New crispr-CAS effector proteins and the use thereof

Abstract

The present invention concerns new subtype V-U1 CRISPR-Cas effector proteins. The invention further relates to the methods of repressing or interfering with the expression of target gene sequences and base editing in cells or in any nucleic acid in vitro.

Description

[0001]NEW CRISPR-CAS EFFECTOR PROTEINS AND THE USE THEREOF FIELD OF INVENTION The invention relates to CRISPR-Cas effector proteins from the subtype V-U1 system and their use in expression control and base editing in cells. BACKGROUND OF INVENTION CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR- associated) systems protect prokaryotes from mobile genetic elements (MGEs) (Barrangou et al., 2007). To target foreign DNA or RNA in a sequence-specific manner Cas effectors utilize guide RNAs (gRNAs) that can consist of single CRISPR RNAs (crRNAs) or crRNAs duplexed with trans-activating crRNAs (tracrRNAs), that can be artificially linked to single- guide RNAs (sgRNAs). Based on Cas complex composition, CRISPR-Cas systems are classified into 2 classes and I-VI types. In class 1 CRISPR-Cas (type I, III, and IV), effector complexes are composed of multiple Cas proteins, while class 2 systems (type II, V, and VI) encompass a single Cas effector (Makarova et al., 2020; Shmakov et al., 2017). DNA targeting class 2 CRISPR-Cas systems, exemplified by Cas9 (type II) and Cas12 (type V) proteins, are of particular interest, due to their deployment for RNA-guided DNA targeting for genome engineering applications (J. Y. Wang et al., 2022; Wang and Doudna, 2023). The Cas12 family containing a single RuvC-like domain is highly diverse both in function and size (400-1,500 aa) (Burstein et al., 2017; Harrington et al., 2018; Karvelis et al., 2020; Pausch et al., 2020; Shmakov et al., 2015; Zetsche et al., 2015). The smallest Cas12 effectors (400-600 aa), initially classified as subtypes V-U1 to V-U5 (Shmakov et al., 2017), are of special interest since they can overcome the DNA cargo packing limitation of adeno- associated viruses (AAVs) which are the preferred delivery vectors for in vivo applications. The necessary requirement for efficient DNA target recognition by CRISPR-Cas effector proteins, including Cas9 and Cas12, is the presence of a short PAM (protospacer adjacent motif) sequence next to the target. To date, the majority of characterized Cas12 family proteins typically recognize 3-4 bp long T-rich PAM sequences introducing the constraints for target site selection for genome editing experiments (Anzalone et al., 2020; Karvelis et al., 2020). The targeting space limitation due to the PAM requirement may become more evident in more advanced genome editing applications relying on homology directed repair (HDR) or base editing as the efficiency and outcome of these approaches are heavily dependent on the proximity of targeted sequence (Anzalone et al., 2020). Therefore, shorter and / or non-T PAM recognizing Cas12 enzymes are highly desirable in genome editing applications to expand the targetable sequences. Additionally, emergence of adenine and cytosine base editing, prime editing and other technologies relying on fusion of additional domains / proteins to Cas effectors for targeted genome engineering increased the demand for smaller RNA-guided systems for their delivery using single AAV or as RNA using LNPs (Anzalone et al., 2020). The current Cas9- and Cas12-based base editors (BEs) can modify the bases across various positions within the protospacer (Anzalone et al., 2020) complicating the use of BEs for precise targeted modifications. Thus, BEs with reduced base editing window and increased base editing efficiency remain of interest. Therefore, small and robust CRISPR-Cas effector proteins remain highly desirable for genome editing and related applications. SUMMARY OF INVENTION The present disclosure provides new CRISPR-Cas effector proteins of the subtype V-U1 system and the use thereof. Directed by guide RNA, new CRISPR-Cas effector proteins bind double stranded DNA (dsDNA) at a target locus without cleaving it. The new CRISPR-Cas effectors use short T- or C-rich PAM sequences, therefore expanding the targetable space for genome engineering. When fused with deaminase, CRISPR-Cas effector proteins of the present disclosure are active in E. coli and human cells and can provide a narrow PAM- proximal editing window for more specific base editing. Accordingly, the present invention provides the following: In a first aspect, the present invention provides a) an isolated CRISPR-Cas effector protein comprising an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 1; b) an isolated CRISPR-Cas effector protein comprising an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 2; c) an isolated CRISPR-Cas effector protein comprising an amino acid sequence as set forth in SEQ ID NO: 3 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%.90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 3; d) an isolated CRISPR-Cas effector protein comprising an amino acid sequence as set forth in SEQ ID NO: 4 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 4; e) an isolated CRISPR-Cas effector protein comprising an amino acid sequence as set forth in SEQ ID NO: 5 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 5; f) an isolated CRISPR-Cas effector protein comprising an amino acid sequence as set forth in SEQ ID NO: 6 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 6; g) an isolated CRISPR-Cas effector protein, comprising an amino acid sequence as set forth in SEQ ID NO: 7 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 7; h) an isolated CRISPR-Cas effector protein comprising an amino acid sequence as set forth in SEQ ID NO: 8 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO: 8. In some embodiments, a composition comprising a CRISPR-Cas effector protein according to the present disclosure and a guide RNA (gRNA) is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising a CRISPR-Cas effector protein according to the present disclosure and a guide RNA (gRNA) is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence. In some embodiments, a polynucleotide encoding said CRISPR-Cas effector protein is provided, as well as an expression vector comprising said polynucleotide, and cell comprising said expression vector. In a second aspect, the present invention provides the isolated CRISPR-Cas effector protein, wherein the CRISPR-Cas effector protein is fused to at least one other protein. Alternatively, the CRISPR-Cas effector protein is fused to one or more proteins. In some embodiments, the CRISPR-Cas effector protein is fused to the at least one other protein that is selected from an enzyme, a ligand, or a marker. In preferred embodiments, the enzyme is a deaminase. In some embodiments, the deaminase is an adenine deaminase. In other embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is fused to the N-terminus of the CRISPR-Cas effector protein. In other embodiments, the deaminase is fused to the C-terminus of the CRISPR-Cas effector protein. In some embodiments, the CRISPR-Cas effector protein is fused to a deaminase, wherein the deaminase comprises an adenine deaminase and a cytidine deaminase. In some embodiments, the adenine deaminase is fused to the N-terminus of the CRISPR-Cas effector protein, and the cytidine deaminase is fused to the C-terminus of the CRISPR-Cas effector protein. In alternative embodiments, the adenine deaminase is fused to the C-terminus of the CRISPR-Cas effector protein, and the cytidine deaminase is fused to the N-terminus of the CRISPR-Cas effector protein. In other embodiments, the adenine deaminase, and the cytidine deaminase are fused at the N-terminus of the CRISPR-Cas effector protein. In yet other embodiments, the adenine deaminase and the cytidine deaminase are fused at the C- terminus of the CRISPR-Cas effector protein. In some embodiments, a composition comprising a CRISPR-Cas effector protein fusion to at least one other protein according to the present disclosure and a guide RNA (gRNA) is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising a CRISPR-Cas effector protein fused to at least one other protein according to the present disclosure and a guide RNA (gRNA) is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence.In some embodiments, a polynucleotide encoding said CRISPR-Cas effector protein is provided, as well as an expression vector comprising said polynucleotide, and cell comprising said expression vector. In a third aspect, the present invention provides a method of repressing or interfering with the expression of a target gene sequence by an organism, a cell, or in a cell-free expression system, comprising exposing double stranded DNA (dsDNA) comprising the target gene sequence to a CRISPR-Cas effector protein according to the present disclosure, and a guide RNA (gRNA) which directs said effector protein to the target gene sequence, wherein targeted binding of the effector protein to the dsDNA results repressing or interfering with the expression of a target gene sequence. In a fourth aspect, the present invention provides a method of repressing or interfering with the function of DNA elements controlling replication by an organism, a cell, or in a cell-free expression system, comprising exposing double stranded DNA (dsDNA) comprising the target gene sequence to a CRISPR-Cas effector protein according to the present disclosure, and a guide RNA (gRNA) which directs said effector protein to the target gene sequence, wherein targeted binding of the effector protein to the dsDNA results repressing or interfering with the function of DNA elements controlling replication. In some embodiments, in the method of the third or fourth aspect, an organism or cell is transfected with an expression vector comprising a polynucleotide encoding the CRISPR- Cas effector protein, under the control of a suitable expression promoter, and (a) is further transfected with a second expression vector which comprises an expression promoter and a sequence under the control of the promoter which encodes the guide RNA (gRNA); or (b) the gRNA is introduced directly into the organism or cell. In some embodiments, in the method of the third or fourth aspect, an organism or cell is transfected with an expression vector comprising a polynucleotide encoding the CRISPR- Cas effector protein, under the control of a suitable expression promoter, the vector further comprising a nucleotide sequence comprising an expression promoter and a sequence under the control of the promoter encoding a guide RNA (gRNA) with a nucleotide sequence which is complementary to a target sequence in double stranded DNA (dsDNA). In a fifth aspect, the present invention provides a method for base-editing a target sequence in an organism, a cell, or in a cell-free system comprising exposing a double stranded DNA (dsDNA) comprising the target sequence to a CRISPR-Cas effector protein according to present disclosure, fused to at least one other protein, and a guide RNA (gRNA) which directs said effector protein to the target sequence. In preferred embodiments, the at least one other protein is an enzyme, wherein the enzyme is a deaminase. In some embodiments, in the method for base-editing, an organism or cell is transfected with an expression vector comprising a polynucleotide encoding the CRISPR-Cas effector protein fused to a deaminase, under the control of a suitable expression promoter, and (a) further transfected with a second expression vector which comprises an expression promoter and a sequence under the control of the promoter which encodes the guide RNA (gRNA); or (b) the gRNA is introduced directly into the organism or cell. In some embodiments, in the method for base-editing the organism or cell is transfected with an expression vector comprising a polynucleotide encoding the CRISPR-Cas effector protein fused to a deaminase, under the control of a suitable expression promoter, the vector further comprising a nucleotide sequence comprising an expression promoter and a sequence under the control of the promoter encoding a guide RNA (gRNA) with a nucleotide sequence which is complementary to a target sequence in double stranded DNA (dsDNA).. In some embodiments, in the method of repressing or interfering with the expression of a target gene