g., cytidine deamination) is fused to a programmable CRISPR Cas enzyme that has been inactivated by mutation so that it no longer has double-stranded DNA cleavage activity. In some embodiments, using a nickase as the programmable Cas protein can enhance the efficiency of CRISPR base editing, as described, for example, in Komor et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature 533:420-424 (2016) and Nishida et al., "Targeted nucleotide editing using hybrid As described in “Prokaryotic and vertebrate adaptive immune systems,” Science 353(6305):aaf8729 (2016) (both of which are incorporated by reference in their entireties), base editing efficiencies have been shown to be improved. It is hypothesized that nickases that nick the non-edited strand of the target locus stimulate endogenous DNA repair pathways—such as mismatch repair or long-patch base excision repair—that preferentially resolve mismatches generated by base editing relative to the desired allele, or that they make the target DNA more accessible to catalytic editing domains.

[0212] Targeted mutagenesis and DNA labeling with nickases and DNA polymerases. The CRISPR systems described herein can be used in conjunction with proteins that act on nicked DNA. One such class of proteins is nick-translation DNA polymerases, such as E. coli DNA polymerase I or Taq DNA polymerase.

[0213] In some embodiments, a CRISPR system (e.g., CRISPR nickase) can be fused to an error-prone DNA polymerase I. This fusion protein can be targeted by an RNA guide to generate a nick at the target DNA site. When the DNA polymerase then initiates DNA synthesis at the nick, downstream nucleotides are substituted and an error-prone polymerase is employed, resulting in mutagenesis of the target locus. Polymerase variants with different processivity, fidelity, and incorporation error bias can be used to affect the properties of the mutants generated. This method, called EvolvR, is described, for example, in Halperin et al., "CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window,” Nature 560, 248-252 (2018), which is incorporated herein by reference in its entirety.

[0214] In some embodiments, CRISPR nickase can be used in nick translation DNA labeling protocols. Nick translation, first described by Rigby et al. in 1977, involves incubating DNA with a DNA nicking enzyme, such as DNase I, which creates one or more nicks in a DNA molecule. Then, a nick translation DNA polymerase, such as DNA polymerase I, is used to incorporate labeled nucleic acid residues at the nicked sites. A method that utilizes the programmability of CRISPR nickase to covalently tag telomere repeat sequences with fluorescent dyes using a variation of the classic nick translation labeling protocol is described in detail, for example, in McCaffery et al., "High-throughput single-molecule telomere characterization," Genome Research 27:1904-1915 (2017) (which is incorporated herein by reference in its entirety). This method achieves haplotype-resolved telomere length analysis at the single molecule level.

[0215] Nucleic Acid Tracking and Labeling Cellular processes depend on a network of molecular interactions between proteins, RNA, and DNA. Accurate detection of protein-DNA and protein-RNA interactions is key to understanding such processes. In vitro proximity labeling techniques use affinity tags combined with reporter groups, such as photoactivatable groups, to label polypeptides and RNAs in vitro near a protein or RNA of interest. After ultraviolet irradiation, the photoactivatable groups react with proteins and other molecules in close proximity to the tagged molecule, thereby labeling them. The labeled interacting molecules can then be recovered and identified. This RNA targeting effector protein can be used, for example, to target probes to selected RNA sequences. Such applications can also be applied to in vivo imaging of disease or difficult-to-culture cell types in animal models. Methods for tracking and labeling nucleic acids are described, for example, in U.S. Pat. No. 8,795,965; WO 2016205764; and WO 2017070605 (each of which is incorporated by reference in its entirety).

[0216] High-throughput screening The CRISPR system described herein can be used to prepare next-generation sequencing (NGS) libraries. For example, to create cost-effective NGS libraries, the CRISPR system can be used to disrupt the coding sequence of target genes, and clones transfected with the CRISPR enzyme can be simultaneously screened by next-generation sequencing (e.g., with the Ion Torrent PGM system). For a detailed description of how to prepare NGS libraries, see, for example, Bell et al., "A high-throughput screening See, for example, “A strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing,” BMC Genomics, 15.1 (2014): 1002, which is incorporated herein by reference in its entirety.

[0217] Engineered Microorganisms Microorganisms (e.g., E. coli, yeast, and microalgae) are widely used in synthetic biology. Advances in synthetic biology have broad utility, including various clinical applications. For example, the programmable CRISPR system described herein can be used to split proteins of toxic domains for targeted cell death, e.g., using cancer-associated RNAs as target transcripts. Furthermore, fusion complexes with appropriate effectors, e.g., kinases or enzymes, can affect pathways involving protein-protein interactions in synthetic biology systems.

[0218] In some embodiments, an RNA guide sequence that targets a phage sequence can be introduced into a microorganism. Accordingly, the present disclosure also provides methods for vaccinating a microorganism (e.g., a production strain) against phage infection.

[0219] In some embodiments, the CRISPR systems provided herein can be used to engineer microorganisms to, for example, improve yields or improve fermentation efficiency. For example, the CRISPR systems described herein can be used to engineer microorganisms such as yeast to produce biofuels or biopolymers from fermentable sugars, or to degrade plant-derived lignocellulose from agricultural waste as a source of fermentable sugars. More specifically, the methods described herein can be used to modify the expression of endogenous genes required for biofuel production and / or to modify endogenous genes that may interfere with biofuel synthesis. These microbial engineering methods are described, for example, in Verwaal et al., "CRISPR / Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae,”Yeast,2017 Sep 8.doi:10.1002 / yea.3278; and Hlavova et al., “Improving microalgae for biotechnology-from Genetics to Synthetic Biology,” Biotechnol. Adv., 2015 Nov 1;33:1194-203 (both of which are incorporated herein by reference in their entireties).

[0220] In some embodiments, the CRISPR system described herein can be used to engineer microorganisms deficient in repair pathways, such as the mesophilic cellulolytic bacterium Clostridium cellulolyticum, a model organism for bioenergy research. In some embodiments, CRISPR nickases can be used to introduce a single nick at a target locus, which may result in insertion by homologous recombination of an exogenously supplied DNA template. For detailed methods on how to use CRISPR nickases to edit repair-deficient microorganisms, see, for example, Xu et al., "Efficient Genome Editing in Clostridium cellulolyticum." via CRISPR-Cas9 Nickase,” Appl Environ Microbiology 81:4423-4431 (2015), which is incorporated herein by reference in its entirety.

[0221] In some embodiments, the CRISPR systems provided herein can be used to induce cell death or dormancy in cells (e.g., microorganisms, such as engineered microorganisms). These methods can be used to induce dormancy or death in numerous cell types, including prokaryotic and eukaryotic cells, including, but not limited to, mammalian cells (e.g., cancer cells or tissue culture cells), protozoa, fungal cells, virus-infected cells, intracellular bacteria-infected cells, intracellular protozoa-infected cells, prion-infected cells, bacteria (e.g., pathogenic and non-pathogenic bacteria), protozoa, and unicellular and multicellular parasites. For example, in the field of synthetic biology, it is highly desirable to have mechanisms to control engineered microorganisms (e.g., bacteria) and prevent their spread or dissemination. The systems described herein can be used as "kill switches" to regulate and / or prevent the spread or dissemination of engineered microorganisms. Furthermore, there is a need in the art for alternatives to current antibiotic treatments.

[0222] The systems described herein can also be used in applications where it is desirable to kill or control a specific microbial population (e.g., bacterial population). For example, the systems described herein can include an RNA guide (e.g., crRNA) that targets a genus-, species-, or strain-specific nucleic acid (e.g., DNA) and delivers it to a cell. Upon complexing and binding to the target nucleic acid, the nuclease activity of the CLUST.029130 (Type VI) CRISPR-Cas effector protein disrupts essential functions within the microorganism, ultimately resulting in dormancy or death. In some embodiments, the method comprises contacting a cell with a system described herein comprising a CLUST.029130 (Type VI) CRISPR-Cas effector protein or a nucleic acid encoding the effector protein, and an RNA guide (e.g., a crRNA) or a nucleic acid encoding an RNA guide, wherein the spacer sequence is complementary to at least 15 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides) of the target nucleic acid.

[0223] Without wishing to be bound by any particular theory, the nuclease activity of CLUST.029130 (Type VI) CRISPR-Cas effector proteins can induce programmed cell death, cytotoxicity, apoptosis, necrosis, necroptosis, cell death, cell cycle arrest, cell anergy, reduced cell growth, or reduced cell proliferation. For example, in bacteria, DNA cleavage by CLUST.029130 (Type VI) CRISPR-Cas effector proteins can be bacteriostatic or bactericidal.

[0224] Applications in plants The CRISPR system described herein has a wide variety of uses in plants. In some embodiments, the CRISPR system can be used to engineer a plant's genome (e.g., to improve production, produce a product with a desired post-translational modification, or introduce a gene to produce an industrial product). In some embodiments, the CRISPR system can be used to introduce a desired trait into a plant (e.g., with or without heritable modifications to the genome) or to regulate the expression of an endogenous gene in a plant cell or whole plant. Plants that can be edited using the CRISPR system (e.g., the Cas12i system) of the present disclosure can be monocotyledonous or dicotyledonous, and include, without limitation, safflower, corn, hemp, rice, sugarcane, canola, sorghum, tobacco, rye, barley, wheat, millet, oat, peanut, potato, switchgrass, turfgrass, soybean, alfalfa, sunflower, cotton, and Arabidopsis. The present disclosure also encompasses plants having traits created by the methods of the present disclosure and / or utilizing the CRISPR systems of the present disclosure.

[0225] In some embodiments, the CRISPR system can be used to identify, edit, and / or silence genes encoding specific proteins, such as allergen proteins (e.g., allergen proteins in peanuts, soybeans, lentils, peas, green beans, and mung beans). Detailed descriptions of methods for identifying, editing, and / or silencing protein-encoding genes are described, for example, in Nicolaou et al., "Molecular diagnosis of peanut and legume allergy," Curr. Opin. Allergy Clin. Immunol., 11(3):222-8 (2011), and WO 2016205764 A1 (both of which are incorporated herein by reference in their entireties).

[0226] gene drive Gene drive is a phenomenon that favors the inheritance of a specific gene or group of genes. Gene drives can be constructed using the CRISPR system described herein. For example, a CRISPR system can be designed to target and destroy a specific allele of a gene, causing cells to copy a second allele and fix the sequence. This copying converts the first allele to the second allele, increasing the likelihood that the second allele will be inherited by offspring. Detailed methods for constructing gene drives using the CRISPR system described herein are described, for example, in Hammond et al., "A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae," Nat. Biotechnol., 2016 Jan;34(1):78-83 (which is incorporated herein by reference in its entirety).

