Cas12a Nickase

Abstract

The present invention relates to the field of gene genome editing. In particular, it relates to the provision and use of Cas12a enzymes with nickase activity and means and methods for modifying genomic loci of interest with Cas12a enzymes with nickase activity.

Description

[Technical field]

[0001] The present invention relates to the field of gene genome editing. In particular, it relates to the provision and use of Cas12a enzymes with nickase activity and means and methods for modifying genomic loci of interest with Cas12a enzymes with nickase activity. [Background technology]

[0002] Over the past few years, variants of CRISPR nucleases that generate single-stranded nicks in DNA rather than double-stranded breaks (DSBs) have emerged as versatile tools for targeted gene editing in cells and organisms. Target-specific nicking has been primarily achieved by Cas9 nickase mutants D10A and H840A (Jinek et al., 2012; Gasiunas et al., 2012). Cas9 D10A cleaves the gRNA-targeted strand, while Cas9 H840A cleaves the non-targeted strand (Jinek et al., 2012; Gasiunas et al., 2012; Cong et al., 2013; Mali et al., 2013).

[0003] Nickases allow for highly specific editing because nicks are primarily repaired via the high-fidelity base excision repair pathway (Dianov and Huebscher, 2013). CRISPR nucleases often induce unexpected cleavage followed by indel formation at genomic sites that share sequence homology with the target site. Such off-target activity can be reduced by introducing paired nickases that efficiently create DSBs by generating two single-stranded breaks proximally on opposite DNA strands. In this dual nickase approach, long overhangs are provided for each of the cut ends instead of blunt ends. This provides improved control over precise gene integration and insertion. Because both nicking enzymes must efficiently nick their target DNA, paired nickases have significantly lower off-target effects compared to double-stranded break Cas systems (Ran et al., 2013; Kuscu et al., 2014).

[0004] Besides reducing off-target editing, nickases can also be exploited to enhance the efficiency of precise gene editing methods such as homology-directed repair (HDR) and base editing. HDR, initiated by double-stranded DNA breaks, is usually accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites (Kosicki et al., 2018; Shin et al., 2017; Tsai et al., 2015; Zhang et al., 2015). Nickases provide an attractive approach to induce high-fidelity HDR without stimulating NHEJ. Base editing similarly allows base substitution at the target site without concomitant indel formation. Because base editors do not usually create DSBs, they minimize the generation of DSB-related by-products (Komor et al., 2016; Gaudelli et al., 2017). DNA base editors (BEs) contain fusions between catalytically inactive Cas nucleases or nickases and base-modifying enzymes that operate on single-stranded DNA (ssDNA) but not on double-stranded DNA (dsDNA). Upon binding to their target locus in DNA, base pairing between the guide RNA and the target DNA strand results in the displacement of a small segment of single-stranded DNA in a so-called "R-loop" (Nishimasu et al., 2014).

[0005] The DNA bases within this single-stranded DNA bubble are modified by deaminase enzymes. To improve editing efficiency, many base editors have been engineered to introduce nicks in the unedited DNA strand, thereby inducing cells to repair the unedited strand using the edited strand as a template (Komor et al., 2016; Nishida et al., 2016; Gaudelli et al., 2017).

[0006] Importantly, nickases, when appropriately adapted, can also play an essential role in the recently developed prime editing technology. Prime editing is a "search and replace" genome editing tool that mediates targeted insertions, deletions, all 12 possible base-base conversions, and combinations thereof, without the need for DSBs or donor templates (Anzalone et al., 2019). Prime editors use reverse transcriptase fused to an RNA-programmable nickase and a guide RNA that is extended with prime editing to directly copy genetic information from an extension on the pegRNA into a target genomic locus. In this approach, Cas9 H840A nickase is used to form a nick in the non-target strand, exposing a 3'-hydroxyl group, which stimulates the reverse transcription of the extension that codes for the edit on the pegRNA directly into the target site. In addition, similar to base editors, third generation prime editors further form nicks in the unedited strand and induce its replacement, further increasing the editing efficiency (Anzalone et al., 2019). As those skilled in the art are well aware, pegRNAs can be designed and optimized according to the desired target cell or construct. For example, prime editing in plants is described in Sretenovic and Qi 2021, and optimized prime editing in monocotyledonous plants is described in Jin et al., 2022.