sequence or in the method of repressing or interfering with the function of DNA elements controlling replication, the CRISPR-Cas effector protein is introduced into the organism or cell simultaneously, sequentially or separately from gRNA. In some embodiments, in the method for base-editing, the CRISPR-Cas effector protein is introduced into the organism or cell simultaneously, sequentially or separately from gRNA. In some embodiments, in the methods of present invention the CRISPR-Cas effector protein is introduced into the cell in the form of an RNA encoding the CRISPR-Cas effector protein. In some embodiments, in the methods of present invention, gRNA is associated with the CRISPR-Cas effector protein before or upon introduction into the organism or cell. In some embodiments, the introducing of the CRISPR-Cas effector protein and / or gRNA into the cell comprises electroporation or microinjection, optionally in combination with the use of liposomes. In other embodiments, the introducing of the CRISPR-Cas effector protein and / or gRNA into the cell is by chemical-based transfection, such as lipofection, calcium phosphate transfection or cationic polymer transfection. In some embodiments, the introducing of the CRISPR-Cas effector protein and / or gRNA into the cell is by delivery with a viral vector. In preferred embodiments, the viral vector is a lentiviral vector, a retroviral vector or an AAV vector. In some embodiments, in the methods of present invention the cell is a prokaryotic cell. In other embodiments, the organism or cell is a eukaryote. In some embodiments, the cell is a non-human animal cell. In other embodiments, the cell is a human cell. DESCRIPTION OF DRAWINGS Figure 1. A non-redundant list of 134 Cas12m family variants identified in diverse bacteria according to Example 1. Figure 2. Characterization of subtype V-M CRISPR-Cas12m systems. (A) Schematic representation of selected subtype V-M CRISPR-Cas12m systems with representative Cas12m effector proteins. Amino acid residues at the predicted RuvC nuclease site are indicated above each cas12m gene. (B) Repeat sequences of CRISPR-Cas12m systems (from top to bottom: SEQ ID Nos: 156-163). (C) Experimental workflow of the biochemical approach used to detect dsDNA binding and examine PAM recognition. (D) WebLogos of the PAM sequences recovered for Cas12m proteins. Figure 3. Cas12m mediated plasmid DNA and bacteriophage interference in E. coli. (A) Results of plasmid DNA interference assay in E. coli. Effective DNA interference of target plasmid resulted in reduction of colony forming units (CFU). KanR - kanamycin resistance gene, ori - DNA replication site. (B) Experimental workflow of plasmid interference assay for superfolder GFP (sfGFP) expression reduction in E. coli. (C) Plasmid DNA expressing sfGFP interference assay in E. coli. Effective DNA binding-based DNA interference of target plasmid resulted in reduction of visible sfGFP expression (blue filter) while CFU remained unchanged (white light). T indicates target, and NT - non-target sequences containing crRNA constructs. (D) Results of bacteriophage plaque assay in E. coli. Effective phage interference resulted in reduction of plaque forming units. S indicates sense and A - antisense respective phage gene DNA strands targeted by Cas12 RNP complexes. Figure 4. GoABE DNA base editing in E. coli and human cells. (A) Experimental workflow of plasmid DNA interference assay for chloramphenicol resistance (CmR) recovery in E. coli. (B) Plasmid DNA interference assay in E. coli. Recovery of the colonies indicates successful targeted base editing. For positive control (Ctrl) E. coli transformation was performed with plasmid encoding intact CmR gene. T indicates target, and NT - non-target sequences containing crRNA constructs. (C) Sanger sequencing analysis of recovered colonies. (D) Experimental workflow of eGFP recovery assay performed in HEK293T cells. (E) Flow cytometry results of eGFP expression in HEK293T cells after 24, 48 and 72 h. (F) Genome base editing in HEK293T cells. DNA targets annotated according to (Richter et al., 2020). Gray areas indicate non-A nucleotides at specific positions across all targets. Sequences in (A): CmR (inactive) – SEQ ID NO: 164 (top) and SEQ ID NO: 165 (bottom), CmR (active) - SEQ ID NO: 166 (top) and SEQ ID NO: 167 (bottom); sequence in (D): eGFP (inactive) - SEQ ID NO: 168 (top) and SEQ ID NO: 169 (bottom), eGFP (active) - SEQ ID NO: 170 (top) and SEQ ID NO: 171 (bottom). Figure 5. GoABE and enAsABE genomic DNA base editing in human cells. DNA targets annotated according to (Richter et al., 2020). GoABE TadA-8e-GoCas12m and enAsABE - TadA-8e-enAsCas12a base editors. Figure 6. Cas12m proteins used in the present disclosure. Figure 7. Sequences of proteins used in this study. Elements in the protein construct names in bold, italic or underlined correspond to respectively marked amino acid sequences. Figure 8. Plasmids used in the present disclosure. Figure 9. RNAs used in the present disclosure. Elements in the RNA construct names in bold, italic or underlined correspond to respectively marked nucleotide sequences. Figure 10. Oligonucleotides and DNA activators used for different cleavage assays in the present disclosure. Elements in the molecule names in bold, italic or underlined correspond to respectively marked nucleotide sequences. Figure 11. Primers used for PAM sequence identification and base editing assays. Elements in the primer names in bold, italic or underlined correspond to respectively marked nucleotide sequences. Figure 12. Target sequences used in the present disclosure. Elements in the sequence names in bold, italic or underlined correspond to respectively marked nucleotide sequences. DETAILED DESCRIPTION OF THE INVENTION In the present disclosure, new CRISPR-Cas effector proteins of subtype V-M have been identified. Accordingly, the present disclosure relates in particular to these CRISPR-Cas effector proteins, effector complexes and methods involving their use in regulation and / or expression of target gene sequences, as well as fused CRISPR-Cas effector proteins and their use in the methods of base editing, in vitro, ex vivo and in vivo (prokaryotic and eukaryotic cells). CRISPR-Cas effector proteins The term “CRISPR-Cas effector protein”, as is used herein, refers to a protein of a CRISPR- Cas system that is RNA-guided to DNA or RNA target. Based on Cas complex composition, CRISPR-Cas systems are classified into 2 classes and I-VI types. The CRISPR-Cas effector proteins provided in the present disclosure are identified as class 2, subtype V-U1 proteins. Alternatively, the subtype V-U1 (subtype V-M) CRISPR Cas effector proteins are called Cas12m. These terms are used interchangeably in the present disclosure. The term “guide ribonucleic acid” “guide RNA”, “gRNA”, “crRNA”, as is used herein, refers to an RNA molecule (or to RNA molecules), that is capable of binding to the CRISPR- Cas effector protein to form the CRISPR-Cas effector complex, and which can guide or direct the effector complex to a target region in a double stranded DNA. The formed CRISPR-Cas effector complex may also be referred to as ribonucleoprotein effector complex (RNP). gRNA comprises at least two parts: (i) a protein-binding segment that allows the gRNA to bind to a CRISPR-Cas effector protein to form an effector complex, and (ii) a polynucleotide-targeting segment comprising a guide sequence (or “spacer sequence”) capable of hybridizing to a target sequence in a target region of a polynucleotide. The sequence on the target nucleic acid that is complementary to the sequence in the gRNA is termed a protospacer. The term “protospacer adjacent motif (PAM)”, as is used herein, refers to an additional nucleic acid sequence that is present on the target nucleic acid and resides adjacent to the protospacer sequence. The PAM size and location varies by CRISPR system and is typically a 2- to 5-bp sequence that may be located upstream or downstream of the protospacer motif. The PAM sequence recognition is required for initiation of gRNA binding to the matching DNA strand through Watson-Crick base pairing. The CRISPR-Cas effector proteins of the present disclosure (the Cas12m effector proteins) target double stranded DNA (dsDNA) in a PAM-specific manner. For efficient dsDNA targeting, the Cas12m effector proteins require a protospacer adjacent motif (PAM) that is upstream of the DNA sequence recognized by gRNA. The present disclosure provides new CRISPR-Cas effector proteins that are advantageously small in size (~600 amino acids), and do not possess double stranded DNA (dsDNA) target cleavage activity but are capable to specifically bind to the target DNA. The first exemplary CRISPR-Cas effector protein (GoCas12m) according to the present disclosure is an isolated CRISPR-Cas effector protein that comprises an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 1. Preferably, the CRISPR-Cas effector protein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, a composition comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence. In some examples, the CRISPR-Cas effector protein GoCas12m may form an effector complex with gRNA the protein-binding segment of which comprises or consists of GUGUCAACGCCAGCGCGGAGGCGUCAAAUCCGCGAC (SEQ ID NO: 9). The protein-binding segment of the gRNA may comprise additional insertions, deletions and / or mismatches which do not affect the efficiency of interaction of gRNA with the CRISPR-Cas effector protein when forming the effector complex. In some examples, the PAM sequence specific to CRISPR-Cas effector protein GoCas12m is 5’-TTN-3’. The second exemplary CRISPR-Cas effector protein (TfCas12m) according to the present disclosure is an isolated CRISPR-Cas effector protein that comprises an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 2. Preferably, the CRISPR-Cas effector protein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, a composition comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence. In some examples, the CRISPR-Cas effector protein TfCas12m may form an effector complex with gRNA the protein-binding segment of which comprises or consists of GUUGCAAUGCCUAGCUCAGAGGUUUAAAGACUGAGAC (SEQ ID NO: 10). The protein-binding segment of the gRNA may comprise additional insertions, deletions and / or mismatches which do not affect the efficiency of interaction of gRNA with the CRISPR-Cas effector protein when forming the effector complex. In some examples, the PAM sequence specific to CRISPR-Cas effector protein TfCas12m is 5’-CCN-3’. In other examples, the PAM sequence specific to CRISPR-Cas effector protein TfCas12m is 5’-TCN-3’. The third exemplary CRISPR-Cas effector protein (MsCas12m) according to the present disclosure is an isolated CRISPR-Cas effector protein that comprises an amino acid sequence as set forth in SEQ ID NO: 3 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 3. Preferably, the CRISPR-Cas effector protein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, a composition comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence. In some examples, the CRISPR-Cas effector protein MsCas12m may form an effector complex with gRNA the protein-binding segment of which comprises or consists of GUAGCAACGCCUAGCUCAGGGGCUCGAAAACUGAGACAA (SEQ ID NO: 11). The protein-binding segment of the gRNA may comprise additional insertions, deletions and / or mismatches which do not affect the efficiency of interaction of gRNA with the CRISPR-Cas effector protein when forming the effector complex. In some examples, the PAM sequence specific to CRISPR-Cas effector protein MsCas12m is 5’-TT-3’ or 5’-TC- 3’. The fourth exemplary CRISPR-Cas effector protein (PpCas12m) according to the present disclosure is an isolated CRISPR-Cas effector protein that comprises an amino acid sequence as set forth in SEQ ID NO: 4 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 4. Preferably, the CRISPR-Cas effector protein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, a composition comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence. In some examples, the CRISPR-Cas effector protein PpCas12m may form an effector complex with gRNA the protein-binding segment of which comprises or consists of GUUGACAUGCGUGCUCCGCCGCUUUGUAGUGGAGAC (SEQ ID NO: 12). The protein-binding segment of the gRNA may comprise