[0227] Pooled Screening As described herein, pooled CRISPR screening is a powerful tool for identifying genes involved in biological mechanisms such as cell proliferation, drug resistance, and viral infection. Cells are bulk-transduced with a library of RNA guide (gRNA)-encoding vectors described herein, and the distribution of gRNAs is measured before and after the application of a selective challenge. Pooled CRISPR screens work well for mechanisms affecting cell survival and proliferation and can be extended to measuring the activity of individual genes (e.g., by using engineered reporter cell lines). Arrayed CRISPR screens, in which only one gene is targeted at a time, allow for the use of RNA-seq as a readout. In some embodiments, the CRISPR system described herein can be used for single-cell CRISPR screens. For a detailed description of pooled CRISPR screening, see, for example, Datlinger et al., "Pooled CRISPR screening with single-cell transcriptome readout," Nat. Methods., 2017 Mar;14(3):297-301, which is incorporated herein by reference in its entirety.

[0228] Saturation mutagenesis ("bashing") The CRISPR system described herein can be used for in situ saturation mutagenesis. In some embodiments, a pooled RNA-guided library can be used to perform in situ saturation mutagenesis of specific genes or regulatory elements. Such methods can reveal the critical minimal features and individual vulnerabilities of those genes or regulatory elements (e.g., enhancers). These methods are described, for example, in Canver et al., "BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis," Nature, 2015 Nov 12;527(7577):192-7 (which is incorporated herein by reference in its entirety).

[0229] Therapeutic Applications CRISPR systems described herein (e.g., Cas12i2) that are active in mammalian cell contexts may have a diverse range of therapeutic applications. Furthermore, ortholog selection is important in assigning nucleases that confer the greatest therapeutic benefit, as each nuclease ortholog may have unique properties (e.g., size, PAM, etc.) that make it advantageous for specific targeting, therapeutic, or delivery modalities.

[0230] Many factors influence the suitability of gene editing as a therapy for a particular disease. In nuclease-based gene therapy, the primary therapeutic editing approaches are considered to be gene disruption and gene correction. In the former, gene disruption generally occurs following an event (e.g., a nuclease-induced targeted double-strand break) that activates the target cell's endogenous non-homologous end-joining DNA repair machinery, resulting in indels that often result in loss-of-function mutations that are intended to benefit the patient. In the latter, gene correction utilizes nuclease activity to induce alternative DNA repair pathways (e.g., homology-directed repair, or HDR) with the aid of template DNA (whether endogenous or exogenous, single-stranded or double-stranded). The templated DNA is either the endogenous correction of a disease-causing mutation or the insertion of a therapeutic transgene into an otherwise selective locus (commonly a safe-harbor locus such as AAVS1). Methods for designing exogenous donor template nucleic acids are described, for example, in International Publication No. WO2016094874 A1, the entire contents of which are expressly incorporated herein by reference. A prerequisite for therapy using any of these editing modalities is an understanding of the genetic regulators of a particular disease; while the disease need not necessarily be a monogenic disease, insight into how mutations can lead to disease progression or outcome is important for providing guidance regarding the potential effectiveness of gene therapy.

[0231] Without wishing to be limited, the CRISPR system described herein can be utilized to treat the following diseases, where specific gene targets are identified, along with relevant references that aid in adapting the Type VI CRISPR system to specific disease areas: cystic fibrosis by targeting CFTR (WO2015157070A2), Duchenne muscular dystrophy and Becker muscular dystrophy by targeting dystrophin (DMD) (WO2016161380A1), alpha-1-antitrypsin deficiency by targeting alpha-1-antitrypsin (A1AT) (WO2016161380A1), and alpha-1-antitrypsin deficiency by targeting alpha-1-antitrypsin (A1AT) (WO2016161380A1). Publication No. 2017165862A1), lysosomal storage disorders such as Pompe disease, also known as glycogen storage disease type II, by targeting acid alpha-glucosidase (GAA), myotonic dystrophy by targeting DMPK, Huntington's disease by targeting HTT, fragile X by targeting FMR1, Friedreich's ataxia by targeting frataxin, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) by targeting C9orf72, inherited chronic kidney disease by targeting ApoL1, and cardiovascular disease and hyperlipidemia by targeting PCSK9, APOC3, ANGPTL3, and LPA (Nature 555,S23-S25(2018)), and congenital blindness such as Leber congenital amaurosis type 10 (LCA10) by targeting CEP290 (Maeder et al., Nat Med. 2019 Feb;25(2):229-233). The majority of the aforementioned diseases are best treated with in vivo gene editing approaches, in which the cell types and tissues involved in the disease must be edited in situ at a dose and efficiency sufficient to produce a therapeutic benefit. While some challenges of in vivo delivery are discussed in the "Delivery of CRISPR Systems" section below, in general, the small gene size of Type VI CRISPR effectors allows for more versatile packaging into payload-limited viral vectors, such as adeno-associated viruses.

[0232] Ex vivo editing, in which cells are removed from a patient's body, then edited, and then transplanted back into the patient, represents a valuable therapeutic opportunity for gene editing technology. The ability to manipulate cells outside the body offers many advantages, ranging from the ability to use techniques that are not suitable for in vivo contexts, such as electroporation and nucleofection, to deliver proteins, DNA, and RNA to cells with high efficiency, to determining toxicity (e.g., from off-target effects), and then successfully selecting and expanding edited cells to produce populations that provide therapeutic benefits. These advantages are offset by the relatively limited number of cell types and populations that can be successfully recovered, processed, and then returned to the body while maintaining functionality. Without wishing to be limited, there are nonetheless serious diseases that are suitable for ex vivo genome editing using the system described herein. For example, sickle cell disease (SCD), as referenced in WO 2015148863A2, and β-thalassemia, as referenced in WO 2015148860A1, are both examples of diseases where understanding of the pathophysiology has enabled several different editing modalities in hematopoietic stem cells for disease treatment. Both β-thalassemia and SCD can be treated by increasing fetal hemoglobin levels through disruption of the BCL11A erythroid enhancer (Psatha et al. Mol Ther Methods (As exemplified by Clin Dev. 2018 Sep 21 using zinc finger nucleases.) Additionally, gene correction methods can be used to revert deleterious mutations in SCD and beta thalassemia. In another example, the addition of beta globin expressed from a safe harbor locus provides another alternative therapeutic strategy to ex vivo gene editing.

[0233] As a corollary to ex vivo editing of hematopoietic stem cells, immune cells can also be edited. In cancer immunotherapy, one treatment modality is to modify immune cells, such as T cells, to recognize and combat cancer, as seen in International Publication No. WO 2015161276A2. To reduce costs while increasing efficacy and accessibility, the creation of "off-the-shelf" allogeneic T cell therapies is attractive, and gene editing has the potential to modify surface antigens to minimize any immunological side effects (Jung et al., Mol Cell. 2018 Aug 31).

[0234] In another embodiment, the present invention is used to target viruses or other pathogens at the double-stranded DNA intermediate stage of their life cycle. Specifically, targeting viruses that persist from initial infection to permanently persistent latent infection can have great therapeutic value. In the following examples, as referenced in International Publication Nos. 2015153789A1, 2015153791A1, and 2017075475A1 for HSV-1 and HSV-2, and International Publication Nos. 2015148670A1 and 2016183236A1 for HIV, the type VI CRISPR system can be used to directly target viral genomes (such as those associated with HSV-1, HSV-2, or HIV), or can be used to edit host cells to reduce or eliminate receptors that enable infection, making them resistant to viruses (HIV).

[0235] In another embodiment, the CRISPR systems described herein can be engineered to utilize the enzymatically inactive Cas12i as a platform onto which protein domains can be attached to achieve additional functions that can confer activities such as transcriptional activation, repression, base editing, and methylation / demethylation.

[0236] Accordingly, the present disclosure provides CRISPR-Cas systems and cells for use in the treatment or prevention of any of the diseases disclosed herein.

[0237] Delivery of the CRISPR system The CRISPR systems described herein, or components thereof, nucleic acid molecules thereof, or nucleic acid molecules encoding or providing such components, can be delivered by various delivery systems, such as vectors, e.g., plasmids, viral delivery vectors, e.g., adeno-associated viruses (AAV), lentiviruses, adenoviruses, and other viral vectors, or by methods such as nucleofection or electroporation of a ribonucleoprotein complex consisting of a type VI effector and its one or more cognate RNA guides. The protein and one or more RNA guides can be packaged into one or more vectors, e.g., plasmids or viral vectors. For bacterial applications, nucleic acids encoding any of the components of the CRISPR systems described herein can be delivered to bacteria using phages. Exemplary phages include, but are not limited to, T4 phage, Mu, λ phage, T5 phage, T7 phage, T3 phage, φ29, M13, MS2, Qβ, and φX174.

[0238] In some embodiments, vectors, such as plasmids or viral vectors, are delivered to the target tissue by, for example, intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration. Such delivery may be by single dose or multiple doses. Those skilled in the art will understand that the actual dosage delivered herein may vary widely depending on a variety of factors, including the choice of vector, the target cell, organism, tissue, the general condition of the subject being treated, the degree of transformation / modification desired, the route and mode of administration, and the type of transformation / modification desired.

[0239] In certain embodiments, delivery is by adeno-associated virus (AAV), e.g., AAV2, AAV8, or AAV9, which is at least 1×10 5A single dose containing particles (also referred to as particle units, pu) of adenovirus or adeno-associated virus can be administered. In some embodiments, the dose is at least about 1 x 10 6 particles, at least about 1 x 10 7 particles, at least about 1 x 10 8 particles, or at least about 1 x 10 9 The adeno-associated virus is a particle. Delivery methods and dosages are described, for example, in International Publication No. 2016205764 and U.S. Patent No. 8,454,972 (both of which are incorporated herein by reference in their entirety). Because the genomic payload of recombinant AAV is limited, the small size of the type VI CRISP-Cas effector protein described herein allows for greater versatility in packaging effectors and RNA guides with appropriate regulatory sequences (e.g., promoters) required for efficient and cell-type-specific expression.

[0240] In some embodiments, delivery is by recombinant adeno-associated virus (rAAV) vector. For example, in some embodiments, modified AAV vectors may be used for delivery. Modified AAV vectors may be based on one or more of several capsid types, including AAV1, AV2, AAV5, AAV6, AAV8, AAV8.2, AAV9, AAV rhlO, modified AAV vectors (e.g., modified AAV2, modified AAV3, modified AAV6) and pseudotyped AAV (e.g., AAV2 / 8, AAV2 / 5 and AAV2 / 6). Exemplary AAV vectors and techniques that can be used to generate rAAV particles are known in the art (see, e.g., Aponte-Ubillus et al. (2018) Appl. Microbiol. Biotechnol. 102(3):1045-54; Zhong et al. (2012) J. Genet. Syndr. Gene Ther. S1:008; West et al. (1987) Virology 160:38-47(1987); Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-60; U.S. Pat. Nos. 4,797,368 and 5,173,414; and WO 2015 / 054653 and WO 93 / 24641, each of which is incorporated by reference).