[0007] Inevitably, the search for versatile base editors and prime editors requires both the proper basic functionality of the nickase itself (high specificity, broad PAM targeting range, stability, low off-target and high on-target activity) as well as the proper steric integration of the nickase domain with other domains and spacers between the effector domain, etc., so that a suitable modular architecture and highly efficient activity at the target site in the selected genome can be achieved.

[0008] Currently, CRISPR-Cas systems are classified into two classes (class 1 and 2), which are further divided into six types (types I to VI). Class 1 (types I, III, and IV) systems use multiple Cas proteins in their CRISPR ribonucleoprotein effector nucleases, while class 2 systems (types II, V, and VI) use a single Cas protein (Nishimasu et al., 2017). In addition to the CRISPR Cas9 system, the CRISPR Cas12a (or Cpf1) system has emerged as a powerful biotechnological tool for mass genome editing applications.

[0009] Cas9 generates blunt-ended DSBs by simultaneously cleaving both DNA strands through the combined activity of two conserved nuclease domains, RuvC and HNH (Jinek et al., 2012; Gasiunas et al., 2012). Cas9 nickase variants can be generated by alanine substitution of key catalytic residues within these domains: the RuvC mutant D10A generates a nick on the targeted strand, while the HNH mutant H840A generates a nick on the non-targeted strand DNA (amino acid numbering of Cas9 from Streptococcus pyogenes, SpCas9; Jinek et al., 2012; Gasiunas et al., 2012; Cong et al., 2013; Mali et al., 2013).

[0010] Recently, it has been described in plant cells that the introduction of paired nicks strongly improves the efficiency of homology-directed repair and allows for precise introduction of donor DNA sequences into the plant genome by reducing random insertions and / or deletions (indels) (WO2021122080A1). Such nickase-based methods can greatly reduce the screening effort.

[0011] A further approach to improve specific and targeted modification of DNA is guide RNAs that are covalently linked to donor nucleotides, thereby enhancing HDR efficiency (WO2017186550A1). Such fusion nucleic acid molecules could be combined with the efficient Cas12a nickase to achieve optimal efficiency and specificity when introducing donor sequences into the target genome.

[0012] In contrast to the previous discovery of Cas9 nickase, target-specific nicking has not yet been achieved for Cas12a, especially in relevant crop plants, and therefore there is a great need to establish suitable Cas12a-based nickase tools.

[0013] Unlike Cas9, Cas12a cleaves both DNA strands sequentially using a single catalytic site located in the RuvC domain, while the Nuc domain plays a role in substrate DNA coordination (Swarts et al., 2017, 2019). This difference in structural organization precludes the design of a true nickase for Cas12a compared to Cas9, the latter CRISPR nuclease having two distinct domains containing two individual active domains, HNH and RuvC, that catalyze the cleavage of the target and non-target strands, respectively.

[0014] In the LbCas12a structure, the RuvC active site is formed by the conserved acidic residues Asp832, Glu925, Asp1180, and Arg1138 (Yamano et al., 2017). In vitro cleavage assays showed that D832A, E925A, and D1180A mutations completely abolish the DNA cleavage activity of LbCas12a, while the R1138A mutant is reported to function as an at least partially active nickase in vitro, as in the case of R1226A AsCas12a (Zetsche et al., 2015; Yamano et al., 2016). As also reported in Yamano et al., 2017, LbCas12a and AsCas12a are structurally and functionally related. Notably, both of these Cas12a variants share an overall domain structure. Another reported nickase variant includes the FnCas12a K1013G / R1014G double mutant, which was reported to cleave only the target strand (WO 2019 / 233990).

[0015] To date, there is no evidence of specific nickase activity in vivo of Cas12a nicking variants, and as a result, there is no generally applicable Cas12a nickase with high and specific nicking activity in vivo in a variety of eukaryotic cells.

[0016] Given the central role of nickases in multiple genome editing tools (HDR, base editing, prime editing), the development of Cas12a variants that exhibit efficient DNA nicking in vivo, including in plants, is critical to exploit the full potential of Cas12a for crop genetic improvement, therapeutic applications, and applications in food and nutritional sciences.