additional insertions, deletions and / or mismatches which do not affect the efficiency of interaction of gRNA with the CRISPR-Cas effector protein when forming the effector complex. In some examples, the PAM sequence specific to CRISPR-Cas effector protein PpCas12m is 5’-CCN-3’. The fifth exemplary CRISPR-Cas effector protein (MkCas12m) according to the present disclosure is an isolated CRISPR-Cas effector protein that comprises an amino acid sequence as set forth in SEQ ID NO: 5 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 5. Preferably, the CRISPR-Cas effector protein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5. In some embodiments, a composition comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence. In some examples, the CRISPR-Cas effector protein MkCas12m may form an effector complex with gRNA the protein-binding segment of which comprises or consists of GUUACAAACCCUGCUCAUUGGGUUGGUUAAUGAGAC (SEQ ID NO: 13). The protein-binding segment of the gRNA may comprise additional insertions, deletions and / or mismatches which do not affect the efficiency of interaction of gRNA with the CRISPR-Cas effector protein when forming the effector complex. In some examples, the PAM sequence specific to CRISPR-Cas effector protein MkCas12m is 5’-TTN-3’. The sixth exemplary CRISPR-Cas effector protein (ApCas12m) according to the present disclosure is an isolated CRISPR-Cas effector protein that comprises an amino acid sequence as set forth in SEQ ID NO: 6 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 6. Preferably, the CRISPR-Cas effector protein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, a composition comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence. In some examples, the CRISPR-Cas effector protein ApCas12m may form an effector complex with gRNA the protein-binding segment of which comprises or consists of GUGACAAAGCCCUGUGCAGCGGGCUCAAAGCUGCGAC (SEQ ID NO: 14). The protein-binding segment of the gRNA may comprise additional insertions, deletions and / or mismatches which do not affect the efficiency of interaction of gRNA with the CRISPR-Cas effector protein when forming the effector complex. The seventh exemplary CRISPR-Cas effector protein (UnCas12m) according to the present disclosure is an isolated CRISPR-Cas effector protein that comprises an amino acid sequence as set forth in SEQ ID NO: 7 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 7. Preferably, the CRISPR-Cas effector protein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7. In some embodiments, a composition comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence. In some examples, the CRISPR-Cas effector protein UnCas12m for may form an effector complex with gRNA the protein-binding segment of which comprises or consists of GUUGCAAUGCCUGGCUCAGGGGUAUAAGAACUGAGAC (SEQ ID NO: 15). The protein-binding segment of the gRNA may comprise additional insertions, deletions and / or mismatches which do not affect the efficiency of interaction of gRNA with the CRISPR-Cas effector protein when forming the effector complex. The eighth exemplary CRISPR-Cas effector protein (MmCas12m) according to the present disclosure is an isolated CRISPR-Cas effector protein that comprises an amino acid sequence as set forth in SEQ ID NO: 8 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 8. Preferably, the CRISPR-Cas effector protein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, a composition comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising the CRISPR-Cas effector protein and a gRNA is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence. In some examples, the CRISPR-Cas effector protein MmCas12m may form an effector complex with gRNA the protein-binding segment of which comprises or consists of GUGUCAUAGCCCAGCUUGGCGGGCGAAGGCCAAGAC (SEQ ID NO: 16). The protein-binding segment of the gRNA may comprise additional insertions, deletions and / or mismatches which do not affect the efficiency of interaction of gRNA with the CRISPR-Cas effector protein when forming the effector complex. In some examples, the PAM sequence specific to CRISPR-Cas effector protein MmCas12m is 5’-TTN-3’. Also disclosed herein are polynucleotides encoding a CRISPR-Cas effector according to the present disclosure. A polynucleotide encoding a CRISPR-Cas effector according to the present disclosure may be comprised in an expression vector, under the control of a suitable expression promoter. The expression vector may further comprise a nucleotide sequence comprising an expression promoter and a sequence under the control of the promoter encoding a guide RNA (gRNA) with a nucleotide sequence which is complementary to a target sequence in double stranded DNA (dsDNA), wherein gRNA is capable of forming an effector complex with a respective CRISPR-Cas effector protein. The present disclosure also provides cells comprising the expression vectors. The present disclosure also relates to DNA and RNA encoding CRISPR-Cas effector proteins, whereas expression of the DNA or RNA can occur in vitro, ex vivo or in vivo. Further provided are the CRISPR-Cas effector proteins according to the present disclosure, wherein the effector proteins are fused to at least one other protein. In some examples, the CRISPR-Cas effector protein is fused to one or more proteins. A CRISPR-Cas effector protein that is fused to one or more other proteins, as used herein, may be referred to as a fusion protein. The at least one protein may be selected from an enzyme, a ligand, or a marker. In some examples, the enzyme is a deaminase. The term “deaminase”, as is used herein, refers to an enzyme that causes removal of an amine group from a compound. A deaminase often is named according to its substrate, e.g., an adenine deaminase, a cytidine deaminase, a guanidine deaminase. A wide selection of deaminases or their variants may be used, deaminases suitable for use in fusion proteins with type V CRISPR-Cas proteins are listed in Liu et al. 2023. Exemplary deaminases may comprise TadA engineered variants (e.g., TadA-8e), APOBEC variants (e.g., rAPOBEC1), AID (e.g., PmCDA1) and other deaminases and their variants. Fusion proteins comprising a CRISPR-Cas effector protein and a cytidine deaminase may also comprise a uracil glycosylase inhibitor (UGI). Fusion proteins comprising a CRISPR-Cas effector protein and an adenine deaminase may also comprise hypoxanthine excision protein N-methylpurine DNA glycosylase (MPG). In some examples, where the CRISPR-Cas effector protein according to the present disclosure is fused to a deaminase, the deaminase is fused to the N-terminus or to the C- terminus of the CRISPR-Cas effector protein. The CRISPR-Cas effector protein may be fused to more than one deaminase. In cases where CRISPR-Cas effector protein is fused to more than one deaminase, the deaminase may comprise an adenine deaminase and a cytidine deaminase. In such fusion protein, for example, the adenine deaminase is fused to the N- terminus of the CRISPR-Cas effector protein, and the cytidine deaminase is fused to the C- terminus of the CRISPR-Cas effector protein. In other examples, the adenine deaminase is fused to the C-terminus of the CRISPR-Cas effector protein, and the cytidine deaminase is fused to the N-terminus of the CRISPR-Cas effector protein. In yet further examples, both the adenine deaminase and the cytidine deaminase are fused to the N-terminus of the CRISPR-Cas effector protein, for example, in tandem order, with or without linker sequences. In other examples, both the adenine deaminase and the cytidine deaminase are fused to the C-terminus of the CRISPR-Cas effector protein, for example, in tandem order, with or without linker sequences. In some examples, the CRISPR-Cas effector protein may be fused to more than one adenine deaminase. In more specific examples, the CRISPR-Cas effector protein is GoCas12m that is fused at its N-terminus or C-terminus to an adenine deaminase and / or a cytidine deaminase. Preferably, GoCas12m is fused at its N-terminus to an adenine deaminase. In other examples, the CRISPR-Cas effector protein selected from TfCas12m, MsCas12m, PpCas12m, MkCas12m, ApCas12m, UnCas12m, MmCas12m is fused at its N-terminus or C-terminus to an adenine deaminase and / or a cytidine deaminase. Preferably, the selected CRISPR-Cas effector protein is fused at its N-terminus to an adenine deaminase. The deaminase may be fused to a CRISPR-Cas effector protein via a linker. A linker may be a short amino acid sequence that may be used for providing more flexibility between the two fused or linked proteins. The linker may include from 2 to about 200 amino acids, preferably the linker includes from 1 to 100 amino acids, from 2 to 50 amino acids, or, more preferably from 4 to about 32 amino acids, more preferably, 4, 16 or 32 amino acids. In some embodiments, a composition comprising a CRISPR-Cas effector protein fused to at least one other protein as described herein and a guide RNA (gRNA) is provided, wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence. In some embodiments, an effector complex comprising a CRISPR-Cas effector protein fused to at least one other protein as described herein and a guide RNA (gRNA) is provided, wherein the gRNA is capable of directing binding of the effector complex to a target sequence. Methods of use The CRISPR-Cas effector proteins according to the present disclosure can be useful in several ways. In one aspect, the present disclosure provides a method of repressing or interfering with the expression of a target gene sequence by an organism, a cell, or in a cell-free expression system, comprising exposing double stranded DNA (dsDNA) comprising the target gene sequence to a CRISPR-Cas effector protein, as disclosed in a previous section herein, and a guide RNA (gRNA) which directs said CRISPR-Cas effector protein to the target gene sequence, wherein targeted binding of the effector protein to the dsDNA results repressing or interfering with the expression of a target gene sequence. The method may also be used for repressing or interfering with the function of DNA elements controlling replication, such as, for example, origin of replication (ori) of a plasmid. In another aspect, the present disclosure provides a method for base-editing a target sequence in an organism, a cell, or in a cell-free system comprising exposing a double stranded DNA (dsDNA) comprising the target sequence to a CRISPR-Cas effector protein fused to a deaminase, as disclosed in the section “CRISPR-Cas effector proteins” herein, and a guide RNA (gRNA) which directs said effector protein to the target sequence. The term “base-editing”, as is used herein, refers to the conversion of one target base or base pair into another, for example A:T to G:C, or C:G to T:A, or conversion of A:T to C:G or T:A, without requiring the creation and repair of double strand breaks. Base editing may be achieved with the help of DNA and RNA base editors that allow the introduction of point mutations at specific sites, in either DNA or RNA. DNA base editors often comprise a fusion of a DNA-binding molecule that is fused to a catalytically active base-modification enzyme that acts on single-stranded DNAs. Said DNA binding molecule often is a catalytically inactive nuclease, such as a clustered regularly interspaced short palindromic repeat (CRISPR)-associated sequence (Cas) nuclease. So far, two types of DNA editors have been developed, a cytosine base editor (CBE) and an adenine base editor (ABE). In order to be used in the methods as disclosed above, CRISPR-Cas effector proteins and CRISPR-Cas effector proteins fused to at least one other protein such as deaminase, of the present disclosure and corresponding gRNA may be delivered into an organism, a cell or a cell-free system. In some examples, in order to provide a target sequence to the CRISPR-Cas effector protein or the CRISPR-Cas effector protein fused to deaminase, and a guide RNA which direct said effector protein to the target sequence, an organism or cell may be transfected with an expression vector comprising a polynucleotide encoding said CRISPR-Cas effector protein or the CRISPR-Cas effector protein fused to deaminase, under the control of a suitable expression promoter, and (a) further transfected with a second expression vector which comprises an expression promoter and a