[0241] In some embodiments, delivery is via a plasmid. The dosage can be a sufficient number of plasmids to elicit a response. In some cases, a suitable amount of plasmid DNA in a plasmid composition can be about 0.1 to about 2 mg. The plasmid will generally include: (i) a promoter; (ii) a sequence encoding a nucleic acid-targeting CRISPR enzyme operably linked to the promoter; (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii). The plasmid can also encode the RNA components of the CRISPR-Cas system, although alternatively, one or more of these may be encoded on a different vector. The frequency of administration is within the purview of a medical or veterinary practitioner (e.g., physician, veterinarian) or skilled in the art.

[0242] In another embodiment, delivery is via liposome or lipofectin formulations, etc., which can be prepared by methods known to those skilled in the art, such as those described in WO2016205764 and U.S. Patent Nos. 5,593,972; 5,589,466; and 5,580,859, each of which is incorporated by reference herein in its entirety.

[0243] In some embodiments, delivery is by nanoparticles or exosomes. For example, exosomes have been shown to be particularly useful for delivery of RNA.

[0244] An additional means of introducing one or more components of this novel CRISPR system into cells is through the use of cell-penetrating peptides (CPPs). In some embodiments, the cell-penetrating peptide is linked to a CRISPR enzyme. In some embodiments, the CRISPR enzyme and / or RNA guides are coupled to one or more CPPs, effectively transporting them into the cell interior (e.g., plant protoplasts). In some embodiments, the CRISPR enzyme and / or one or more RNA guides are encoded by one or more circular or non-circular DNA molecules that are coupled to one or more CPPs for cellular delivery.

[0245] CPPs are short peptides of less than 35 amino acids derived from either proteins or chimeric sequences that have the ability to transport biomolecules across cell membranes in a receptor-independent manner. CPPs can be cationic peptides, peptides with hydrophobic sequences, amphipathic peptides, peptides with proline-rich antimicrobial sequences, and chimeric or bipartite peptides. Examples of CPPs include Tat (a nuclear transcription activator protein required for HIV type 1 viral replication), penetratin, Kaposi's fibroblast growth factor (FGF) signal peptide sequence, integrin β3 signal peptide sequence, polyarginine peptide Arg sequence, guanine-rich molecular transporter, and sweet arrow peptide. CPPs and methods for their use are described, for example, in Haellbrink et al., "Prediction of cell-penetrating peptides," Methods Mol. Biol., 2015;1324:39-58; Ramakrishna et al., "Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA," Genome Res., 2014 Jun;24(6):1020-7; and WO 2016205764 A1 (each of which is incorporated by reference herein in its entirety).

[0246] Delivery of the Type VI CRISPR system as a ribonucleoprotein complex by electroporation or nucleofection, in which purified Cas12i protein is pre-incubated with an RNA guide and electroporated (or nucleofected) into the cells of interest, is another method to efficiently introduce the CRISPR system into cells for gene editing. This is particularly useful for ex vivo genome editing and the development of cell therapies; such methods are described in detail in Roth et al., "Reprogramming human T cell function and specificity with non-viral Genome targeting,” Nature, 2018 Jul;559(7714):405-409.

[0247] Various delivery methods for the CRISPR systems described herein are also described in, for example, U.S. Pat. No. 8,795,965, EP 3,009,511, WO 2016205764, and WO 2017070605, each of which is herein incorporated by reference in its entirety.

[0248] kit The present disclosure also encompasses kits for carrying out various methods of the present disclosure that utilize the CRISPR system described herein. One exemplary kit of the present disclosure includes (a) one or more nucleic acids encoding a CRISPR-associated protein and a cognate crRNA, and / or (b) a ribonucleoprotein complex of a CRISPR-associated protein and a cognate crRNA. In some embodiments, the kit includes a Cas12i protein and a Cas12i guide RNA. As described above, the complex of the protein and the guide RNA has editing activity, such as single-stranded subunit (SSB) formation, double-stranded subunit (DSB) formation, CRISPR interference, nucleobase modification, DNA methylation or demethylation, or chromatin modification. In certain embodiments, the CRISPR-associated protein is a mutant, such as one with reduced endonuclease activity.

[0249] Kits of the present disclosure also optionally include additional reagents, including one or more of a reaction buffer, a wash buffer, one or more control materials (e.g., a substrate or a nucleic acid encoding a CRISPR system component), etc. Kits of the present disclosure also optionally include instructions for practicing the methods of the present disclosure using the materials provided in the kit. The instructions may be provided in physical form, e.g., as printed documents physically packaged with other items of the kit, and / or in digital form, e.g., as digitally published documents downloadable from a website or provided on a computer-readable medium. [Example]

[0250] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0251] Example 1: Identification of the minimal components of the CLUST.029130 (Type VI) CRISPR-Cas system (Figures 1-3) This protein family represents large single effectors associated with CRISPR systems found in uncultured metagenomic sequences from freshwater environments (Table 3). The CLUST.029130 (Type VI) effector, designated Cas12i, includes exemplary proteins detailed in Tables 3 and 4. Exemplary direct repeat sequences for these systems are shown in Table 5.

[0252] NCBI(Benson et al.(2013)GenBank.Nucleic Acids Res.41,D36-42;Pruitt et al.(2012)NCBI Reference Sequences(RefSeq):current status, new features and genome annotation policy. Nucleic Acids Res. 40, D130-135), NCBI whole genome sequencing (WGS), and DOE JGI Integrated Microbial Genomes (Markowitz et al. (2012) IMG: the Integrated Microbial By downloading and aggregating genome and metagenomic sequences from the Genomes database and comparative analysis system (Nucleic Acids Res. 40, D115-122), we constructed a database of 293,985 putative CRISPR-Cas systems, within which we identified novel nuclease systems. This pipeline engineering approach expands the search space for novel CRISPR effector discovery and reduces bias by implementing minimal filtering at intermediate stages.

[0253] The classification tree shown in Figure 1A-1B was constructed by comparing sequence profiles extracted from multiple alignments of easily alignable Cas12 proteins. Profile comparisons were performed using HHsearch (Soeding et al. (2005) Protein homology detection by HMM-HMM comparison. Bioinforma. Oxf. Engl. 21, 951-960); the scores between two profiles were normalized by the minimum of their own scores and converted to a distance matrix on a natural logarithmic scale. From this distance matrix, a UPGMA dendrogram was reconstructed. The tree at a depth of two distance units (e compared to the own scores) was then reconstructed. -2D = 0.02), but typically reliably recovers profile similarity and can serve as a guide for subtype classification ( Shmakov et al., 2017 ).

[0254] The domain organization of Cas12i, shown in Figures 2A and 2B, indicates that the effector contains the active catalytic residues of the RuvC nuclease domain. Additionally, the predicted secondary structures of the most frequent direct repeats for type VI loci, shown in Figure 3, indicate a stem-loop structure that is conserved in the crRNAs of many exemplary type VI CRISPR-Cas systems.

[0255] [Table 3]

[0256] [Table 4-1]

[0257] [Table 4-2]

[0258] [Table 4-3]

[0259] [Table 4-4]

[0260] [Table 4-5]

[0261] [Table 4-6]

[0262] [Table 4-7]

[0263] [Table 4-8]

[0264] [Table 4-9]

[0265] [Table 4-10]

[0266] [Table 5A]

[0267] [Table 5B-1]

[0268] [Table 5B-2]

[0269] Example 2: In vivo bacterial validation of the engineered CLUST.029130 (Type VI) CRISPR-Cas system (Figures 4A-10B) Having identified the minimal components of the Type VI CRISPR-Cas system, we selected two systems for functional validation, one containing an effector designated Cas12i1 (SEQ ID NO: 3) and the other containing an effector designated Cas12i2 (SEQ ID NO: 5).

[0270] method Gene synthesis and oligo library cloning Effector plasmids were generated by cloning E. coli codon-optimized protein sequences for CRISPR effectors and accessory proteins into pET-28a(+) (EMD-Millipore). Cas genes (including 150 nt of terminal CDS coding sequence) or non-coding sequences flanking the CRISPR array were synthesized (Genscript) into pACYC184 (New England Biolabs) to generate non-coding plasmids (Figure 4A). Effector mutant (e.g., D513A or A513D) plasmids were cloned by site-directed mutagenesis using the indicated primers in the sequence listing: sequence changes were first introduced into PCR fragments, which were then reassembled into plasmids using NEBuilder HiFi DNA Assembly Master Mix or NEB Gibson Assembly Master Mix (New England Biolabs) according to the manufacturer's instructions.

[0271] For the pooled spacer library, we first computationally designed an Oligonucleotide Library Synthesis (OLS) pool (Agilent) to express a minimal CRISPR array of "repeat-spacer-repeat" sequences. The "repeat" elements were derived from consensus direct repeat sequences found in effector-associated CRISPR arrays, and the "spacers" represented approximately 8,900 sequences targeting the pACYC184 plasmid and essential E. coli genes, or negative controls with no targeting sequences. The spacer length was determined by the most common spacer length found in endogenous CRISPR arrays. The minimal CRISPR array was flanked by unique PCR priming sites that enabled amplification of specific libraries from the larger oligo synthesis pool.

[0272] We then cloned the minimal CRISPR array library into effector plasmids to create an effector plasmid library. We added unique molecular identifiers, flanking restriction sites, and the J23119 promoter for array expression on the oligo library using PCR (NEBNext High-Fidelity 2x PCR Master Mix), and then assembled the complete effector plasmid library with its targeting array using NEB Golden Gate Assembly Master Mix (New England Biolabs). This represented the "input library" for the screen.

[0273] In vivo E. coli screen We performed in vivo screens using electrocompetent E. cloni EXPRESS BL21(DE3) Escherichia coli (E. coli) cells (Lucigen) unless otherwise indicated. Competent cells were co-transformed with effector and / or non-coding plasmids (Figure 4B). Cells were electroporated with the "input library" using a Gene Pulser Xcell® (Bio-Rad) according to the manufacturer's protocol in 1.0 mm cuvettes. Cells were plated onto bioassay plates containing both chloramphenicol (Fisher) and kanamycin (Alfa Aesar). After 11 hours of growth, we confirmed sufficient library representation by estimating approximate colony counts and harvested the cells.

[0274] An "output library" was generated by extracting the plasmid DNA fraction from the harvested cells using a QIAprep® Spin miniprep kit (Qiagen), while total RNA = 17 nt was recovered by lysing the harvested cells in Direct-zol® (Zymo Research) followed by extraction using a Direct-zol RNA miniprep kit (Zymo Research).