[0017] The application of CRISPR-Cas is very challenging in wheat, one of the most important crop plants worldwide but difficult to genetically modify, but efficient methods for precise introduction of donor DNA sequences into the wheat genome have been developed in recent years (WO2021122081A1). Therefore, an efficient and specific Cas12a nickase may also have great potential for improving precise genetic modification in wheat. Summary of the Invention [Means for solving the problem]

[0018] Thus, the overarching objective is to design and identify, through rational design and directed evolution approaches, one or more Cas12a nickase variants that enable the in vitro and particularly in vivo generation of nicks (or pairs of nicks) in a wide range of prokaryotic and eukaryotic chromosomal DNA, and which Cas12a nickases should possess highly specific nickase activity and low off-target activity, as well as overall robustness and stability that provide a highly versatile and broadly applicable genome nicking tool to be used in a variety of genome modification settings, including base editing, prime editing and paired nickase assays.

[0019] definition Broad nickase activity, as used herein, refers to the ability to efficiently generate specific single-stranded DNA breaks (nicks) both in vitro and in vivo, with minimal or no residual nuclease activity, preferably the residual nuclease activity in vitro and / or in vivo is less than about 20%, more preferably less than about 15%, even more preferably less than about 10%, and most preferably less than about 5% of the total enzyme activity, where the total enzyme activity is the sum of the nickase activity and nuclease activity of a given Cas12a enzyme or catalytically active fragment thereof having nickase activity, and the nickase activity and nuclease activity of a given Cas12a enzyme or catalytically active fragment thereof having nickase activity are determined and compared with the method in the same detection system and / or in a suitable cell-based and / or in vitro system, using suitable and reasonable reaction conditions and further using the same target site under the same conditions within the reasonable constraints of said cell-based and / or in vitro system. Those skilled in the art are well aware of a variety of different suitable methods for determining the nickase and nuclease activity of Cas12a enzyme, including the methods disclosed herein. The term "nuclease activity" as used herein refers to nucleolytic activity, and while one nuclease effector can generate a double-stranded break, for a nickase, two individual nicks (either by the same or by at least two different nickases) are required to achieve a double-stranded break. Target strand (TS) nickase activity as used herein refers to nickase activity as described above, and at least 90% of the nicking occurs on the target strand. Non-target strand (NTS) nickase activity as used herein refers to nickase activity as described above, and at least 90% of the nicking occurs on the non-target strand.

[0020] A target site, as used herein, refers to both strands of double stranded DNA to which a guide RNA anneals, i.e. the target strand and the complementary non-target strand; a target site is a stretch of DNA to which a guide RNA has suitable complementarity with a target strand; in embodiments, at least two compatible guide RNAs are designed to allow the coordinated action of one or at least two Cas enzymes; a target site refers to at least two stretches of DNA, for each of which one guide RNA has complementarity with a target strand, further comprising any DNA sequence between said at least two stretches of DNA (see also FIG. 7A); said at least two stretches of DNA to each of which one guide RNA has complementarity may also overlap or be identical.

[0021] "At or near a target site," as used herein, refers to a portion of DNA up to 10 bp, up to 20 bp, up to 30 bp, or up to 40 bp that is within the target site or adjacent to the target site, including in both directions.

[0022] "Donor repair template", or "donor template", or "donor DNA", or simply "donor", refers to a nucleic acid template that can be provided to enable and mediate HDR, which can be used to achieve error-free modification of a target locus and / or introduction of a foreign nucleic acid sequence, such as a transgene. At least one donor repair template can include or encode a double-stranded and / or single-stranded nucleic acid sequence. At least one donor repair template can include or encode an RNA and / or DNA sequence. At least one donor repair template can include or encode a symmetric or asymmetric homology arm. In certain embodiments, at least one donor repair template can further include at least one chemically modified base and / or backbone, such as a fluorescent marker and / or a phosphorothioate modified backbone. The design and use of donor repair templates for various purposes is well known to those skilled in the art.

[0023] The term "disease state associated target site" as used herein refers to any target site where a particular allele, variant or mutation actually or potentially results in, affects or may be a risk factor for at least one physical and / or psychiatric disease, illness, disorder or adverse condition or property, or the progression or prognosis thereof. A disease state associated target site may be, for example, a target site that contains a missense or nonsense mutation in a protein-coding gene or it may be a target site that contains various polymorphisms, such as single nucleotide polymorphisms, that correlate with what may be a risk factor for the development of a particular disease.