sequence under the control of the promoter which encodes the guide RNA (gRNA); or (b) the gRNA is introduced directly into the organism or cell. Alternatively, the organism or cell may be transfected with an expression vector a polynucleotide encoding said CRISPR-Cas effector protein or the CRISPR-Cas effector protein fused to deaminase, under the control of a suitable expression promoter, the vector further comprising a nucleotide sequence comprising an expression promoter and a sequence under the control of the promoter encoding a guide RNA (gRNA) with a nucleotide sequence which is complementary to a target sequence in double stranded DNA (dsDNA). The CRISPR-Cas effector protein or CRISPR-Cas effector protein fused to a deaminase may be introduced into the organism or cell simultaneously, sequentially or separately from gRNA. In some examples, the CRISPR-Cas effector protein or CRISPR-Cas effector protein fused to a deaminase is introduced into the cell in the form of an RNA encoding the CRISPR-Cas effector protein. In some examples, gRNA is associated with the CRISPR-Cas effector protein before or upon introduction into the organism or cell. The effector complex of the present disclosure may be produced in vitro, ex vivo or in vivo. In particular, the method can comprise assembly of the effector complex from the RNA and the protein described herein in cells or in vitro in a cell-free system. Where the effector complex is produced in cells, the method may comprise providing the following in the cell: (i) the gRNA described herein, and DNA encoding the CRISPR-Cas effector protein described herein; (ii) the CRISPR-Cas effector protein described herein, and DNA encoding the gRNA described herein; or (iii) DNA encoding the CRISPR-Cas effector protein described herein and DNA encoding the gRNA described herein; (iv) RNA (mRNA) encoding the CRISPR-Cas effector protein described herein and the gRNA described herein; or (v) RNA (mRNA) encoding the CRISPR-Cas effector protein described herein and DNA encoding the gRNA described herein. Depending on how the CRISPR-Cas effector protein or effector complex is to be delivered to the cell, the protein may also comprise additional peptides such as nuclear localization signal, cell penetrating peptide, or similar. Where the effector complex is produced in vitro in a cell-free system, the method may comprise in vitro expression of DNA encoding the RNA, in vitro expression of DNA encoding the protein, or in vitro expression of both DNA encoding the RNA and DNA encoding the protein. The CRISPR-Cas effector protein and / or gRNA may be introduced into the cell by electroporation or microinjection, optionally in combination with the use of liposomes. Alternatively, the introducing of the CRISPR-Cas effector protein and / or gRNA into the cell may be by chemical-based transfection, such as lipofection, calcium phosphate transfection or cationic polymer transfection. The introducing of the CRISPR-Cas effector protein and / or gRNA into the cell may be by delivery with a viral vector, including lentiviral vectors, retroviral vectors and AAV vectors. In particular, delivery systems based on those already described for the CRISPR Cas9 and Cas12 systems can be utilised (see e.g., https: / / blog.addgene.org / crispr-101-mammalian- expression-systems-and-delivery-methods). The methods of the present disclosure have utility in an organism, cell or cell-free system. The cell may be a prokaryotic cell or eukaryotic cell. Where the cell is a eukaryotic cell, it may be a non-human cell or a human cell. The disclosure will now be described in more detail, by way of example only, with reference to the following experimental work. EXAMPLES Materials and Methods Identification of CRISPR-Cas V-U1 family members. The initial search of CRISPR-Cas subtype V-U1 candidates, was conducted in the NCBI nr (Sayers et al., 2021), IMG / M (Chen et al., 2021), and IMG / VR (Roux et al., 2021) databases using online versions of BLASTP (E-value < 1e-5) (Camacho et al., 2009) and a previously identified subtype V-U1 family effector protein from Gordonia otitidis NBRC 100426 (GAB36148.1) (Makarova et al., 2020; Shmakov et al., 2017) as a query. Next, from the retrieved candidates, we selected the Cas12m variant from Thermanaerosceptrum fracticalcis DRI-13 (QNB45423.1) and performed a second round of search using the same strategy. The retrieved proteins from both searches were combined and variants larger than 300 amino acids (aa) were retained for further analysis. The obtained Cas12m candidates were then clustered using online version of MMseqs2 (0.95 identity, 0.8 coverage) (Gabler et al., 2020), and only the largest variant was retained from each cluster, resulting in a non-redundant list of 134 Cas12m protein variants. Nucleotide sequences up to 2.5 kb upstream and downstream of the Cas12m encoding genes were analyzed using an online version of the CRISPRCasFinder tool (Couvin et al., 2018) to detect CRISPR region. Finally, 8 CRISPR-Cas subtype V-M systems were selected for further analysis (Figure 6). Cloning of Cas12m expression vectors. The genes of selected Cas12m proteins were synthesized and cloned into pBAD expression vectors (Twist Biosciences) fused with N- terminal Twin-Strep-10×His-MBP purification tag encoding sequences (pTK200, pTK203, pTK216-217). Cas12m expression vectors containing a minimal CRISPR region, consisting of two repeats and a single spacer between them, were cloned using Golden Gate strategy (pTK205-208, pTK213-214, pTK218-219). Specifically, the fragments of the minimal CRISPR region with the native adjacent sequences (up to 300 bp downstream the last repeat) were ordered as gBlocks (IDT). A plasmid encoding Cas12m and fragments of its respective CRISPR region was incubated with FD BpiI (Thermo Fisher Scientific) and T4 DNA ligase (Thermo Fisher Scientific) in 1× FastDigest buffer supplemented with 0.5 mM ATP (Thermo Fisher Scientific) for 30 cycles at 37 °C / 5 min and 16 °C / 5 min, followed by 65 °C 10 min. The plasmids were propagated in E. coli DH5ɑ and purified using GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific). Descriptions of plasmids of the present disclosure are provided in Figure 8. Expression and purification of Cas12 effectors and their RNP complexes. For Cas12m and Cas12a protein purification, E. coli DH10B strain was transformed with expression vectors (pMBP-AsCas12a (gift from Jennifer Doudna, Addgene plasmid #113430), pGB060 (produced from pMBP-AsCas12a by introducing D908A modification, using Phusion Site- Directed Mutagenesis Kit (Thermo Fisher Scientific), pTK200, pTK203). The cells were grown in LB broth supplemented with ampicillin (100 μg / ml) at 37 °C until OD600reached 0.8. The protein expression was induced with 0.2% L-arabinose and the cells were grown for additional 16h at 16 °C. Cells were pelleted by centrifugation, resuspended in 20 mM Tris-HCl (pH 8.0 at 25 °C), 1 M NaCl, 5 mM 2-mercaptoethanol, 25 mM imidazole and 5% (v / v) glycerol buffer, and disrupted by sonication. Cell debris was removed by centrifugation and the remaining supernatant was loaded on Ni2+-charged HiTrap chelating HP column (GE Healthcare). The proteins of interest were eluted by increasing imidazole concentration from 25 mM to 500 mM in 20 mM Tris-HCl (pH 8.0 at 25 °C), 500 mM NaCl, 5 mM 2- mercaptoethanol and 5% (v / v) glycerol buffer. The fractions containing the protein of interest were pooled and the 10×His-MBP tag was cleaved by incubating with TEV protease overnight at 4 °C. To remove the cleaved 10×His-MBP tag and TEV protease, reaction mixtures were loaded onto a HiTrap heparin HP 5 column (GE Healthcare) and a linear gradient of increasing NaCl concentration (250-1000 mM) was used for elution. The collected fractions with Cas12 proteins were dialyzed against 20 mM Tris-HCl (pH 8.0 at 25 °C), 0.5 M NaCl, 2 mM DTT and 50% (v / v) glycerol buffer, and stored at -20^°C. For Cas12m RNP complex purification, E. coli DH10B strain was transformed with expression vectors encoding Cas12m and their respective minimal CRISPR region (pTK205-208, pTK213-214, pTK218-219). The cells were grown in LB broth supplemented with ampicillin (100 μg / ml) at 37 °C until OD600 reached 0.8. The protein expression was induced with 0.2% L-arabinose and the cells were grown for additional 16h at 16 °C. Cells were pelleted by centrifugation, resuspended in 20 mM Tris-HCl (pH 8.0 at 25 °C), 100 mM NaCl, 5 mM 2-mercaptoethanol, 25 mM imidazole, 5% (v / v) glycerol and 1× HaltTMProtease Inhibitor Cocktail (Thermo Fisher Scientific) containing buffer and disrupted by sonication. Cell debris was removed by centrifugation and the remaining supernatant was loaded on Ni2+-charged HiTrap chelating HP column (GE Healthcare). The Cas12m RNP complexes were eluted by increasing imidazole concentration from 25 mM to 500 mM in 20 mM Tris-HCl (pH 8.0 at 25 °C), 100 mM NaCl, 5 mM 2-mercaptoethanol and 5% (v / v) glycerol buffer. The collected fractions containing Cas12m RNP were dialyzed against 20 mM Tris-HCl (pH 8.0 at 25 °C), 100 mM NaCl, 2 mM DTT, and 50% (v / v) glycerol buffer and stored at -20 °C. Extraction and sequencing of Cas12m bound RNA species. Cas12m RNP samples (100 ul) were treated with 5 ul (20 mg / ml) of Proteinase K (Thermo Fisher Scientific) for 45 min at 37 °C in 1 ml of 10 mM Tris-HCl (pH 7.5 at 37 °C), 100 mM NaCl, 5 mM MgCl2, 1 mM DTT and 1 mM EDTA buffer. Then 2.5 ul (1 U / µl) of DNase I (Thermo Fisher Scientific) was added and the samples were incubated for additional 30 min at 37 °C. RNA was purified using GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Fisher Scientific). The extracted RNA (1 µg) was treated with RiboCop rRNA Depletion Kit (Lexogen) and end- repaired with T4 Polynucleotide Kinase (20 U) in 1× reaction buffer A (Thermo Fisher Scientific) supplemented with 1 mM ATP (Thermo Fisher Scientific) for 1h at 37 °C. The reaction products were purified using RNA Clean & Concentrator-5 kit (Zymo Research) and 11-36 ng of RNA was used for library generation with the Small RNA-Seq Library Prep Kit (Lexogen). The obtained libraries were quantified by Qubit 4 Fluorometer (Thermo Fisher Scientific), pooled in an equimolar ratio, and paired-end sequenced (2 × 75 bp) using the MiniSeq High Output Reagent Kit, 150 cycles (Illumina) on a MiniSeq System (Illumina). The adapter sequences and paired-end reads shorter than 15 bp were filtered using Cutadapt (v2.8) (Martin, 2011). The remaining reads were mapped to the corresponding Cas12m and CRISPR expression plasmids (pTK205-208, pTK213-214, pTK218-219) using BWA-MEM (v0.7.17-r1188) (Li and Durbin, 2010). Paired-end reads with mapping Phred score < 30 were filtered out with SAMtools (v1.10) (Li et al., 2009). Custom Python script (Karvelis, 2023, https: / / doi.org / 10.5281 / zenodo.8354294) was used to extract and visualize entire RNA sequences (≤ 65 nt) from paired-end alignments. RNA synthesis. Templates for in vitro transcription of Cas12m pre-crRNAs were generated by PCR of respective Cas12m systems minimal CRISPR region with addition of a T7 promoter sequence at the 5’-end. Similarly, templates for in vitro transcription of RNA targets and crRNAs were generated by PCR of respective oligo duplexes. Obtained RNA templates were transcribed using TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) and purified using GeneJET RNA Cleanup and Concentration Kit (Thermo Fisher Scientific). Sequences of the RNAs used in this study are available in Figure 9. Pre-crRNA processing assays. Pre-crRNA processing was verified using in vitro synthesized putative pre-crRNAs and purified Cas12m proteins. Reaction mixtures contained 500 nM pre-crRNA, 2000 nM (or as indicated) Cas12m RNP complex in 10 mM Tris-HCl (pH 7.5 at 37^°C), 1 mM EDTA, 1 mM DTT, 100 mM NaCl and 10 mM MgCl2buffer. Reactions were incubated at 37 °C for 1h, followed by protein inactivation with the addition of Proteinase K (final conc.1 mg / ml) and incubation at 50 °C for 10 min. The final quench of the reaction was performed by adding EDTA (15 nM final conc.) and 2× RNA loading dye (Thermo Fisher Scientific), and incubating at 75 °C for 10 min. Reaction products were resolved using denaturing urea-PAGE (15% acrylamide:bis-acrylamide 29:1, 8.5 M urea, 0.5× TBE) gel stained with SYBR Gold Nucleic Acid Stain (Invitrogen) and imaged on an