[0275] Next-generation sequencing libraries for DNA depletion signals were prepared by PCR on both the input and output libraries using custom primers containing barcodes and handles that flank the CRISPR array cassettes in the effector plasmid library and are compatible with Illumina sequencing chemistry. The libraries were then normalized, pooled, and loaded onto a Nextseq 550 (Illumina) to assess effector activity.

[0276] Bacterial Screen Sequencing Analysis Next-generation sequencing data for the screen input and output libraries were demultiplexed using Illumina bcl2fastq. Reads in the resulting fastq file for each sample contained CRISPR array elements for the screening plasmid library. The array orientation was determined using the direct repeat sequence of the CRISPR array, and corresponding targets were determined by mapping the spacer sequence to the source (pACYC184 or E. coli essential genes) or negative control sequence (GFP). For each sample, the total number of reads (r) for each unique array element in a given plasmid library was calculated. a ) were counted and normalized as follows: (r a +1) / total number of reads for all library array elements. The depletion score was calculated by dividing the normalized output reads for a given array element by the normalized input reads.

[0277] To identify specific parameters driving enzymatic activity and bacterial cell death, we used next-generation sequencing (NGS) to quantify and compare the representation of individual CRISPR arrays (i.e., repeat-spacer-repeat) in the PCR products of the input and output plasmid libraries. We defined the depletion fold for each CRISPR array as the normalized input read count divided by the normalized output read count (plus 1 to avoid division by zero). An array was considered "strongly depleted" if the depletion fold was greater than 3. When calculating array depletion fold across biological replicates, we took the maximum depletion fold value for a given CRISPR array across all experiments (i.e., a strongly depleted array must be strongly depleted in all biological replicates). For each spacer target, we created a matrix containing the array depletion fold and the following features: target strand, transcript targeting, ORI targeting, target sequence motif, flanking sequence motif, and target secondary structure. We investigated the extent to which different features in this matrix explain target depletion for Type VI systems, thereby obtaining a broad survey of functional parameters within a single screen.

[0278] result Figures 5A-5D show the locations of the strongly depleted targets for pACYC184 and Cas12i1 and Cas12i2 targeting essential genes in E. coli (E. cloni®). Notably, the locations of the strongly depleted targets appear to be dispersed throughout the potential target space.

[0279] We found that the dsDNA interference activity of the type VI effectors, Cas12i1 (1094aa) and Cas12i2 (1054aa), was abolished by mutation of the conserved aspartic acid in the RuvC I motif (Figures 6A and 6B). The RuvC-dependent dsDNA interference activity of Cas12i indicated no requirement for non-coding sequences adjacent to the CRISPR array or cas genes (Figures 7A and 7B), and the minimal VI interference module contains only the effector and crRNA (Figures 8A and 8B).

[0280] Analysis of target-flanking sequences corresponding to the highly depleted arrays from the in vivo screen indicates that Cas12i-mediated dsDNA interference is PAM-dependent. Specifically, we found that both Cas12i1 and Cas12i2 exhibited a 5'TTN PAM preference (Figures 9A-9B and 10A-10B). These results suggest that the compact Cas12i effector possesses autonomous PAM-dependent dsDNA interference capabilities.

[0281] Example 3: Characterization of the Biochemical Mechanism of the Engineered CLUST.029130 (Type VI) CRISPR-Cas System (Figures 11A-13, 15-17B) Cas12i processes pre-crRNA in vivo To investigate crRNA biogenesis for the type VI CRISPR-Cas system, we purified and sequenced small RNAs from E. coli expressing Cas12i and a minimal CRISPR array library from a bacterial screen. Figures 11A and 11B show the cumulative RNA-sequencing reads, revealing strong consensus forms of Cas12i1 and Cas12i2 mature crRNAs, respectively, as well as the distribution of spacer lengths. The most common spacer length observed was 21, with length variation ranging from 16 nt to 22 nt.

[0282] For Type VI CRISPR-Cas systems including Cas12i1, the mature crRNA can take the form 5'-AUUUUUGUGCCCAUCGUUGGCAC[spacer]-3' (SEQ ID NO: 100).

[0283] For Type VI CRISPR-Cas systems including Cas12i2, the mature crRNA can take the form 5'-AGAAAUCCGUCUUUCAUUGACGG[spacer]-3' (SEQ ID NO: 101).

[0284] Sequencing of small RNAs from the in vivo bacterial screen was performed by extracting total RNA from the recovered bacteria using Direct-zol RNA MiniPrep Plus with TRI Reagent (Zymo Research). Ribosomal RNA was removed using the Ribo-Zero rRNA Removal Kit for Bacteria, followed by RNA Clean and The resulting ribosomal RNA-depleted total RNA was treated with ATP-free T4 PNK for 3 hours to enrich for 3'-P ends, after which ATP was added and the reaction was incubated for an additional hour to enrich for 5'-OH ends. The sample was then column purified, incubated with RNA 5' polyphosphatase (Lucigen), column purified again, and prepared for next-generation sequencing using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs). The library was subjected to paired-end sequencing on a Nextseq 550 (Illumina), and the resulting paired-end alignments were analyzed using Geneious 11.0.2 (Biomatters).

[0285] Cas12i effector purification The effector vector was transformed into Escherichia coli (E. coli) NiCo21(DE3) (New England BioLabs) for expression under the T7 promoter. Transformed cells were initially grown overnight in 3 mL of Luria Broth (Sigma) plus 50 μg / mL kanamycin, followed by inoculation of 1 mL of the overnight culture into 1 L of Terrific Broth medium (Sigma) plus 50 μg / mL kanamycin. Cells were grown at 37°C to an OD600 of 1–1.5, and then protein expression was induced with 0.2 mM IPTG. The culture was then grown at 20°C for an additional 14–18 hours. The culture was harvested, pelleted by centrifugation, and resuspended in 80 mL of lysis buffer (50 mM HEPES pH 7.6, 0.5 M NaCl, 10 mM imidazole, 14 mM 2-mercaptoethanol, and 5% glycerol) plus protease inhibitors (Sigma). Cells were lysed using a cell disruptor (Constant System Limited), and the lysate was clarified by two 20-minute centrifugations at 28,000 × g at 4 °C. The lysate was loaded onto a 5 mL HisTrap FF column (GE Life Sciences) and then purified by FPLC (AKTA Pure, GE Life Sciences) using an imidazole gradient from 10 mM to 250 mM. Cas12i1 was purified in low-salt buffer (50 mM HEPES-KOH pH 7.8, 500 mM KCl, 10 mM MgCl2, 14 mM 2-mercaptoethanol, and 5% glycerol). After purification, fractions were run on an SDS-PAGE gel, and fractions containing the appropriate size protein were pooled and concentrated using a 10 kD Amicon Ultra-15 centrifugal unit. Protein concentration was determined using a Qubit protein assay (Thermo Fisher).

[0286] Cas12i processes pre-crRNA in vitro To determine whether Cas12i1 has autonomous crRNA biogenesis capabilities, we incubated effector proteins purified from E. coli with pre-crRNA expressed from a minimal CRISPR array (repeat-spacer-repeat-spacer-repeat). We observed that purified Cas12i1 processed the pre-crRNA into fragments consistent with the mature crRNA identified from in vivo small RNA sequencing, suggesting that Cas12i1 has autonomous pre-crRNA processing capabilities (Figure 12).

[0287] The Cas12i1 pre-crRNA processing assay was performed at 37°C for 30 minutes at a final pre-crRNA concentration of 100 nM in cleavage buffer. The reaction was performed in a Cas12i-optimized cleavage buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT, 10 mM MgCl2, 50 μg / ml BSA). The reaction was quenched by adding 1 μg / μL proteinase K (Ambion) and incubated at 37°C for 15 minutes. After adding 50 mM EDTA, the reaction was mixed with an equal volume of 2x TBE-urea sample buffer (Invitrogen) and denatured at 65°C for 3 minutes. Samples were analyzed on a 15% TBE-urea gel (Invitrogen). The gel was stained with SYBR Gold nucleic acid stain (Invitrogen) for 5 minutes and imaged on a Gel Doc EZ (Biorad). The gel containing the labeled pre-crRNA was first imaged on an Odyssey CLx scanner (LI-COR Biosciences) and then SYBR stained.

[0288] Cas12i1 DNA manipulation using potently depleted arrays To explore the mechanism of Cas12i1's interference activity, we selected a highly depleted CRISPR array sequence from an in vivo negative selection screen and created a pre-crRNA with a DR-spacer-DR-spacer-DR sequence. The pre-crRNA was designed to target Cas12i1 to 128-nt ssDNA and dsDNA substrates containing a target sequence complementary to the second spacer of the pre-crRNA. We observed that the Cas12i1 binary complex consisting of the effector protein and pre-crRNA cleaved 100 nM of target ssDNA until saturation at a complex concentration of 62.5 nM (Figure 13). Further degradation of the cleaved ssDNA down to short fragments or single nucleotides was observed at higher complex concentrations, suggesting collateral ssDNA cleavage activated by the binary complex binding to the ssDNA target (Figure 13).

[0289] To explore the dsDNA interference activity of Cas12i, we targeted the Cas12i1 binary complex to a target dsDNA substrate containing a 5'-terminal label on the non-spacer complementary strand. To comprehensively evaluate both dsDNA cleavage and nicking activities, the resulting dsDNA cleavage reaction was divided into three fractions for different analyses. The first two fractions were quenched and analyzed under denaturing or non-denaturing gel electrophoresis conditions, respectively. The third fraction was treated with 0.1 U of S1 nuclease to convert any dsDNA nicks into double-strand breaks, quenched, and analyzed by non-denaturing gel electrophoresis.

[0290] We observed dose-dependent cleavage under denaturing conditions, suggesting either target nicking or dsDNA cleavage (Figure 15). Under non-denaturing conditions without S1 nuclease treatment, we observed a dose-dependent increase in primary products that migrated with electrophoretic mobilities slightly lower than those of the input dsDNA, suggesting nicked dsDNA products (Figure 16). Incubation of these products with S1 nuclease converted the upward-shifted band into a smaller dsDNA product, indicating S1-mediated conversion of nicked dsDNA to double-stranded breaks (Figure 16). We also observed fewer dsDNA cleavage products at higher concentrations and longer incubation times, indicating that Cas12i1 is a dsDNA nuclease that cleaves the spacer-complementary ("SC") and non-spacer-complementary ("NSC") strands of target dsDNA with substantially different efficiencies (Figure 17A).