[0024] The term "guide RNA" may refer to any RNA that includes a Cas protein binding region and a targeting region, and can guide a Cas protein to a target nucleotide sequence that is sufficiently complementary to the targeting region of the guide RNA, as long as the target nucleotide sequence is adjacent to a suitable PAM sequence for the respective Cas protein. With respect to the Cas12a system, the terms "guide RNA", "crRNA", "gRNA" or "sgRNA" are used interchangeably. With respect to systems and / or methods that use two guide RNA molecules in a natural environment as known in the art, such as crRNA and tracrRNA, the term guide RNA refers to both RNA molecules. Having described the CRISPR effector system comprising the Cas enzyme and the cognate guide RNA (crRNA, or crRNA::tracrRNA), the skilled artisan therefore knows which type of guide RNA is used for which type of Cas enzyme, e.g., the Cas12a system uses a single crRNA, while the Cas12e system, like the Cas9 system, uses a crRNA::tracrRNA duplex, however, the crRNA::tracrRNA duplex can be mimicked by a synthetic single guide RNA molecule. Furthermore, the skilled artisan is well aware of designing, expressing / synthesizing and adapting guide RNAs for the required purpose. In particular, the mutations to the (n)Cas12a enzyme and its (n)Cas12 ortholog provided herein do not affect the overall design and interaction pattern of the cognate guide RNA for a given nCas12a enzyme or nCas12 ortholog. In embodiments related to a prime editor or prime editor complex, the guide RNA may be a pegRNA (prime editing guide RNA) and may further comprise a primer binding site (PBS) and / or a reverse transcriptase template sequence. The design of guide RNAs, including pegRNAs, suitable for a variety of different Cas systems is well known to those skilled in the art.

[0025] "Identity," when used in reference to a comparison of two or more nucleic acid or amino acid molecules, means that the sequences of the molecules share a certain degree of sequence similarity and that the sequences are partially identical.

[0026] Enzyme variants can be defined by their sequence identity when compared to the parent enzyme. Sequence identity is usually indicated as "% sequence identity" or "% identity". In a first step, to determine the percent identity between two amino acid sequences, a pairwise sequence alignment is made between the two sequences, and the two sequences are aligned over their entire length (i.e., pairwise global alignment). The alignment is made using a program that implements the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably using the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) with the program default parameters (gap open=10.0, gap extension=0.5 and matrix=EBLOSUM62). The preferred alignment for the purposes of the present invention is the alignment that allows the maximum sequence identity to be determined.

[0027] The following example is intended to illustrate two nucleotide sequences, but the same calculations apply to protein sequences. Seq A: AAGATACTG Length: 9 bases Seq B: GATCTGA Length: 7 bases

[0028] The shorter sequence is therefore sequence B.

[0029] Generating a pairwise global alignment showing both sequences over their full length gives rise to the following: [ka]

[0030] The symbol "I" in the alignment indicates an identical residue (meaning a base in the case of DNA or an amino acid in the case of proteins). The number of identical residues is six.

[0031] The symbol "-" in the alignment indicates a gap. The number of gaps introduced by the alignment within Seq B is 1. The number of gaps introduced by the alignment at the boundaries of Seq B is 2 and at the boundaries of Seq A is 1.

[0032] The alignment length is 10, showing sequences aligned over their entire length.

[0033] According to the present invention, the generation of a pairwise alignment showing a shorter sequence over its full length results in: [ka]

[0034] According to the present invention, the generation of a pairwise alignment showing sequence A over its entire length results in: [ka]

[0035] According to the present invention, the generation of a pairwise alignment showing sequence B over its entire length results in: [ka]

[0036] The alignment length showing the shorter sequence over its entire length is 8 (there is one gap included in the alignment length of the shorter sequence).

[0037] Therefore, the alignment length showing Seq A over its entire length would be 9 (meaning that Seq A is a sequence of the present invention).

[0038] Therefore, the alignment length showing Seq B over its entire length would be 8 (meaning that Seq B is a sequence of the invention).