Amersham Typhoon phosphorimager (GE Healthcare). Cell-free Cas12m RNP complex production. Cell-free Cas12m RNP complexes were obtained using PURExpress In Vitro Protein Synthesis Kit (NEB) cell-free in vitro transcription / translation (IVTT) system. DNA templates for Cas12m production were generated by PCR from the pBAD expression vectors encoding Cas12m proteins (Twist Biosciences, pTK205-208, pTK213-214, pTK218-219). crRNAs targeting 20 bp sequence in the plasmid library (pTZ57) adjacent to a 7 bp (7N) randomized PAM (protospacer adjacent motif) sequence (Karvelis et al., 2019, 2015) were assembled by PCR from oligonucleotides (Figure 10). IVTT reactions were carried out from Cas12m and crRNA encoding DNA constructs (125 ng each) in 12.5 µl IVTT reaction mixture for 4 h at 37 °C. Crude mixtures of such prepared RNP complexes later were used for the following dsDNA cleavage and binding activity determination assays. dsDNA cleavage activity determination using plasmid PAM library. To test DNA cleavage activity of Cas12m RNP complexes, the previously developed dsDNA cleavage assay using 7N PAM plasmid library was adopted (Gasiunas et al., 2020; Karvelis et al., 2019, 2015) with the following modifications. Five µl of IVTT-prepared crude reaction mixture with Cas12m RNP complex were incubated with 500 ng of pTZ57 7N PAM library in 50 μl reaction buffer containing 10 mM Tris-HCl (pH 7.5 at 37^°C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2for 1 h at 37 °C. After incubation, DNA ends were repaired by adding 0.5 μl (2.5 U) of T4 DNA polymerase (Thermo Fisher Scientific), 0.5 μl of 10 mM dNTP mix (Thermo Fisher Scientific) followed by incubation for 20 min at 11^°C and 10 min at 75^°C. Next, 3′-dA overhangs were added by incubating the mixture with 0.5 μl (2.5 U) of DreamTaq polymerase (Thermo Fisher Scientific) and 0.5 μl of 10 mM dATP (Thermo Fisher Scientific) for 30 min at 72^°C. RNA was degraded by incubating for 15 min at 37^°C with 0.5 μl of 10 mg / ml of RNase A (Thermo Fisher Scientific), followed by DNA purification using GeneJet PCR Purification Kit (Thermo Fisher Scientific). Next, 100 ng of purified DNA products were ligated with 100 ng of dsDNA adapter containing a 3′-dT overhang (Figure 10) for 1 h at 22^°C using 1 μl (5 U) of T4 DNA ligase (Thermo Fisher Scientific) in 25 μl reaction volume. The ligation products (2 μl) were PCR amplified with Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific) using TK-737 and an equimolar mixture of TK-735, 765-771 oligonucleotides (Figure 11) for 17 cycles in 20 μl reaction volume. In the second round of PCR P5 and P7 adapters for Illumina sequencing were added using the Lexogen PCR Add on Kit (Lexogen) with the i76 nt Index Set (Lexogen). For this 3 μl of the first round PCR products were used as a template in 30 μl total reaction volume for 17 cycles. Indexed DNA samples were pooled in an equimolar ratio, gel purified with GeneJet Gel Purification Kit (Thermo Fisher Scientific) and quantified by Qubit 4 Fluorometer (Thermo Fisher Scientific). Lastly, samples were single-end sequenced (1 × 150 bp) using the MiniSeq High Output Reagent Kit, 150 cycles (Illumina) on a MiniSeq System (Illumina). dsDNA cleavage activity was evaluated by examining the adapter ligation at the targeted sequence in the plasmid library. The reads containing the adapter ligated at the 0-30 bp target positions next to the 7N region were counted using a custom Python script (Karvelis, 2023, https: / / doi.org / 10.5281 / zenodo.8354294). dsDNA binding activity determination using plasmid PAM library. To test the DNA binding activity of Cas12m RNP complexes, the previously developed 7N PAM plasmid library- based dsDNA cleavage assay was modified to include treatment with restriction enzyme, similar to the previously developed assay (Wimmer et al., 2022). Briefly, 2 ul of IVTT- reaction mixture with Cas12m RNP complex were incubated with 40 ng of 7N PAM library in 20 μl reaction buffer containing 10 mM Tris-HCl (pH 7.5 at 37^°C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 for 1 h at 37 °C. After incubation, the samples were diluted 10 times, and 40 μl of each sample was further incubated with 1 μl of FastDigest Eam1105I (Thermo Fisher Scientific) for 1 h at 37 °C, followed by additional incubation for 20 min at 65^°C. Next, samples were treated with 1 ul (20 mg / ml) of Proteinase K (Thermo Fisher Scientific) for 30 min at 50 °C. RNA was removed by incubating with 1 μl of 10 mg / ml RNase A (Thermo Fisher Scientific) for 15 min at 37^°C and for 5 min at 95^°C. The resulting reaction mixture of PAM plasmid library (5 μl) was PCR amplified with Phusion Hot Start II DNA polymerase (Thermo Fisher Scientific) using TK-737 and an equimolar mixture of TK-736, 772-778 oligonucleotides (Figure 11) for 25 cycles in 50 μl reaction volume and purified using the GeneJet PCR Purification Kit (Thermo Fisher Scientific). In the second round of PCR P5 and P7 adapters for Illumina sequencing were added using the Lexogen PCR Add on Kit (Lexogen) with the i76 nt Index Set (Lexogen). Two ng of the products from the first round of PCR were used as a template in 30 μl total reaction volume for 25 cycles. Indexed DNA samples were pooled in an equimolar ratio, gel purified with GeneJet Gel Purification Kit (Thermo Fisher Scientific) and quantified by Qubit 4 Fluorometer (Thermo Fisher Scientific). Lastly, samples were single-end sequenced (1 × 150 bp) using the MiniSeq High Output Reagent Kit, 150 cycles (Illumina) on a MiniSeq System (Illumina). The obtained sequences were used for PAM extraction, frequency calculation, and normalization to the original 7N PAM library to account for inherent biases. To reduce the influence of background noise resulting from the incomplete cleavage of all available targets by Eam1105I, only PAM sequences with > 5-fold enrichment compared to the sequenced initial library were extracted and represented in WebLogo format (Crooks, 2004). All sequence manipulations and analyses were performed using a custom Python script (Karvelis, 2023, https: / / doi.org / 10.5281 / zenodo.8354294). RNP complex assembly. Purified Cas12m protein (1 µM) was combined with its corresponding crRNA in 1:1 molar ratio in complex assembly buffer (10 mM Tris-HCl (pH 7.5 at 37 °C), 100 mM NaCl, 1 mM EDTA, 1 mM DTT and 10 mM MgCl2) and allowed to incubate for 30 min at 37 °C. DNA substrate generation. The 5’-ends of oligonucleotides were first radiolabeled using T4 PNK (Thermo Fisher Scientific) and [γ-32P]ATP (PerkinElmer). The dsDNA substrates were generated by annealing two oligonucleotides with complementary sequences, one of which had a radioactive label introduced at the 5’-end. Annealing was performed at 95 °C followed by a slow cooling to room temperature. The sequences of the DNA target substrates are provided in Figure 10. DNA cleavage assay. Reactions with oligo duplexes or ssDNA oligonucleotides were carried out by mixing labeled DNA samples with preassembled Cas12m or Cas12a RNP complexes and incubating for 1 h at 37 °C. Reaction mixtures contained 1 nM labeled oligo duplex, 100 nM Cas12 RNP complex, and 10 mM Tris-HCl (pH 7.5 at 37^°C), 1 mM EDTA, 1 mM DTT, 100 mM NaCl and 10 mM MgCl2in a 50 µl final volume. Aliquots of 6 μl were removed from the reaction mixture and quenched with 10 μl of loading dye (95% (v / v) formamide, 0.01% Bromophenol Blue and 25 mM EDTA). Reaction products were analyzed by denaturing gel electrophoresis (20% polyacrylamide containing 8.5 M urea in 0.5× TBE buffer), followed by gels being dried and visualized by phosphor imaging. RNA substrate generation. The 5’-ends of transcribed target RNA molecules were first dephosphorylated using FastAP (Thermo Fisher Scientific) and then radiolabeled using T4 PNK (Thermo Fisher Scientific) and [γ-32P]ATP (PerkinElmer). The sequences of RNA targets are provided in Figure 9. RNA cleavage assay. Reactions were carried out by mixing labeled RNA samples with preassembled Cas12m or Cas12a RNP complex and incubating for 1 h at 37 °C. Reaction mixtures contained 1 nM labeled RNA, 100 nM Cas12m RNP complex, and 10 mM Tris- HCl (pH 7.5 at 37^°C), 1 mM EDTA, 1 mM DTT, 100 mM NaCl and 10 mM MgCl2 in a 20 µl final volume. Aliquots of 10 μl were removed from the reaction mixture and quenched with 10 μl of 2×RNA gel loading dye (Thermo Fisher Scientific). Reaction products were analyzed by denaturing gel electrophoresis (15% polyacrylamide containing 8.5 M urea in 0.5× TBE buffer), followed by drying gels and visualizing by phosphor imaging. Trans-cleavage assay. Target-activated trans-cleavage activity was measured by performing the fluorophore quencher-labeled reporter assays. Reaction mixtures contained 100 nM preassembled Cas12m RNP complex, 25 nM ssDNA or dsDNA activator, and 100 nM DNaseAlert or RNaseAlert reporters (IDT), respectively. Activator dsDNA duplex was prepared by annealing 1.15-fold molar excess of the non-target strand to the target strand. Reactions were performed in 10 mM Tris-HCl (pH 7.5 at 37^°C), 1 mM EDTA, 1 mM DTT, 100 mM NaCl and 10 mM MgCl2 buffer in a 50 µl final volume. Reactions were carried out in ClarioStar fluorescence plate-reader (BMG Labtech) for 2h at 37 °C, with fluorescence measurements taken every 2 min (DNaseAlert reporter - excitation filter: 533 nm / 8 bandpass, emission filter: 559 nm / 8 bandpass; RNaseAlert reporter - excitation filter: 484 nm / 20 bandpass, emission filter: 529 nm / 20 bandpass). The sequences of the DNA activators are listed in Figure 10. Plasmid interference assay. Plasmid interference assays were performed in E. coli DH10B strain bearing Cas12m (pTK147, pRZ169 and pTK200, pTK203, pTK216-217) and crRNA guide sequence (pRZ166-168, pRZ170-173, pRZ184-188 and pRZ194-201) containing plasmids . The cells were grown at 37 °C overnight, diluted 100× in LB medium, and additionally grown to OD600 of ~0.5-0.6. Following that, a third chemical transformation was performed with 250 ng of target encoding plasmids (pKP8 for pRZ169, pTK147, pTK200, pKP9 for pTK203, and pKP11 for pTK216-217). The co-transformed cells were further diluted by serial 10× fold dilutions and grown at 37 °C overnight on plates containing L-arabinose (0.2%), glucose (1%), carbenicillin (100 µg / ml), chloramphenicol (15 µg / ml) and kanamycin (100 µg / ml). The efficiency of interference calculations for a given condition was performed by normalizing the mean of colony-forming units (CFU) for a condition to the mean CFU of a non-targeting control: mean(CFU_condition) / mean(CFU_negative_control). All interference assays were performed in biological triplicates. Target sequences can be found in Figure 12. GFP silencing assay. Similarly to plasmid interference assays, silencing assays were performed in E. coli DH10B strain bearing Cas12m (pTK147, pRZ169 and pTK200) and crRNA (pRZ166-167, pGB109-120) encoding plasmids. The cells were grown at 37 °C overnight, diluted 40× in LB medium, and additionally grown to OD600 of ~0.5-0.6. A third chemical transformation was performed with 250 ng of target sequences containing plasmid (pGB108) which was obtained by cloning superfolder GFP (sfGFP) encoding biobrick (gift from iGEM, catalog no. K515105) into pSG4K5 (gift from Xiao Wang, Addgene plasmid #74492). The co-transformed cells were further diluted by serial 10× fold dilutions and grown at 37 °C for 24 h on plates containing L-arabinose (0.2%), glucose (1%), streptomycin (50 µg / ml), carbenicillin (100 µg / ml), chloramphenicol (25 µg / ml) and kanamycin (50 µg / ml). Plates were visualized by fluorescence imaging (488 nm laser; blue filter; Amersham Typhoon scanner) and white light imaging (white light epi-illumination; Uvitec Firereader V10). GFP silencing assays were performed in biological triplicates. Target sequences can be found in Figure 12 Bacteriophage plaque assay. In this study, ER2267, DH10B and BL21 (DE3) E. coli strains were used for phages M13mp18, λvir and T4, and VpaE1 respectively. Bacteriophage assays were conducted following a modified double agar overlay protocol. Different E. coli strains containing both Cas12m effector (pTK147, pRZ169 and pTK200) and crRNA (pRZ166- 167, pGB085-100) encoding plasmids were grown overnight at 37 °C, 200 rpm. One mL of saturated overnight culture was mixed with 4 ml molten 0.75% (w / v) agar LB for the top layer, spread onto a corresponding LB agar plate (1.50% (w / v) agar, 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 0.5% (w / v) NaCl, 100 µg / ml carbenicillin, 25 µg / ml chloramphenicol, different L-arabinose concentrations (0.01% for VpaE1 and 0.001% for the rest)) and allowed to completely solidify. The selected phages were further diluted by serial 10× fold dilutions with LB (with 5 mM MgCl2addition for λvir). 