[0291] The observation of nicking activity with 5' labeling of the spacer-complementary strand of the dsDNA substrate suggests that Cas12i1 preferentially nicks the opposite DNA strand of the crRNA-target DNA hybrid. To verify this bias in DNA strand cleavage by Cas12i1, we created dsDNA substrates labeled with IR800 dye at either the 5' end of the spacer-complementary strand or the 5' end of the non-spacer-complementary strand. At low concentrations of effector complex, we observed cleavage only of the NSC strand of the DNA duplex, whereas at high concentrations of effector complex, cleavage of both the NSC and SC strands was observed (Figures 17A-B). Comparing SYBR staining, which labels all nucleic acid products, with strand-specific labeling using IR800 dye reveals differences in the rate of strand product formation compared to overall cleavage product accumulation. These results suggest that an ordered sequence of events leads to dsDNA interference, whereby the Cas12i1 binary complex first nicks the NSC strand and then cleaves the SC strand less efficiently, resulting in dsDNA cleavage. Collectively, these findings demonstrate that Cas12i is an effector capable of autonomous pre-crRNA processing, ssDNA targeted and collateral cleavage, and dsDNA cleavage. This catalytic sequence is similar to that of Cas12a and Cas12b, except for a significant bias toward non-spacer complementary strand cleavage, resulting in preferential dsDNA nicking.

[0292] crRNA and substrate RNA preparation Single-stranded DNA oligo templates for crRNA and substrate RNA were ordered from IDT. Double-stranded in vitro transcription (IVT) template DNA was generated by PCR amplification of the substrate RNA and pre-crRNA templates using NEBNEXT Hifi 2x Master Mix (New England Biolabs). Double-stranded DNA templates for mature cr-RNA were generated by annealing a T7 primer to the template followed by extension using DNA polymerase I, large (Klenow) fragment (New England Biolabs). Annealing was performed by incubating at 95°C for 5 minutes, followed by a -5°C / min ramp to 4°C. In vitro transcription was performed by incubating the dsDNA template with T7 RNA polymerase at 37°C for 3 hours using the HiScribe T7 Quick High Yield RNA Kit (New England Biolabs). After incubation, the IVT samples were treated with Turbo DNase® (Thermo Scientific) and then purified using the RNA Clean & Concentrator Kit (Zymo Research). Mature cr-RNA prepared from IVT was treated with calf intestinal alkaline phosphatase (Thermo Fisher) or RNA 5'-polyphosphatase (Lucigen) at 37°C for 2 hours to generate 5'-hydroxyl or 5'-monophosphate, respectively, followed by cleanup with the RNA Clean & Concentrator kit (Zymo Research). The concentration was measured using a Nanodrop 2000 (Thermo Fisher).

[0293] The pre-crRNA sequences used for biochemical characterization Cas12i are included in Table 6. Oligonucleotide templates and primers for preparation of crRNA are included in Table 9.

[0294] Preparation of IR-800-labeled substrate RNA and DNA The RNA substrate from the IVT was treated with calf intestinal alkaline phosphatase (Thermo Fisher) at 37°C for 30 min to convert the 5'-triphosphate to a 5'-terminal hydroxyl group, and purified using the RNA Clean & Concentrator kit (Zymo DNA and RNA substrates were purified using a 5'EndTag Labeling Kit (Vector Labs). A thiol end group was added to the 5'-terminal hydroxyl group of the DNA and RNA substrates using the 5'EndTag Labeling Kit (Vector Labs), and the substrates were then labeled with IRDye 800CW maleimide (LI-COR Biosciences). Substrates were purified using a DNA Clean & Concentrator Kit or an RNA Clean & Concentrator Kit (Zymo Research). Labeled dsDNA substrates were generated by labeling the non-target (non-spacer-complementary) ssDNA strand, annealing it with a primer, and then extending it with DNA polymerase I, large (Klenow) fragment (New England Biolabs) at 25°C for 15 minutes. These substrates were purified using a DNA Clean & Concentrator Kit (Zymo Research). Concentrations were measured using a Nanodrop 2000 (Thermo Fisher).

[0295] The RNA and DNA substrate sequences used for the biochemical characterization of Cas12i are included in Tables 7 and 8.

[0296] Cas12i-mediated target cleavage assay ssDNA: Optimized cleavage buffer (50 mM Tris-HCl pH 8.0, 50 mM Cas12i target cleavage assays with ssDNA were performed in a 500-well plate containing 1000 kJ / ml of RNase A (NaCl, 1 mM DTT, 10 mM MgCl2, 50 μg / ml BSA). Binary complexes were formed by incubating a 1:2 molar ratio of Cas12i:pre-crRNA at 37°C for 10 minutes and then transferring to ice. All further complex dilutions were performed on ice, keeping the protein:RNA ratio constant. This complex was added to 100 nM IR800-labeled substrate and incubated at 37°C for 30 minutes. Reactions were treated with RNase cocktail and proteinase K and analyzed as described above.

[0297] dsDNA: The dsDNA target cleavage assay was set up in optimized cleavage buffer for 1 hour at 37°C. The binary complex was formed as described above and added to 100 nM dsDNA substrate. The reaction was first treated with an RNase cocktail by incubation at 37°C for 15 minutes. Next, it was treated with proteinase K by incubation at 37°C for 15 minutes. To detect dsDNA cleavage products, the reaction was analyzed on a 15% TBE-urea gel as described above. To detect Cas12i nicking activity, the reaction was SPRI-purified after proteinase K treatment and divided into three fractions. One fraction was analyzed on a 15% TBE-urea gel as described above. The other fraction was mixed with 5x high-density TBE sample buffer and analyzed on a non-denaturing 4-20% TBE gel to detect nicked dsDNA products. The final fraction was incubated with 0.01 U / µL S1 nuclease (Thermo Scientific) for 1 hour at 50°C to convert nicks to double-strand breaks, then mixed with 5x high-density TBE sample buffer and analyzed on a non-denaturing 4-20% TBE gel. All gels were imaged on an Odyssey CLx scanner, followed by 5 minutes of SYBR staining and imaging on a Gel Doc imager.

[0298] To identify the nicked strand, dsDNA was prepared by labeling either the target strand (complementary to the crRNA) or the non-target strand (non-spacer complementary, same sequence as the crRNA). Cleavage reactions were performed as described. The labeled strand was then annealed with the corresponding primer and extended with DNA polymerase I, large (Klenow) fragment (New England Biolabs) for 15 min at 25°C. The dsDNA substrate was then purified using SPRI purification.

[0299] [Table 6]

[0300] [Table 7]

[0301] [Table 8]

[0302] [Table 9]

[0303] Example 4: In vitro pooled screening for rapid evaluation of CRISPR-Cas systems (Figures 20-25) As described herein, in vitro pooled screening serves as an efficient, high-throughput method for conducting biochemical evaluations. In summary, we begin with in vitro reconstitution of the CRISPR-Cas system (Figure 20). In one embodiment, effector proteins are generated using a dsDNA template containing a T7-RNA polymerase promoter to drive expression of one or more effector proteins, and in vitro transcription and translation reagents to generate the proteins for the reaction. In another embodiment, a minimal CRISPR array and tracrRNA contain a T7 promoter sequence added in either the top-strand or bottom-strand transcription direction using PCR to explore all possible RNA orientations. As shown in Figure 20, the apo version contains only the effector; the binary version contains the effector protein and the T7-transcribed minimal CRISPR array; and the binary + tracrRNA version adds any T7-transcribed tracrRNA elements to the complex for incubation.

[0304] In one embodiment, the endonucleolytic activity of the CRISPR-Cas system is the primary biochemical activity assayed. Figure 21 shows one form of ssDNA and dsDNA substrate, where the target sequence is flanked on both sides by six degenerate bases to create a pool of possible PAM sequences that can gate ssDNA and dsDNA cleavage activity. In addition to the PAM sequence, the substrate contains 5' and 3' fiducial marks designed to provide unique sequences that facilitate downstream next-generation sequencing library preparation protocols that selectively enrich the substrate ssDNA or dsDNA and facilitate mapping of cleavage products. In one embodiment, the dsDNA substrate is generated by 5' to 3' second-strand synthesis using a short DNA primer and DNA polymerase I. Similar reactions can be performed using pools of different targets in minimal CRISPR arrays, as well as libraries of different ssDNA and dsDNA sequences.

[0305] The CRISPR-Cas cleavage reaction is performed by mixing and incubating the preformed Apo / binary / binary-tracrRNA complex with either a targeting or non-targeting substrate. While other methods, such as gel electrophoresis, are possible, a useful embodiment for cleavage capture with the highest sensitivity and base pair resolution is next-generation sequencing of the ssDNA or dsDNA substrate after incubation with the effector complex. Figure 22 shows a schematic illustrating library preparation for enrichment of ssDNA substrates. By annealing primers to well-defined sequences within the fiducial marks, second-strand synthesis and end repair occur, generating dsDNA fragments representing both cut and uncut ssDNA. These newly formed dsDNA molecules then serve as substrates for adapter ligation, followed by selective PCR using one primer (I5 / P5) complementary to the ligated adapter and another (I7 / P7) complementary to the 3' fiducial of the original ssDNA substrate. This ultimately generates a sequencing library containing both full-length and cleaved and degraded ssDNA products, as shown in Figure 24A. dsDNA readout NGS library preparation begins without the need for primer annealing and second-strand synthesis, so end repair and subsequent adapter ligation can be performed directly. Figure 23 describes a general outline of library preparation that labels both cleaved / degraded and uncleaved fragments, similar to ssDNA preparation. Notably, either end of the dsDNA cleaved fragments can be enriched based on the selection of PCR primers. In one embodiment shown in Figure 24A, a readout dsDNA-engineered next-generation sequencing library can be prepared with a first primer complementary to a handle ligated to the 5' end of the full-length or cleaved substrate (and containing the I5 / P5 sequence) and a second primer complementary to the 3' reference sequence of the substrate (and containing the I7 / P7 sequence).In one embodiment, shown in Figure 24B, a DNA engineering next-generation sequencing library for readout can be prepared with a first primer complementary to the 5' reference sequence of the substrate (and containing the I5 / P5 sequence) and a second primer complementary to a handle ligated to the 3' end of the full-length or cleaved substrate (and containing the I7 / P7 sequence). Target and substrate lengths can be extracted from NGS reads obtained from an RNA / ssDNA / dsDNA engineering experiment, as illustrated in Figures 25A-25B, respectively. The extracted target and substrate lengths can be used to determine the presence of RNA / ssDNA / dsDNA nicking or cleavage.

[0306] Example 5: Characterization of dsDNA cleavage activity of the V-I1 type CRISPR-Cas system (Figures 26-32) Having computationally identified the minimal components of a type VI CRISPR-Cas system, we investigated double-stranded DNA (dsDNA) cleavage activity from a type V-I1 system containing the effector Cas12i1.

[0307] IVTT expression of Cas12i1 in a complex with top-strand-expressed crRNA targeting dsDNA resulted in a truncated target length population not present in the apo (effector only) control, as shown in Figures 26A-B. A library prepared using a 5' ligation adapter and selecting for a 3' reference (as illustrated in Figure 24A) showed a cleavage product at position +24 within the target sequence that was not present in the apo control. This result indicates nicking of either the non-target dsDNA strand or both strands of the dsDNA between nucleotides +24 and +25 relative to the PAM. Target length analysis showed a peak at +24, indicating truncation of the target between nucleotides +24 and +25 (Figure 27A). This truncated target sequence population matched the substrate length, indicating cleavage of the non-target dsDNA strand between nucleotides +24 and +25 of the target sequence (Figure 28A).

[0308] A library prepared using a 3' ligation adapter and selecting for a 5' reference (as illustrated in Figure 24B) showed a cleavage product at position -9 within the target sequence (+19 considering the 28-nt target) that was not present in the control. This result indicates nicking of either the target dsDNA strand or both strands of the dsDNA between nucleotides +19 and +20 relative to the PAM. Target length analysis showed a peak at -9 nucleotides from the PAM (28-nt full-length target), indicating truncation of the target between nucleotides +19 and +20 (Figure 27B). This truncated target sequence population matches the substrate length, indicating cleavage of the target dsDNA strand between nucleotides +19 and +20 of the target sequence (Figure 28B).

[0309] Sequence motif analysis of substrates exhibiting non-target strand cleavage between +24 / +25 nucleotides relative to the PAM revealed a 5' TTN PAM motif to the left of the target sequence for Cas12i1 (Figure 29). No PAM sequence requirement was observed for the right side of the Cas12i1 target. In summary, in vitro screening of Cas12i1 showed predominant nicking between +24 / +25 nucleotides on the non-target strand relative to the TTN PAM, with a significant proportion of these products converted to double-stranded breaks with 5-nt 3' overhangs by cleavage of the target strand between +19 / +20 nucleotides relative to the PAM (Figure 30).

[0310] Targeting Cas12i1 in complexes with non-targeting crRNAs expressed by the top strand did not result in relative dsDNA manipulation, indicating that Cas12i1 cleavage specificity is conferred by the crRNA spacer (Figures 31A-31B). Cas12i1 exhibited no cleavage activity in the presence of bottom-strand crRNAs targeting dsDNA substrates, indicating that top-strand orientation of the crRNA is required for active Cas12i1 complex formation (Figures 32A-32B).

[0311] Example 6: Characterization of the dsDNA cleavage activity of the V-I2 CRISPR-Cas system (Figures 33-39) Having computationally identified the minimal components of a type VI CRISPR-Cas system, we investigated double-stranded DNA (dsDNA) cleavage activity from a type V-I2 system containing the effector Cas12i2.

[0312] IVTT expression of Cas12i2 in complexes with top-strand-expressed crRNA targeting dsDNA resulted in a truncated target length population not present in the apo (effector only) control, as shown in Figures 33A-B. A library prepared using a 5' ligation adapter and selecting for the 3' reference (as illustrated in Figure 24A) showed a cleavage product at position +24 within the target sequence that was not present in the Apo control. This result indicates nicking of either the non-target dsDNA strand or both strands of the dsDNA between nucleotides +24 and +25 relative to the PAM. Target length analysis showed a peak at +24, indicating truncation of the target between nucleotides +24 and +25 (Figure 34A). This truncated target sequence population matched the substrate length, indicating cleavage of the non-target dsDNA strand between nucleotides +24 and +25 of the target sequence (Figure 35A).

[0313] The library prepared using the 3' ligation adapter and selecting for the 5' reference (as illustrated in Figure 33B) showed a cleavage product at position -7 within the target sequence (+24 considering the 31 nt target) that was not present in the Apo control. This result indicates nicking of either the target dsDNA strand or both strands of the dsDNA between nucleotides +24 and +25 relative to the PAM. Target length analysis showed a peak at -7 nucleotides from the PAM (28 nt full-length target), indicating truncation of the target between nucleotides +24 and +25 (Figure 34B). This truncated target sequence population matches the substrate length, indicating cleavage of the target dsDNA strand between nucleotides +24 and +25 of the target sequence (Figure 35B).

[0314] Sequence motif analysis of substrates exhibiting non-target strand cleavage between +24 / +25 nucleotides relative to the PAM revealed a 5'TTN PAM motif to the left of the target sequence for Cas12i2 (Figure 36). No PAM sequence requirement was observed for the right side of the Cas12i2 target. In summary, in vitro screening of Cas12i2 showed predominant nicking between +24 / +25 nucleotides on the non-target strand relative to the TTN PAM, and a significant proportion of these products were converted to double-stranded breaks with blunt-ended nicking by cleavage of the target strand between +24 / +25 nucleotides relative to the PAM (Figure 37).

[0315] Targeting Cas12i2 in complexes with top-stranded non-targeting crRNA did not result in relative dsDNA manipulation, indicating that Cas12i2 cleavage specificity is conferred by the crRNA spacer (Figures 38A-B). Cas12i2 exhibited no cleavage activity in the presence of bottom-stranded crRNA targeting dsDNA substrates, indicating that top-stranded crRNA is required for active Cas12i2 complex formation (Figures 39A-B).

[0316] Example 7: The CLUST.029130 (Type VI) CRISPR-Cas system can be used for gene silencing in vitro To rapidly validate the activity of novel CRISPR-Cas systems, we developed an in vitro gene silencing assay (Figures 18A and 18B) that mimics in vivo gene silencing activity and simultaneously assesses various mechanisms of activity and functional parameters in an unbiased manner outside the native cellular environment.

[0317] First, we supplemented the reconstituted IVTT (in vitro transcription and translation) system with Escherichia coli (E. coli) RNA polymerase core enzyme, allowing gene expression (protein synthesis) to occur not only from the T7 promoter but also from any E. coli (E. coli) promoter as long as the corresponding E. coli sigma factor is present.

[0318] Second, to facilitate rapid, high-throughput experimental methods, we directly used linear DNA templates generated from PCR reactions. These linear DNA templates included those encoding the type VI effector, RNA guide, and E. coli sigma factor 28. Incubation of these DNA templates with reconstituted IVTT reagents resulted in coexpression of the type VI effector and RNA guide and the formation of RNPs (ribonucleoprotein complexes). E. coli sigma factor 28 was also expressed for subsequent expression of GFP and RFP, as described below.

[0319] Third, linear or plasmid DNA encoding GFP expressed from the sigma factor 28 promoter was included in the incubation reaction as a target substrate to ensure that newly synthesized RNPs immediately reached the target substrate. As an internal control, non-target linear DNA encoding RFP expressed from the sigma factor 28 promoter was also included. The RNA polymerase core enzyme alone does not recognize the sigma factor 28 promoter until sufficient sigma factor 28 protein has been synthesized. This delay in GFP and RFP expression could allow newly synthesized RNPs to interfere with the GFP target substrate, resulting in reduced GFP expression and depletion of GFP fluorescence. On the other hand, RFP expression was not negatively affected, which serves as an internal control for protein synthesis and fluorescence measurements.

[0320] Certain important advantages of the in vitro gene silencing assays described herein include: (1) Modularity—Reconstituted IVTT is a synthetic system composed of individually purified components, allowing assays to be custom designed for various controls and activities. Each component of the CRISPR-Cas system is encoded on a separate linear DNA template, allowing for the rapid assay of different effector, effector mutant, and RNA guide combinations; (2) Complexity - The assay includes all essential components of RNA transcription and protein synthesis, allowing for testing of diverse interference mechanisms, including DNA and RNA cleavage and transcription-dependent interference, in a single, one-pot reaction. The assay's kinetic fluorescence readout provides significantly more data points than endpoint activity assays; (3) Sensitivity—This assay combines effector and RNA-guided synthesis with substrate interference, allowing newly synthesized RNPs (ribonucleoprotein complexes of effector proteins and RNA guides) to immediately interact with the substrate in the same reaction. There is no separate purification step, so potentially small amounts of RNP are sufficient to generate a signal. Furthermore, interference with GFP expression is amplified due to coupled GFP transcription and translation, which can generate over 100 GFP proteins per DNA template. (4) Efficiency—The assay is designed to be highly compatible with high-throughput platforms. Due to its modularity, all components of the assay can be added in 96-, 384-, and 1536-well formats using commonly available liquid handling equipment, and fluorescence can be measured using commonly available plate fluorometers. (5) Relevance—This assay tests the ability of a CRISPR-Cas effector protein to interfere with gene expression during transcription and translation in a system engineered in vitro outside its native cellular environment. It is possible that highly active CRISPR-Cas effectors measured by this gene silencing assay may also be highly efficient for gene editing in mammalian cells.

[0321] This assay was used to measure the gene silencing effect of the Cas12i effector complex as described herein when targeting GFP encoded on plasmid DNA. Multiple type VI RNA guides were designed, one containing a spacer sequence complementary to the template strand of the GFP sequence and the other containing a spacer sequence complementary to the coding strand of the GFP sequence. The extent of gene silencing by the Cas12i1 effector protein was then compared with mutants Cas12i1 D647A, Cas12i1 E894A, and Cas12i1 D948A.

[0322] Figure 19A shows the fold depletion of each of the four tested Cas12i effectors when complexed with an RNA guide complementary to the template strand. In this case, the non-target strand that is preferentially nicked is the coding strand. Cas12i1 shows approximately 2-fold depletion of GFP expression after 400 minutes, while each of the three mutant forms shows a lower degree of depletion.

[0323] Figure 19B shows the fold depletion of each of the four tested Cas12i1 effectors when complexed with an RNA guide complementary to the coding strand. In this case, the non-target strand that is preferentially nicked is the template strand. In this configuration, the ability of RNA polymerase to generate functional RNA transcripts appears to be significantly impaired by Cas12i1, with a depletion of more than four-fold in the case of Cas12i. The gene silencing abilities of the three mutant forms appear to be significantly reduced.

[0324] Taken together, the data shown in Figures 19A and 19B demonstrate that this assay is effective for detecting the gene silencing activity of Cas12i1 when using RNA guides that target both the coding and template strands. The significantly higher depletion observed when targeting the coding strand compared with the template strand suggests that Cas12i1 interferes with GFP expression by preferentially nicking the non-target strand. All three Cas12i1 mutants substitute alanine (A) for the putative catalytic residues (aspartic acid (D) and glutamic acid (E)). The reduced silencing activity of these Cas12i1 mutants further supports the notion that DNA strand cleavage, rather than simple binding, underlies the mechanism of Cas12i1 gene silencing.