2 ul of each dilution were spotted onto the top agar and grown for 16-20 h at 37 °C. After overnight incubation, the effect was evaluated by counting plaque-forming units (PFUs). If PFUs were not enumerable, but clearings were observed at high phage concentrations, the most concentrated dilution at which no plaques / clearings were observed was counted as 1 PFU. Efficiency of plating (EOP) calculations for a given condition were performed by normalizing the mean of PFU for a condition to the mean PFU of a non-targeting control: mean(PFU_condition) / mean(PFU_negative_control). Plaque assays were performed in biological triplicates. Target sequences can be found in Figure 12. Cloning of Cas12 base editors expression vectors. Adenosine base editor (ABE) was selected for further Cas12m activity studies in E. coli and human cells. Here, initial enAsABE8e plasmid (here renamed as pTK221) with optimized deaminase TadA-8e paired with enhanced (en) AsCas12a gene (enAsABE) (Richter et al., 2020) was used (gift from David R. Liu, Addgene plasmid #138506). For TadA-8e-GoCas12m (GoABE) expression in human cells, enAsCas12a was replaced with codon-optimized GoCas12m gene (Twist Bioscience) (pTK225) using BamHI and PaeI restriction enzymes (Thermo Fisher Scientific). crRNA encoding constructs were cloned into the pX458-derived plasmid (gift from Feng Zhang, Addgene plasmid #48138) under U6 promoter (pBD1-2 and pGB125- 126), using Gibson assembly. For base editors activity studies in E. coli cells, unaltered enAsABE and GoABE genes and respective crRNA were cloned into modified pETDuet1 (MilliporeSigma) expression vector with Bsp1407I, XhoI and BpiI (Thermo Fisher Scientific) restriction enzymes (pGB129-132). Chloramphenicol resistance recovery assay. Chloramphenicol resistance recovery assays were performed in E. coli BW25141 (λDE3) strain (gift from David R. Edgell) bearing GoABE or enAsABE base editors and crRNA guide sequence containing plasmids (pGB129-132) and inactive chloramphenicol resistance gene containing plasmid (pGB121), obtained from pACYC-184 (New England Biolabs) parental plasmid control by whole plasmid PCR introducing premature stop codon. The cells were grown at 37 °C overnight, diluted 100× in LB medium, and grown to OD600 of ~0.4. Following that, the cells were induced with 0.5 mM of IPTG and grown additionally overnight at 37 °C, 200 rpm. After overnight induction, the co-transformed cells were further diluted by serial 10× fold dilutions and grown at 37 °C overnight on plates containing carbenicillin (100 µg / ml), chloramphenicol (25 µg / ml), and tetracycline (10 µg / ml). Base editing efficiency on target plasmid was evaluated by Sanger sequencing. All recovery assays were performed in biological triplicates. Target sequences can be found in Figure 12. HEK cultivation. HEK293T cells obtained from ATCC (catalog number CRL-3216) were cultivated in DMEM medium (Gibco) supplemented with 10% FBS (Gibco), penicillin (100 U / ml) and streptomycin (100 µg / ml), and grown at 37^°C and 5% CO2. The cells were transfected at 70-80% confluency and grown for 72 hours unless specified otherwise. GFP recovery assay. For GFP recovery assay experiments of GoABE and enAsABE on inactive enhanced GFP (eGFP) targets, HEK293T cells were seeded at 1.4 × 10^5 cells per well in 24-well plates. Twenty-four hours after seeding, cells were co-transfected with 400 ng base editor variant plasmids (pTK221, pTK225), 200 ng crRNA expression plasmids (pBD1-2 and pGB125-126), and 400 ng inactive eGFP encoding plasmid (pGB122) obtained from eGFP encoding construct (pRZ174; gift from Toni Cathomen) by quick change mutagenesis, using 2.5 μl TurboFect Transfection Reagent (Thermo Fisher Scientific). The medium was changed at 24 h post-transfection and flow cytometry analysis was performed at 24, 48 and 72 hours post-transfection with Partec CyFlow Space flow cytometer (Sysmex Partec, Goerlitz, Germany). Data analysis and visualisation were performed with FlowJo software (FlowJo LLC, Ashland, OR, USA). Target sequences can be found in Figure 12. Genome base editing assay. For base editing experiments of GoABE and enAsABE on genomic DNA targets, HEK293T cells were seeded at 30,000 cells per well in 96-well plates. Twenty-four hours after seeding, cells were co-transfected with 150^ng of the base editor (pTK221 and pTK225) and 50^ng of crRNA (pBD1-2, pBD5-6, pBD9-10, pBD17-18 and pBD23-26) encoding plasmids using 0.5 µl Turbofect transfection reagent (Thermo Fisher Scientific). For all base editing experiments, cells were cultured for 72 hours, then washed with 1× PBS (Thermo Fisher Scientific), followed by cell lysis with 30^µl QuickExtract solution (Lucigen). Next, two rounds of PCR were performed: first to amplify the DNA region surrounding each target site, and second to add the sequence adapters required for Illumina sequencing. Briefly, 1 µl of cell lysate was used in the primary PCR with primers specific to the targeted genomic locus that were 5′ tailed with Illumina sequences (Figure 11), in a final volume of 20 µl, using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). A total of 1.5 µl first round PCR product in a final volume of 30 µL was used as a template for the second round of PCR to index and add P5 and P7 adapters required for Illumina sequencing. Samples were pooled in an equimolar ratio and purified from agarose gel using a GeneJET Gel extraction kit (Thermo Fisher Scientific). Barcoded and purified DNA samples were quantified with Qubit 4 Fluorometer (Thermo Fisher Scientific), analyzed using BioAnalyzer (Agilent), and pair-end sequenced (2 × 75 bp) using the MiniSeq High Output Reagent Kit, 150 cycles (Illumina) on a MiniSeq System (Illumina). Base editing efficiency was analyzed using CRISPResso2 (Clement et al., 2019). Target sequences can be found in Figure 12. Example 1 Identification and characterization of subtype V-M (V-U1) CRISPR-Cas12m effectors. Protein BLAST search in sequence databases using the V-U1 Cas12m protein from Gordonia otitidis (Go) (Shmakov et al., 2017 ) as an initial query resulted in a non-redundant list of 134 Cas12m family variants identified in diverse bacteria (Figure 1). For experimental characterization, we selected 8 candidates that are encoded adjacent to the CRISPR region that show a different arrangement of putative RuvC active site: 4 variants contain typical D- E-D, 2 – H-D-D, and 2 – N-D-D (Figure 2A; Figure 6). Analysis of the repeat sequences from the CRISPR regions showed high sequence conservation between orthologous CRISPR-Cas12m systems and indicated the potential formation of secondary structures within the repeat (Figure 2B), which have also been observed in other type V systems (Pausch et al., 2020; Yan et al., 2019; Zetsche et al., 2015). To experimentally characterize the Cas12m effectors, we first determined the guide RNA requirements. The Cas12m ribonucleoprotein (RNP) complexes were heterologously expressed in E. coli cells from the plasmids harboring the cas12m gene and an engineered minimal CRISPR region containing a single spacer. The RNA isolated from the purified RNP complexes was sequenced, revealing the enrichment of crRNAs derived from the CRISPR region. Examination of the 5’- crRNA ends suggested the processing of precursor CRISPR RNA (pre-crRNA) within the repeat sequences. Sequence analysis of enriched RNAs derived from regions adjacent to the CRISPR did not reveal complementarity to the repeat sequences, ruling out the presence of the trans-activating RNAs (tracrRNAs) and indicating that Cas12m proteins form the complexes with and are guided by single crRNAs. The repeat sequences of the respective crRNAs as determined above are provided in Fig. 9. Example 2 Cas12m effectors bind DNA targets in a PAM-dependent manner. For efficient dsDNA targeting, Cas12 effectors require a protospacer adjacent motif (PAM) next to the DNA sequence recognized by guide RNA (gRNA) (Karvelis et al., 2020; Zetsche et al., 2015). To test whether Cas12m proteins guided by a single crRNA can recognize and cleave dsDNA in a PAM-dependent manner, we adopted the protospacer / transposon adjacent motif (PAM / TAM) identification assay previously developed for the characterization of Cas9 / Cas12 and TnpB nucleases (Karvelis et al., 2021, 2020, 2019). Firstly, Cas12m proteins and crRNAs, encoded in PCR-generated DNA fragments, were expressed using a cell-free in vitro transcription-translation (IVTT) system. Next, IVTT mixture with produced Cas12m complexes was incubated with the plasmid library, engineered to contain a 7 bp (7N) randomized region next to the target covering all possible PAM sequences. The DNA ends that would be generated after target DNA cleavage were repaired with T4 polymerase and used for adapter ligation, PCR amplification, and sequencing. The DNA cleavage activity was assessed by scanning the target sequence for increased frequencies of adapter ligation events, which would indicate the cleavage by Cas12m effectors at the target site. Interestingly, adapter ligation frequencies were similar for all samples, including the negative control containing no Cas12m complex, indicating the absence of dsDNA target cleavage activity of Cas12m effectors. The lack of detectable dsDNA cleavage for all tested Cas12m variants prompted investigation whether selected Cas12m effectors can specifically bind to DNA. To test the dsDNA binding activity of Cas12m, the 7N plasmid library was incubated with an IVTT mixture of Cas12m complexes followed by the addition of restriction enzyme (RE) Eam1105I (Figure 2C). Since both Cas12m and Eam1105I recognition sites overlap, binding events of Cas12m RNP complexes would prevent its cleavage by RE. After PCR amplification and sequencing, we detected that Cas12m variants were able to prevent DNA cleavage by Eam1105I of the targets containing T- and, advantageously, C-rich PAM sequences (Figure 2D), demonstrating the PAM-dependent dsDNA binding by Cas12m effectors. In addition, we assembled Ms and GoCas12m effector complexes from purified components and incubated them with the target-containing DNA and RNA substrates in vitro, showing the absence of targeted nucleic acid cleavage (and target-activated collateral DNA and RNA cleavage activity which was previously reported for other Cas12 proteins (Chen et al., 2018; Harrington et al., 2018; Karvelis et al., 2020; Yan et al., 2019 ). Together, these data reveal an evolutionary conserved PAM-dependent dsDNA binding activity in the absence of RuvC-mediated cleavage by Cas12m effectors. Example 3 CRISPR-Cas12m provides plasmid DNA interference in E. coli. The demonstration of PAM- dependent dsDNA binding by Cas12m effectors raised the question if subtype V-M CRISPR-Cas can function as a defense system and confer resistance to MGEs by targeted DNA binding rather than cleavage. We selected 4 variants representing Cas12m effectors with different RuvC active site residues to test their ability to interfere with plasmid DNA propagation in E. coli. Briefly, E. coli cells expressing Cas12m and crRNA were transformed with a pSC101-based plasmid containing the targeted sequences next to the identified PAM sequences either in the origin of replication (ori), plasmid backbone, or the kanamycin resistance gene (KanR) (Figure 3A) and grown on kanamycin-supplemented agar plates. Transformation efficiency was assessed by counting colony forming units (CFU) after serial dilution of transformants and represented as the fold change in CFU when compared to the non-targeting control. Additionally, Acidaminococcus sp. Cas12a (AsCas12a) and nuclease-deficient dAsCas12a variants (Zetsche et al., 2015) were included as a reference. As expected, the AsCas12a nuclease provided efficient plasmid interference in E. coli for all tested targets, while cleavage-deficient dAsCas12a had no effect on transformation efficiency when compared to the non-targeting control (Figure 3A). In contrast to dAsCas12a, plasmid