[0325] Example 8: The CLUST.029130 (Type VI) CRISPR-Cas system can be used with fluorescent reporters for specific detection of nucleic acid species The nuclease activity of the Cas12i protein (i.e., nonspecific collateral DNase activity activated by target ssDNA substrates complementary to the crRNA spacer) makes these effectors promising candidates for use in detecting nucleic acid species. Some of these methods have been previously described (e.g., East-Seletsky et al. "Two distinct RNase activities of CRISPR-C2c2"). enable guide-RNA processing and RNA detection,” Nature. 2016 Oct 13;538(7624):270-273), Gootenberg et al. (2017), Chen (2018) and Gootenberg et al. (2018) "Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6," Science 15 Feb 2018:eaaq0179, described the general RNA detection principle using Cas13a (East-Seletsky et al. (2016)), which was supplemented by amplification and optimization of additional Cas13a enzymes to increase detection sensitivity (Gootenberg et al. (2017)), and more recently by the inclusion of additional RNA targets, orthologous and paralogous enzymes, and Csm6 activators to achieve multiplexed nucleic acid detection with increased detection sensitivity (Gootenberg et al. (2018)). The addition of Cas12i to this toolkit provides an additional channel of orthogonal activity for nucleic acid detection.

[0326] Given that dye-labeled collateral DNA was efficiently cleaved at low target ssDNA concentrations and that background nuclease activity was limited with non-targeting substrates, the in vitro biochemical activity of Cas12i1 suggests that it may be promising for sensitive nucleic acid detection applications (Figure 14). Adapting Cas12i1 for sensitive nucleic acid detection applications requires several steps, including but not limited to optimizing the substrate for sensitive readout of collateral activity and identifying the per-base mismatch tolerance between the spacer and target substrate.

[0327] Identification of optimal substrates for nucleic acid detection may be informed by performing next-generation sequencing (NGS) on the cleavage products of Cas12i collateral activity on both DNA substrates. It may be necessary to titrate the enzyme concentration or adjust the incubation time to generate cleavage fragments that are still large enough to be prepared into next-generation sequencing libraries. NGS data reveals the enzyme cleavage site and adjacent base preferences. It has been demonstrated that individual effectors within the Cas13a and b families have different dinucleotide base preferences for RNA cleavage, resulting in significantly different cleavage magnitudes and signal-to-noise ratios (Gootenberg et al. (2018)). Collateral NGS data therefore provide better insight into Cas12i preferences. A separate experimental approach to identifying dinucleotide preferences for Cas12i collateral cleavage is to create collateral DNA substrates with degenerate Ns at consecutive positions, providing a wider sequence space than defined sequences. Library preparation and analysis of NGS data can similarly identify base preferences for cleavage. To confirm this preference, collateral substrates containing synthesized short DNA fragments with 5'- and 3'-terminal fluorophore / quencher pairs can be introduced into the cleavage reaction to assess the signal-to-noise ratio. Further optimization of the length of the collateral DNA substrates can be performed to determine whether Cas12i1 has a length preference.

[0328] Having identified preferred substrates, another important parameter to determine is the mismatch tolerance of the Cas12i system, as it has implications for guide design, which affects the enzyme's ability to discriminate between single base pair mismatches. Mismatch tolerance may be determined by designing a panel of targets bearing different positions and types of mismatches (e.g., insertions / deletions, single base pair mismatches, adjacent double mismatches, distant double mismatches, triple mismatches, and more). Mismatch tolerance may also be measured by assessing the amount of collateral DNA cleavage for targets containing varying amounts of mismatches. As an example, the collateral DNA substrate may be a short ssDNA probe containing a fluorophore and a quencher on opposing sides. For reactions involving Cas12i effectors, RNA guides, and target substrates containing different numbers of mismatches, insertions, and deletions in the target sequence, successful activation of the Cas12i system by targeting the modified target DNA sequence will result in collateral cleavage of the fluorescent probe. The resulting fluorescence measurements, representing the cleaved collateral substrate, can then be used to estimate the effect of target modification on the efficiency of collateral cleavage by Cas12i by subtracting the background using a negative control sample and normalizing to the signal from the perfectly matched target. The resulting map of mismatch, insertion, and deletion tolerance by the Cas12i enzyme versus target length relative to the PAM can be used to design RNA guides optimal for discriminating between different DNA sequences or genotypes for specific detection or differentiation between different nucleic acid species. Using a fluorometric cleavage readout and a preferred collateral substrate, the location and type of mismatch for which the enzyme is most sensitive can be determined by comparing the fluorescence activity to the perfectly matched sequence.

[0329] This optimization process can be further applied to other Cas12i orthologs to generate other systems that may have different properties, for example, orthogonal dinucleotide preferences for collateral cleavage can aid in generating distinct detection channels.

[0330] Example 9. CLUST.029130 (Type VI) CRISPR Cas system can be used for paired nicking to achieve highly specific dsDNA engineering The CLUST.029130 effector Cas12i has the ability to manipulate dsDNA through nicking of the non-target strand (Figures 15, 16, and 17A-17B). Catalytically inactive Cas12i can also be fused to a FokI nuclease domain to create a fusion protein capable of binding and nicking dsDNA. Some of these methods have been described previously. Ran et al. (2013) "Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity" Science 29 Aug 2013 describes the general principles and optimization of double nicking using Cas9; Guilinger et al. (2014) "Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification" Science 25 Apr 2014 describes the principles of double nicking using a FokI-dCas9 fusion.

[0331] The use of paired Cas12i nickases allows for highly specific dsDNA engineering as follows: a first Cas12i complex carrying a crRNA targeting one strand of a dsDNA target region is introduced together with a second Cas12i-crRNA complex targeting the opposite strand of the dsDNA to achieve a dsDNA cleavage reaction. By targeting the Cas12i complexes to different dsDNA strands, the first and second Cas12i complexes cleave opposing dsDNA strands, resulting in a double-strand break.

[0332] To optimize the efficiency of double-nicking to form double-stranded breaks in dsDNA, pairs of crRNA spacer sequences are selected whose predicted nuclease cleavage sites are separated by different lengths. Cleavage of the top and bottom strands of dsDNA by paired Cas12i nickases with different target substitutions generates sequence overhangs of different lengths, resulting in different efficiencies in forming double-stranded breaks. Paired nickase targets can be selected with specific orientations to generate either 3' or 5' overhangs or blunt-ended (zero overhang length) double-stranded breaks.

[0333] For nicking applications in which the Cas12i1 and Cas12i2-WT enzymes contain a 5' TTN PAM, a paired nickase target orientation with the PAM "out" (the PAM is on the outside of the paired target) results in a 5' overhang, while a paired nickase target with the PAM on the inside of the target pair results in a 3' overhang. In some instances, the 3' and 5' overhangs range from 1 to 200 nt. In some instances, the 3' and 5' overhangs are 20 to 100 nt.

[0334] Autonomous pre-crRNA processing, coupled with the ability to target two separate genomic loci from a single crRNA transcript, facilitates Cas12i delivery for double-nicking applications (Figure 12). In this regard, Cas12i and a CRISPR array containing two spacer sequences that target Cas12i for nicking opposite strands of dsDNA can be expressed from a single viral vector or plasmid. Cas12i and the CRISPR array can also be delivered in separate plasmids or viral vectors. Cas12i protein then processes the CRISPR array into two cognate crRNAs, which result in the formation of a paired nicking complex. Viral vectors can include phage or adeno-associated viruses for delivery to bacteria or mammalian cells, respectively.

[0335] In addition to viral or plasmid delivery methods, paired nicking complexes can be directly delivered using nanoparticles or other direct protein delivery methods, such that complexes containing both paired crRNA elements are co-delivered. Furthermore, proteins can be delivered to cells by viral vectors or directly, followed by direct delivery of CRISPR arrays containing two paired spacers for double nicking. In some cases, for direct delivery of RNA, the RNA may be conjugated to at least one sugar moiety, such as N-acetylgalactosamine (GalNAc), particularly triantennary GalNAc.

[0336] Example 10: Adaptation of CLUST.029130 (Type VI) CRISPR Cas system effectors for eukaryotic and mammalian activity To develop the CLUST.029130 (Type VI) CRISPR Cas system for eukaryotic applications, constructs encoding protein effectors were first codon-optimized for expression in mammalian cells, with optional addition of specific localization tags to either the N-terminus or C-terminus, or both, of the effector protein. Such localization tags can include sequences such as nuclear localization signal (NLS) sequences that localize the effector to the nucleus for modification of genomic DNA. These sequences are described in the "Functional Mutations" section above. Some examples of non-naturally occurring nucleotide sequences engineered to encode mammalian codon-optimized Cas12i effectors containing localization tags are provided in Table 10. Other accessory proteins, such as fluorescent proteins, may also be added. It has been demonstrated that the addition of robust "superfolding" proteins, such as superfolding green fluorescent protein (GFP), can increase the activity of CRISPR enzymes in mammalian cells when attached to an effector (Abudayyeh et al. (2017) Nature 550(7675):280-4, and Cox et al. (2017) Science 358(6366):1019-27).

[0337] The codon-optimized sequences encoding Cas12i and the added accessory proteins and localization signals were then cloned into eukaryotic expression vectors with appropriate 5' Kozak eukaryotic translation initiation sequences, eukaryotic promoters, and polyadenylation signals. In mammalian expression vectors, these promoters can include, for example, general promoters such as CMV, EF1a, EFS, CAG, and SV40, as well as cell-type-specific RNA polymerase II promoters such as Syn and CamKIIa for neuronal expression and thyroxine-binding globulin (TBG) for hepatocyte expression, to name a few. Similarly, useful polyadenylation signals include, but are not limited to, SV40, hGH, and BGH. Additional transcript stabilization or transcript nuclear export elements, such as WPRE, can be used to increase expression of such constructs. RNA polymerase III promoters, such as H1 or U6, can be used to express pre-crRNA or mature crRNA.

[0338] Depending on the application and packaging method, the eukaryotic expression vector may be a lentiviral plasmid backbone, an adeno-associated virus (AAV) plasmid backbone, or a similar plasmid backbone that can be used in recombinant viral vector production. Notably, the small size of the CLUST.029130 (Type VI) CRISPR Cas effector protein, such as the Cas12i protein, makes it ideally suited for packaging into a single adeno-associated virus particle along with its crRNA and appropriate regulatory sequences; the 4.7 kb packaging size limit of AAV may preclude the use of large effectors, especially when large, cell-type-specific promoters are used to control expression.

[0339] After adapting the sequences, delivery vectors, and methods for eukaryotic and mammalian use, different Cas12i constructs were characterized for performance as described herein. Initial characterization was performed by lipofection of DNA constructs expressing the minimal components of the Cas12i system, adapted for eukaryotic use as described above. In one embodiment, the Cas12i effector is mammalian codon-optimized, and a nucleoplasmin nuclear localization sequence (npNLS) is added to the C-terminus of the protein. Effector expression is driven by the elongation factor 1 alpha short (EFS) promoter and terminated using a bGH poly(A) signal (Table 10). Expression of the cognate RNA guide for the Cas12i system was performed using a double-stranded linear PCR product containing a U6 promoter, as adapted from (Ran et al. "Genome engineering using the CRISPR-Cas9 system," Nat Protoc. 2013 Nov;8(11):2281-2308). This approach is well suited for testing large numbers of sgRNAs through plasmid cloning and sequence verification (Figure 40). The effector plasmid and U6-guided PCR fragment were co-transfected into 293T cells at a molar ratio of approximately 1:2 between the plasmid and PCR product, with 400 ng of effector plasmid and 30 ng of U6-guided PCR product for a 24-well plate format. The resulting gene editing events were assessed using next-generation sequencing of the targeted PCR amplicons around the target site (Hsu et al., "DNA targeting specificity of RNA-guided Cas9 nucleases," Nat Biotechnol. 2013 Sep;31(9):827-32).

[0340] Initial evaluation of Cas12i2 yielded 13% indel activity at the VEGFA locus at target sites containing a TTC PAM. We tested different RNA guide designs as described in Figure 41, where the strongest indel efficiency was achieved using pre-crRNA, and the indel rate decreased with shorter spacer lengths. Examination of the indels generated by Cas12i2 reveals that the predominant position of indels is centered at +20 relative to the PAM sequence.

[0341] Multiplexing of Type VI effectors is achieved using effector-capable pre-crRNA processing, where multiple targets containing different sequences can be programmed onto a single RNA guide. In this way, multiple gene or DNA targets can be simultaneously manipulated for therapeutic applications. One embodiment of the RNA guide design is a pre-crRNA expressed from a CRISPR array consisting of a target sequence interleaved with unprocessed DR sequences, repeated to enable simultaneous targeting of one, two, or more gene loci by endogenous pre-crRNA processing of the effector.

[0342] In addition to testing various construct configurations and accessory sequences against individual targets, a pooled library-based approach is used to determine 1) any targeting dependency of a particular Cas12i protein in mammalian cells, and 2) the effect of mismatch positions and combinations along the length of the targeting crRNA. Briefly, pooled libraries contain plasmids expressing target DNA containing different flanking sequences and mismatches with one or more guides used in the screening experiment, such that successful target recognition and cleavage results in depletion of sequences from the library. Furthermore, targeted indel sequencing or unbiased genome-wide cleavage assays can be used to assess the specificity of the CLUST.029130 (Type VI) CRISPR-Cas system (Hsu et al. (2013), Tsai et al. "GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nat Biotechnol. 2015 Feb;33(2):187-197, Kim et al. "Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells," Nat Methods. 2015 Mar;12(3):237-43, Tsai et al., "CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets," Nat Methods. 2017 Jun;14(6):607-614).

[0343] Additionally, mutations are engineered to expand the functional scope of the Cas12i protein. In some embodiments, catalytically inactive Cas12i proteins can be generated in which conserved residues in the RuvC domain are mutated to alanine (e.g., the D647A mutation for Cas12i1 and the D599A mutation for Cas12i2). Catalytically inactive Cas12i versions (referred to as dCas12i) retain their programmable DNA-binding activity but are no longer able to cleave target or collateral ssDNA or dsDNA. Direct applications of dCas12i include immunoprecipitation and transcriptional repression. Additional functionality can be provided by adding other domains to the dCas12i protein.

[0344] The activities of these domains include, but are not limited to, DNA base modification (e.g., ecTAD and its evolved forms, APOBEC), DNA methylation (m 6 These include mitochondrial domains (e.g., KRAB, VP64), localization factors (e.g., KDEL retention sequence, mitochondrial targeting signal), and transcriptional modifiers (e.g., KRAB, VP64). Additionally, domains such as light-gating (cryptochrome) and chemical-inducible components (e.g., FKBP-FRB chemical-induced dimerization) can be added to provide further control.

[0345] Optimizing the activity of such fusion proteins requires a systematic approach to compare linkers connecting dCas12i to additional domains. These linkers can include, but are not limited to, flexible glycine-serine (GS) linkers in various combinations and lengths, non-flexible linkers such as the α-helix-forming EAAAK sequence, XTEN linkers (Schellenberger V, et al. Nat. Biotechnol. 2009;27:1186-1190), and various combinations thereof (see Table 11). Different designs are then assayed in parallel on the same crRNA-target complex and functional readout to determine which produces the desired properties.

[0346] To adapt Cas12i for use in targeted DNA base modification (see, e.g., Gaudelli et al. (2017) "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage," Science, 25 Oct 2017), we started with the Cas12i ortholog and NLS combination that yielded the highest intrinsic mammalian DNA cleavage activity and mutated conserved residues in the RuvC domain to create a catalytically inactive enzyme (dCas12i). We then created a fusion protein using a linker between the dCas12i-NLS and the base-editing domain. Initially, this domain will consist of the ecTadA(wt) / ecTadA*(7.10) heterodimer (hereafter referred to as the dCas12i-TadA heterodimer), previously engineered for hyperactivity and modification of dsDNA A·T dinucleotides to G·C (Table 11). Given the possible structural differences between the small Cas12i and the previously characterized Cas9 effectors, alternative linker designs and lengths may lead to optimal designs of base-editing fusion proteins.

[0347] To assess the activity of dCas12i-derived base editors, HEK 293T cells are transiently transfected with the dCas12i-TadA heterodimer construct, a plasmid expressing crRNA, and, optionally, a reporter plasmid if targeting a reporter rather than the endogenous locus. Forty-eight hours after transient transfection, the cells are harvested, and DNA is extracted and prepared for next-generation sequencing. Analysis of the base composition of the locus in samples containing targeting crRNA compared with a negative control non-targeting crRNA provides information about editing efficiency, and analysis of broader changes in the transcriptome provides information about off-target activity.

[0348] One particular advantage of developing a DNA base editing system using Cas12i is that its small size, smaller than existing Cas9 and Cas12a effectors, allows for easier packaging of the dCas12i-TadA heterodimer together with its crRNA and regulatory elements into AAV without the need for protein truncation. This all-in-one AAV vector allows for higher efficacy of in vivo base editing in tissues, which is particularly relevant as a path to therapeutic applications of Cas12i.

[0349] In addition to editing using Cas12i and RNA guides, additional template DNA sequences can be co-delivered in vectors such as AAV viral vectors or as linear single- or double-stranded DNA fragments. For template DNA insertion via homology-directed repair (HDR), a template sequence is designed that includes the payload sequence to be inserted into the locus of interest and flanking sequences homologous to endogenous sequences adjacent to the desired insertion site. In some cases, for insertion of short DNA payloads (e.g., less than 1 kb in length), the flanking homologous sequences may be short (e.g., in the range of 15 to 200 nt in length). In other cases, for insertion of long DNA payloads (e.g., 1 kb or longer), long homologous flanking sequences are required to promote efficient HDR (e.g., greater than 200 nt in length). Excision of the target genomic locus for HDR between sequences homologous to the template DNA flanking regions can significantly increase HDR frequency. Cas12i cleavage events that promote HDR include, but are not limited to, dsDNA cleavage, double nicking, and single-strand nicking activity.

[0350] The dsDNA fragment may contain an overhang sequence complementary to the overhang generated by double-nicking using Cas12i. Pairing of the insert with the double-nicked overhang and subsequent ligation by endogenous DNA repair mechanisms results in seamless insertion of the template DNA at the double-nicked site.

[0351] [Table 10-1]

[0352] [Table 10-2]

[0353] [Table 11]

[0354] These results suggest that compact type VI CRISPR family members can be engineered for activity in eukaryotic cells, specifically for genome editing in mammalian cells. Functional mammalian type VI effectors enable the development of additional technologies based on further engineering on the DNA-binding platform.

[0355] Example 11. Type VI CRISPR-Cas systems can be used to provide genotypic gating of genome replication, viral propagation, plasmid propagation, cell death, or cell dormancy. Hybridization of Type VI CRISPR-Cas effector proteins and crRNA to specific ssDNA or dsDNA targets results in nicking or cleavage of the substrate. The dependence of such activity on the presence of specific DNA targets in cells is valuable because it allows for targeting of specific genomic material or cell populations based on specific underlying genotypes. Numerous applications exist for such control of genome replication, cell death, or cell dormancy in both eukaryotic, prokaryotic, and viral / plasmid settings.

[0356] For prokaryotic, viral, and plasmid applications, type VI CRISPR-Cas systems (e.g., comprising a type VI effector and an RNA guide) can be delivered (e.g., in vitro or in vivo) to halt genome replication and / or induce cell death or dormancy in specific prokaryotic populations (e.g., bacterial populations) in a genotype-specific manner. For example, a type VI CRISPR-Cas system can include one or more RNA guides that specifically target a particular virus, plasmid, or prokaryotic genus, species, or strain. As shown in Figures 5A-5D, cleavage, nicking, or interference with the E. coli genome or plasmid DNA that confers antibiotic resistance in E. coli by a type VI system results in the specific depletion of E. coli containing those sequences. Specific targeting of viruses, plasmids, or prokaryotes can also be used to target unwanted bacteria (e.g., Clostridium difficile). This has many therapeutic benefits, as it can be used to induce the death or dormancy of pathogenic bacteria such as Clostridium difficile. Additionally, the Type VI system provided herein may be used to target prokaryotic cells with specific genotypes. Among the microbial diversity that colonizes humans, only a small number of bacterial strains are capable of inducing pathogenicity. Furthermore, even among pathogenic strains such as Clostridium difficile, not all members of the bacterial population are always present in an active, disease-causing state. Therefore, targeting Type VI systems based on the genotype of viruses, plasmids, or prokaryotic cells allows for specific control over which genomes or cell populations are targeted without disrupting the entire microbiome.

[0357] In addition, bacterial strains can be easily engineered using genetic circuits or environmentally controlled expression elements to generate genetic kill switches that limit the growth, colonization, and / or excretion of engineered bacterial strains. For example, expression of type VI effectors and specific crRNAs can be controlled using promoters derived from the regulatory regions of genes encoding proteins expressed in response to external stimuli, such as cold-sensitive proteins (PcspA), heat shock proteins (Hsp), and chemically inducible systems (Tet, Lac, AraC). Controlled expression of one or more elements of a type VI system allows for expression of a fully functional system only upon exposure to an environmental stimulus, resulting in the genotype-specific DNA interference activity of the system and ultimately inducing cell death or dormancy. Kill switches containing Cas12i effectors, such as those described herein, may be advantageous over traditional kill switch designs, such as toxin / antitoxin systems (e.g., CcdB / CcdA type II toxin / antitoxin systems), because they are independent of relative protein expression rates that can be affected by leaky expression from promoters (e.g., environmental stimulus-dependent promoters), thereby allowing for more precise control of the kill switch.

[0358] Other embodiments While the present invention has been described with a detailed description thereof, it should be understood that the foregoing description is illustrative and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

[Claim 1] The invention as set forth in the drawings.

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