interference was observed for the TfCas12m and GoCas12m variants. The functional targets for TfCas12m and GoCas12m were located in ori and also in KanR for GoCas12m, indicating Cas12m-mediated plasmid interference activity is utilized by hindering plasmid replication initiation or acting as a roadblock for transcription of the essential gene for cell survival. To confirm that binding by Cas12m can block the transcription, we adopted the assay, based on superfolder (sf) GFP expression (Figure 3B). Briefly, the E. coli cells expressing GoCas12m, dAsCas12a, or AsCas12a complex components targeting the sfGFP encoding sequence were transformed with the sfGFP expression plasmid and the transformation efficiency and sfGFP fluorescence levels in the cells were assessed. Serial dilution of the transformants revealed plasmid interference in the cells containing AsCas12a. In contrast to AsCas12a, the cells expressing GoCas12m and dAsCas12a showed no reduction in transformation efficiency compared to non-targeting controls (Figure 3C), supporting the observation that only essential gene targeting by nuclease-deficient Cas12m effectors provides the interference activity (Figure 3A). However, the sfGFP fluorescence readout revealed reduced expression of sfGFP in the cells targeted by GoCas12m and dAsCas12a within the promoter or encoding region of sfGFP (Figure 2C) confirming binding-induced transcription inhibition. Example 4 CRISPR-Cas12m protects E. coli from bacteriophages. To test whether Cas12m effectors could also confer immunity against bacteriophages, we performed a phage plaque assay using GoCas12m. Complexes were directed to either sense or antisense strands within essential genes of M13mp18, lambda-vir (λ), VpaE1, and T4 phages. To assay the influence of epigenetic modifications on effector binding, T4 phage variants containing glycosylated (WT), 5-hydroxymethylated (147) or unmodified (C) cytosines in their genome were used. Efficiency of plating (EOP) was evaluated and results were expressed as fold change in EOP compared to non-targeted Cas12 controls (Figure 3D). Similarly to the results of the plasmid transformation assay, the AsCas12a effector provided a reduction in plaque formation with variable efficiency for almost all phages tested except T4 (WT) (Figure 3D). The limited AsCas12a immunity against T4 (WT) is most likely due to the naturally occurring glycosylation of cytosines, as the T4 phage variants containing hydroxymethyl or unmodified cytosines were efficiently targeted. dAsCas12a was unable to prevent plaque formation for all phage variants tested. However, GoCas12m efficiently protected E. coli from M13mp18, λ, and unmodified cytosine-containing T4 phages (Figure 3D), providing an anti-phage immunity function. Overall, these results confirm that compact Cas12m effectors are capable of providing binding-based DNA interference without DNase activity and support the defence function of the subtype V-M CRISPR-Cas system. Example 5 Harnessing GoCas12m for adenine base editing in E. coli and human cells. We engineered the GoABE variant by fusing human codon-optimized GoCa12m at the N-terminal end with TadA-8e adenine deaminase (Richter et al., 2020) and tested its base editing potential in E. coli and human cells. To evaluate GoABE activity in E. coli, we first used a selection plasmid containing the chloramphenicol resistance gene (CmR) inactivated by a premature stop codon (Figure 4A). GoABE-induced A-to-G conversion within the stop codon, resulting in restoration of CmR function, was evaluated by growing E. coli cells on Cm supplemented media. The results showed that E. coli cells expressing the GoABE targeting inactivated CmR exhibited efficient growth, indicating the restoration of CmR function (Figure 4B). Similar results were observed using engineered enAsCas12a (Kleinstiver et al., 2019) fused to TadA-8e (enAsABE), for which adenine base editing activity was previously confirmed (Richter et al., 2020). As expected, sequencing of the plasmids extracted from single colonies revealed targeted A-to-G editing for both GoABE and enAsABE (Figure 4C). Next, we probed whether the engineered GoABE is active in human cells. First, we used the reporter plasmid containing a stop codon within the gene encoding enhanced GFP (eGFP) (Figure 4D). After co-transfection of the plasmids encoding GoABE, gRNA, and reporter into HEK293T cells we performed flow cytometry analysis of cells after 24, 48, and 72 hours (h) using enAsABE as a reference to validate the eGFP reporter assay and compare base editing activity. The results revealed an increase in eGFP-positive cells after co- transfection of GoABE and enAsABE with reporter-targeting crRNAs (up to 40% and 47% after 48 h, respectively) when compared to the non-targeting ABE constructs, indicating efficient A-to-G base editing (Figure 4E). Therefore, advantageously, GoABE was active in human cells and could induce base editing. We next tested whether endogenous genomic sites could be modified using GoABE. We selected five targets previously examined for editing with enAsABE (Richter et al., 2020) and measured A-to-G conversion after 72 h. The results showed that A-to-G editing reached up to 19% and 20% for GoABE and enAsABE, respectively (Figures 4F and 5), indicating the efficient base editing activity of GoABE. Surprisingly, while the A-to-G conversion levels were similar between the two base editors, the editing windows were different (Figures 4F and S10). GoABE showed a preference for A-to-G conversion at PAM-proximal positions, which remained inaccessible to enAsABE. GoABE was capable of editing the A bases within a distinct and narrow base editing window, specifically, it was able to efficiently edit adenines at positions as close to PAM sequence as positions 3 to 5. Advantageously, the adenines at the PAM-distal positions were not affected, putting GoABE in a position for use as a base editor with a distinct base editing window and, therefore, decreased bystander editing. Taken together, these data demonstrate that nuclease-deficient Cas12m proteins, as exemplified by GoCas12m, can be adopted as a platform for base editing in E. coli and human cells, providing a unique editing window and size advantage among type V family effectors. This is advantageous, having in mind that to date, only a small fraction of compact Cas12 enzymes characterized to date demonstrate activity in human cells. Additionally, Cas12m effector proteins (TfCas12m, MsCas12m, PpCas12m, MkCas12m, ApCas12m, UnCas12m, MmCas12m) are engineered into respective Cas12m-ABE variants by fusing a respective human codon-optimized Ca12m effector protein at the N-terminal end with TadA-8e adenine deaminase and base editing experimentsin E. coli and human cells are performed using the respective materials and methods as described in the section “Materials and Methods”. Use of Cas12m effector proteins (GoCas12m, TfCas12m, MsCas12m, PpCas12m, MkCas12m, ApCas12m, UnCas12m, MmCas12m) in cytidine editing in E.coli and human cells. For this purpose, the respective Cas12m-CBE variants are constructed by fusing a respective human codon-optimized Cas12m effector protein at the N-terminal end with cytidine deaminase (rAPOBEC1) and, Uracil glycosylase inhibitor (UGI) at the C-terminal and base editing experiments in E. coli and human cells are performed using the respective materials and methods. Table of sequences SEQ Description Sequence ID NO:GoCas12mMTRVTVQTAGVHYKWQMPDQLTQQLRLAHDLREDLVTLEYEYEDAVKAVWSSYPAVAALEAQVAELDERASELASTVKEEKSRQRTKRPSHPAVAQ LAETRAQLKAAKASRREAIASVRDEATERLRTISDERYAAQKQLYRDY CTDGLLYWATFNAVLDHHKTAVKRIAAHRKQGRAAQLRHHRWDGTGTI SVQLQRQATDPARTPAIIADADTGKWRSSLIVPWVNPDVWDTMDRASR RKAGRVVIRMRCGSSRNPDGTKTSEWIDVPVQQHRMLPADADITAAQL TVRREGADLRATIGITAKIPDQGEVDEGPTIAVHLGWRSSDHGTVVAT WRSTEPLDIPETLRGVITTQSAERTVGSIVVPHRIEQRVHHHATVASH RDLAVDSIRDTLVAWLTEHGPQPHPYDGDPITAASVQRWKAPRRFAWL ALQWRDTPPPEGADIAETLEAWRRADKKLWLESEHGRGRALRHRTDLH RQVAAYFAGVAGRIVVDDSDIAQIAGTAKHSELLTDVDRQIARRRAIA APGMLRAAIVAAATRDEVPTTTVSHTGLSRVHAACGHENPADDRYLMQ PVLCDGCGRTYDTDLSATILMLQRASAATSNTfCas12mMETAATKNYLALSFGCLSPTRGEEYLLDQIKKKHDLWNKLVEKDREHREKVRQVMVFESETTKKIKELEEELNSLREEIKNQRKTKRTGKVDLTDQ KARIEEIKPQLKQLKEKFKEERSFIFEARKQELAQLEKERWAVVKELG KGSGLYWCNLEDVVNSYDIGRKKAKAAGGEMRFHRWDGTGKVTVRFQK GLPVNEMFSCTNNLLQIDPVDKDAWYNPVRAIRRKKSRTRVRLRACSE NKKPLFIELPVVLHREIPEDALIRTASVIREKVGMRYRYKLNLVLEIL GENTNRILPALEGTAAIDLGWRTVKDGLRVACLVDDKGHSEELILDND VLHEFNKIKDLQSIRDNLFNETKAKLMELLKTLELPDEAKERTSHMAN WRSQQKMLRLHQYWRENRLPGDDEVWEVLEYWRKREIHLYEWQENLRD QVLRRRKEIYRIFAAKITRKYKTIVLEEFTLNKTVQKPNPEEGPAGTL PANRNRFIAAISEFRNELANACRKNHVEFTYVPAENTTITCHKCGHKE KFDAAAQIIHTCSTCGELWDQDYNAAKNLLAFSQKGGVKMsCas12mMPFGKKARHVKAYQFGADAPQEGMEAVLEQHRLRTDYYNALVEMELRQREERTALLANLAAESGLESPNQVYERLKAAGEKGIRKHPEYVAARERQ KALYGHPRLLELQSRQREERNALRRSFGAKGLYSSNYLDVERAFDKAR QSPELRFRRYSPHEGRLAVLYTEGLPMREIGSDTRVQLPLPDPIIYRD RATRRKHQRVLMKFRVRSVERQPLWITVPVYLHRELPDGVCREVSLHW HRVADRLRWTVSVVVEVEGPPVASPTGRGAVAVDLGWRRVEGGLRAGF WVGEDGAGGEIALSEGDLKQFSKVEDLRSIRDQHLNALKEALAAWLEA PPAPLPDWLAEETKTLPQWRSPARFAALFRRWQSERVHADEAAYGLLE GWHKRDRHLWQYEANLREQMILRRREQYRVLAATLARQYDALIVEDFN LRAAAELDQGGSDLPDAARRYRTIASPSTLRDALVNAFAQRGKPVRKL NPAHTTTDCHACGGALVGDPAKELRLYCPTCERFYDQDENAARNLLRR AQEVQAQVPpCas12mMIAPTRTEKYLYLVPDEQVQPVTTIVRKYGLLSPLDWDCPDYPAGDAFEHLFLQNKLWNDLVTIEREHRAKYRELIGSDEETAQMDTEIASIKDRL SVLDEGRKKLRVEHRKKKCPEIDCLDENIKKLKSELKAVASKAKETRA AAKDRIRAAGNDIENLEKDRQAAVIKAYNNSGLWWGNYNAVLESYKKA RIKALKDGAELKYHRFDGSGRFTNQIQGGMSVQDLLEGNRNVASLRLV SSGELGDISGKKPPSLDLQSVGSRRDSREYGILAITLYTGTDEQSKKF RRTLSFPVILHRPLPEGATLKSLSVHRKRVGTDFVWSVVFTFTTDCPT YDQRSSTGNRCGLNLGWKKQAGGGLRVATIYDGSDARHITLPQAIIDG LDYVNGDLQGRIDSAANENHAWLLEQWGGDELPESLQELRSMLRRSKR PHPAKFAKAVIAWRNYPEYLGDARDEAEQRRKATKRLTIEMAHKREKL LRRRMDFYRNTAKQLTSVYDVICLDKMDLRRLALLEKGDGTPNELTKI ARKQRQQAAISELRECLSKAAAKNGTQIEQVSTASSATCSACKGKMEQ VDGIMWRCRECRALVDQDINAAANLFREVLMkCas12mMIRTYKYSLKAPENFAEDCEDELRRMNDLWNRLIEIDRQRERSFKDLCRSTSAEYAAAQDEIEALREPIDNLYDAIRAERIATRSKEPSDELRARR DELLGRRKALWEICKAIQKAIPKESQAPINEVYKTNVKLARQQSGCFW GNYNAVIESFETAKSKAIKDGGRLHFKSFDGSGRFVNQIQGGMTVTEL LAGSHSQAQLTNLVTTNKTKGRFAFTAFTGKDDAGKRFRRQLFSEINY HRPIPADGVIKAVEVVKVPHDGKQKYKWHACFTVALPEVDIKHPKRNI AGVNLGWRQFGGRLRVAVVVDDAGKKTEYFVPAELVSKFEAAETIQKA ADDARNEMLSWLRTFYQDNRDEAPQEWRESIQGLLRNRPSVDAANHLM TIWRECVFAQEESRRYAAWLKSDAALRRSYTGCRQNAVKWREEIYRHI AKELAERYAVLAVTDTPLSTMSRTKAKDDLAVDNALPESARRNRVIAA IYSLKEWIGKQAAKTGSTVETITGKMTATCHKCGYVAEKRLRGSQYAT CKSCGSELELDENAAINCRNHASGAVLISDKPEKTGRFQRAKMAENDF ARKIGDNASPLVTApCas12mMSEQLDDTPEQPNEVEETKKRKQRNKGKHPARIWSVFSRYLVSGREHFDKQVLLAHRFRNKLVELELQRRAAANVVIAQASSELQPLIDALAAAEQ VLEVSLQELKAVRAKHRRRAESAAQRDAVTNARTARNQASKALSKARK DAFASEAAQVGLWLAEEHHFQAVLAARHAFINDGLYWPTATDVQDRAR AMRKGAPPVFRRFGGAEQAGRIAVQIQKSTDKSQSEGGITFEEAFSCS HGFFRLEKKPGRDPLPEIADQPDYKSKRQQLLTYARAWLRVGSEGKGA RAKPCWVVADVLLTRQAPKTARIVQVYLDHSVIGDRERWRLSLVLTNQ EGWPKPNRASGCMVGIDLGWRLLDTGELRVAYACGADGQHHELRLPAS LVKVWRRPDRIQQERDNLFNDVKARLLEWLKGREDLPDWLKEQAEHLH LWKSSTRLSRLVDHWAGRDINWSSQRRIAGDEEILASLRGWVKRNLHL RDYQYHEREQLAAHRLDVYRKWADGLARLYQTAVLEDADWRDLARLPS PEDDAVNETARYNQRMASPGLLASVITNMFAITSRVECANTTRECWRC GHTEAFDAEAQLIRVCPGCGDACDQDESAARVLLARGQALNQSQVAEA APSSUnCas12mMPFRKPAEPRDNRVYEYGCLPPTAGFDDMLDQLRGRNTFWNCLVEIERRQRIEVRSLLQPPESKVPALQQELEEARVAIKAERKRQRSRNADVSDL RAYIKDLQQQLKSARAEDKAARERIKEASQPRLDELEAERLEQVKAAQ HNSNLYWCNYDDVLASYGVARVRAMREGTEIKFHRFDGTGKVSVRYQQ GLPVADVFGADTRLQIRPVDPRAWTSPIRGERRRLARTVVRIRIGSHE DRSPVWLELPMVMHRPLPEGGGIRNAAVVRERIADSWRYRLVITVATP QQEIVTHQDRNRIAIDVGWRKLDEGLRVAYWVDDLGQQGQVLLANEVI EQFRKVDDLKSIRDLHFNATRVALVDWLQHVVVPDWLAERCKTLSFWQ SQARLSALVRDWEGQRFDADDEMLAGLQAWWVKDRHLWQWESHLRDQV GRHRRELYRRFAADLVRRYRQVVLEDFDLRRVIEHPKAEVGTGGGIPG AWYRKTAAISSLRLTIEHTCSREGVEVLKVNPSYSTLTCHVCGQIDQF NAAAELVHKCSQCGLLWDQDANAAAVLLQRASQIEAVSEQKTSCEEST TPPTDGGMmCas12mMTTMTVHTMGVHYKWQIPEVLRQQLWLAHNLREDLVSLQLAYDDDLKAIWSSYPDVAQAEDTMAAAEADAVALSERVKQARIEARSKKISTELTQQ LRDAKKRLKDARQARRDAIAVVKDDAAERRKARSDQLAADQKALYGQY CRDGDLYWASFNTVLDHHKTAVKRIAAQRASGKPATLRHHRFDGSGTI AVQLQRQAGAPPRTPMVLADEAGKYRNVLHIPGWTDPDVWEQMTRSQC RQSGRVTVRMRCGSTDGQPQWIDLPVQVHRWLPADADITGAELVVTRV AGIYRAKLCVTARIGDTEPVTSGPTVALHLGWRSTEEGTAVATWRSDA PLDIPFGLRTVMRVDAAGTSGIIVVPATIERRLTRTENIASSRSLALD ALRDKVVGWLSDNDAPTYRDAPLEAATVKQWKSPQRFASLAHAWKDNG TEISDILWAWFSLDRKQWAQQENGRRKALGHRDDLYRQIAAVISDQAG HVLVDDTSVAELSARAMERTELPTEVQQKIDRRRDHAAPGGLRASVVA AMTRDGVPVTIVAAADFTRTHSRCGHVNPADDRYLSNPVRCDGCGAMY DQDRSFVTLMLRAATAPSNP References: 1. 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Claims

CLAIMS:

1. An isolated CRISPR-Cas effector protein comprising: a) an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence of at least 55% identity to SEQ ID NO: 1, b) an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence of at least 55% identity to SEQ ID NO: 2, c) an amino acid sequence as set forth in SEQ ID NO: 3 or a sequence of at least 55% identity to SEQ ID NO: 3, d) an amino acid sequence as set forth in SEQ ID NO: 4 or a sequence of at least 55% identity to SEQ ID NO: 4, e) an amino acid sequence as set forth in SEQ ID NO: 5 or a sequence of at least 55% identity to SEQ ID NO: 5, f) an amino acid sequence as set forth in SEQ ID NO: 6 or a sequence of at least 55% identity to SEQ ID NO: 6, g) an amino acid sequence as set forth in SEQ ID NO: 7 or a sequence of at least 55% identity to SEQ ID NO: 7, or h) an amino acid sequence as set forth in SEQ ID NO: 8 or a sequence of at least 55% identity to SEQ ID NO:

8.

2. The isolated CRISPR-Cas effector protein according to claim 1, comprising an amino acid sequence as set forth in SEQ ID NO: 1 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO:

1.

3. The isolated CRISPR-Cas effector protein according to claim 1, comprising an amino acid sequence as set forth in SEQ ID NO: 2 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO:

2.

4. The isolated CRISPR-Cas effector protein according to claim 1, comprising an amino acid sequence as set forth in SEQ ID NO: 3 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO:

3.

5. The isolated CRISPR-Cas effector protein according to claim 1, comprising an amino acid sequence as set forth in SEQ ID NO: 4 or a sequence of at least 55%,60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO:

4.

6. The isolated CRISPR-Cas effector protein according to claim 1, comprising an amino acid sequence as set forth in SEQ ID NO: 5 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO:

5.

7. The isolated CRISPR-Cas effector protein according to claim 1, comprising an amino acid sequence as set forth in SEQ ID NO: 6 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO:

6.

8. The isolated CRISPR-Cas effector protein according to claim 1, comprising an amino acid sequence as set forth in SEQ ID NO: 7 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO:

7.

9. The isolated CRISPR-Cas effector protein according to claim 1, comprising an amino acid sequence as set forth in SEQ ID NO: 8 or a sequence of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, 97%, 98%, 99% identity to SEQ ID NO:

8.

10. A composition comprising an effector CRISPR-Cas effector protein according to any one of claims 1 to 9 and a guide RNA (gRNA), wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence.

11. An effector complex, the effector complex comprising a CRISPR-Cas effector protein according to any one of claims 1 to 9 and a guide RNA (gRNA), wherein the gRNA is capable of directing binding of the effector complex to a target sequence.

12. A polynucleotide encoding a CRISPR-Cas effector protein according to any one of claims 1 to 9.

13. An expression vector comprising a polynucleotide according to claim 12, under the control of a suitable expression promoter.

14. The expression vector according to claim 13, further comprising a nucleotide sequence comprising an expression promoter and a sequence under the control of the promoter encoding a guide RNA (gRNA) with a nucleotide sequence which is complementary to a target sequence in double stranded DNA (dsDNA).

15. A cell comprising the expression vector according to claim 13 or 14.

16. The isolated CRISPR-Cas effector protein according to any one of claims 1 to 9, wherein the effector protein is fused to at least one other protein.

17. The isolated CRISPR-Cas effector protein according to claim 16, wherein the effector protein is fused to one or more proteins.

18. The CRISPR-Cas effector protein according to claim 16 or 17, wherein the at least one other protein is selected from an enzyme, a ligand, or a marker.

19. The CRISPR-Cas effector protein according to claim 18, wherein the enzyme is a deaminase.

20. The CRISPR-Cas effector protein according to claim 19, wherein the deaminase is an adenine deaminase.

21. The CRISPR-Cas effector protein according to claim 19, wherein the deaminase is a cytidine deaminase.

22. The CRISPR-Cas effector protein according to any one of claims 19 to 21, wherein the deaminase is fused to the N-terminus of the CRISPR-Cas effector protein.

23. The CRISPR-Cas effector protein according to any one of claims 19 to 21, wherein the deaminase is fused to the C-terminus of the CRISPR-Cas effector protein.

24. The CRISPR-Cas effector protein according to claim 19, wherein the deaminase comprises an adenine deaminase and a cytidine deaminase.

25. The CRISPR-Cas effector protein according to claim 24, wherein the adenine deaminase is fused to the N-terminus of the CRISPR-Cas effector protein, and the cytidine deaminase is fused to the C-terminus of the CRISPR-Cas effector protein.

26. The CRISPR-Cas effector protein according to claim 24, wherein the adenine deaminase is fused to the C-terminus of the CRISPR-Cas effector protein, and the cytidine deaminase is fused to the N-terminus of the CRISPR-Cas effector protein.

27. The CRISPR-Cas effector protein according to claim 24, wherein the adenine deaminase, and the cytidine deaminase are fused to the N-terminus of the CRISPR- Cas effector protein.

28. CRISPR-Cas effector protein according to claim 24, wherein the adenine deaminase and the cytidine deaminase are fused to the C-terminus of the CRISPR-Cas effector protein.

29. A composition comprising an effector CRISPR-Cas effector protein according to any one of claims 16 to 28 and a guide RNA (gRNA), wherein the gRNA is capable of forming a complex with the CRISPR-Cas effector protein and directing binding of said complex to a target sequence.

30. An effector complex, the effector complex comprising a CRISPR-Cas effector protein according to any one of claims 16 to 28 and a guide RNA (gRNA), wherein the gRNA is capable of directing binding of the effector complex to a target sequence.

31. A polynucleotide encoding a CRISPR-Cas effector protein according to any one of claims 16 to 28.

32. An expression vector comprising a polynucleotide according to claim 31, under the control of a suitable expression promoter.

33. The expression vector according to claim 32, further comprising a nucleotide sequence comprising an expression promoter and a sequence under the control of the promoter encoding a guide RNA (gRNA) with a nucleotide sequence which is complementary to a target sequence in double stranded DNA (dsDNA).

34. A cell comprising the expression vector according to claim 32 or 33.

35. A method of repressing or interfering with the expression of a target gene sequence by an organism, a cell, or in a cell-free expression system, comprising exposing double stranded DNA (dsDNA) comprising the target gene sequence to a CRISPR- Cas effector protein according to any one of claims 1 to 9, and a guide RNA (gRNA) which directs said effector protein to the target gene sequence, wherein targeted binding of the effector protein to the dsDNA results repressing or interfering with the expression of a target gene sequence.

36. A method of repressing or interfering with the function of DNA elements controlling replication by an organism, a cell, or in a cell-free expression system, comprising exposing double stranded DNA (dsDNA) comprising the target gene sequence to a CRISPR-Cas effector protein according to any one of claims 1 to 9, and a guide RNA (gRNA) which directs said effector protein to the target gene sequence, wherein targeted binding of the effector protein to the dsDNA results repressing or interfering with the function of DNA elements controlling replication.

37. The method according to claim 35 or 36, wherein an organism or cell is transfected with an expression vector of claim 13, and (a) further transfected with a second expression vector which comprises an expression promoter and a sequence under the control of the promoter which encodes the guide RNA (gRNA); or (b) the gRNA is introduced directly into the organism or cell.

38. The method according to claim 35 or 36, wherein the organism or cell is transfected with an expression vector of claim 14.

39. A method for base-editing a target sequence in an organism, a cell, or in a cell-free system comprising exposing a double stranded DNA (dsDNA) comprising the target sequence to a CRISPR-Cas effector protein according to any one of claims 16 to 28, and a guide RNA (gRNA) which directs said effector protein to the target sequence.

40. The method according to claim 39, wherein an organism or cell is transfected with an expression vector of claim 32, and (a) further transfected with a second expression vector which comprises an expression promoter and a sequence under the control of the promoter which encodes the guide RNA (gRNA); or (b) the gRNA is introduced directly into the organism or cell.

41. The method according to claim 39, wherein the organism or cell is transfected with an expression vector of claim 33.

42. The method according to any one of claims 35 to 41, wherein the CRISPR-Cas effector protein is introduced into the organism or cell simultaneously, sequentially or separately from gRNA.

43. The method according to claim 42, wherein the CRISPR-Cas effector protein is introduced into the cell in the form of an RNA encoding the CRISPR-Cas effector protein.

44. The method according to any one of claims 35 to 43, wherein gRNA is associated with the CRISPR-Cas effector protein before or upon introduction into the organism or cell.

45. The method according to any one of claims 35 to 44, wherein the introducing of the CRISPR-Cas effector protein and / or gRNA into the cell comprises electroporation or microinjection, optionally in combination with the use of liposomes.

46. The method according to any one of claims 35 to 44, wherein the introducing of the CRISPR-Cas effector protein and / or gRNA into the cell is by chemical-based transfection, such as lipofection, calcium phosphate transfection or cationic polymer transfection.

47. The method according to any one of claims 35 to 44, wherein the introducing of the CRISPR-Cas effector protein and / or gRNA into the cell is by delivery with a viral vector.

48. The method of claim 47, wherein the viral vector is a lentiviral vector, a retroviral vector or an AAV vector.

49. The method according to any one of claims 35 to 48, wherein the cell is a prokaryotic cell.

50. The method according to any one of claims 35 to 48, wherein the organism or cell is a eukaryote.

51. The method according to claim 50, wherein the cell is a non-human animal cell.

52. The method according to claim 50, where in the cell is a human cell.

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