[0039] After aligning the two sequences, in a second step, an identity value is determined from the resulting alignment. For the purposes of this description, the percent identity is calculated by %-identity=(identical residues / length of the alignment region showing each of the sequences of the present invention over its entire length)*100. Thus, the sequence identity associated with the comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues by the length of the alignment region showing each of the sequences of the present invention over its entire length. This value is multiplied by 100 to obtain the "%-identity". According to the example shown above, the %-identity is (6 / 9)*100=66.7% when Seq A is the sequence of the present invention, and (6 / 8)*100=75% when Seq B is the sequence of the present invention.

[0040] "Indel" is a term for the random insertion or deletion of bases in the genome of an organism associated with the repair of DSBs by NHEJ. It is classified among small genetic changes and measures from 1 to 10,000 base pairs in length. As used herein, it refers to the random insertion or deletion of bases in or near the target site (e.g., less than 1000bp, less than 900bp, less than 800bp, less than 700bp, less than 600bp, less than 500bp, less than 400bp, less than 300bp, less than 250bp, less than 200bp, less than 150bp, less than 100bp, less than 50bp, less than 40bp, less than 30bp, less than 25bp, less than 20bp, less than 15bp, less than 10bp or less than 5bp upstream and / or downstream).

[0041] The term in vitro, as used herein, refers to the state or nature of a method or application or procedure that is not carried out inside a living cell, preferably in a cell-free system. In vitro methods, applications or procedures are typically carried out with biological material, such as nucleic acids, polypeptides, etc., that have been purified from cells and / or artificially processed or synthesized, usually in a reaction tube or reaction compartment containing a suitable buffer system and suitable reaction components.

[0042] The term in vivo, as used herein, refers to the state or nature of a method, application or procedure that involves the manipulation of at least one living cell (including cells grown in cell culture), such as the introduction of CRISPR components into a living cell and potential genome nicking, double-strand breaks and / or modifications in said cell. An in vivo method, application or procedure may be followed, for example, by in vitro analysis of purified DNA after cell lysis. Thus, in vivo, as used herein, does not necessarily mean that the method is performed within a living organism, and an in vivo method may be performed in an in vitro environment, such as an in vitro cell culture.

[0043] The term ex vivo, as used herein, refers to the state or nature of a method, application or procedure that involves living cells and / or living tissue that have been extracted from an organism, and that may be reinserted into the organism from which they were extracted following the ex vivo method, application or procedure.

[0044] The term "offset," as used herein, refers to the number of base pairs between the binding sites of two guide RNAs designed to allow for the coordinated action of one or at least two Cas enzymes (see FIG. 7A, which shows an exemplary offset of +5 bp). [Brief description of the drawings]

[0045] [Figure 1](Figure 1) shows an excerpt from an alignment of the full-length sequences of SEQ ID NOs: 1-12, created by CLUSTAL Omega (version 1.2.4) multiple sequence alignment. In particular, Figure 1 shows the sequence identified herein as the "core lid domain" highlighted in bold starting at position L927 and ending at position V942 with respect to LbCas12a (SEQ ID NO: 1) as the reference sequence, said reference core lid domain sequence is further highlighted by underlining. The catalytically active E925 of LbCas12a, which is completely conserved in all Cas12a orthologs / homologs shown (others not shown, e.g., FnCas12a from UniProt accession A0Q7Q2), is highlighted by underlining. The following parameters were used for the alignment: Input parameters: output guide tree=true; output distance matrix=false; Dealign input sequences=false; mBed-like clustering guide tree=true; mBed-like clustering iterations=true; number of iterations=0; max guide tree iterations=-1; max HMM iterations=-1; output alignment format=clustal_num: output order=aligned; sequence type=protein. The displayed sequences are included in the order shown as SEQ ID NOs: 123-134, respectively. [Diagram 2] (Figure 2) shows a schematic diagram of the LbCas12a domain structure and a schematic 2D model of the approximate protein structure in contact with the crRNA and target DNA. PI: PAM interaction domain, BH: bridge helix. The stars in the domain overview and model diagram represent the approximate location of the RuvC lid mutation according to the present invention. [Diagram 3](FIG. 3) shows a model diagram of the E. coli GFP / RFP detection assay used to analyze nickase activity (by detecting paired nicking) and nuclease activity in vivo. The Cas12a vectors shown represent either a Cas12a variant library or one or more specific Cas12a variants. "sgRNA1" refers to a sequence encoding a guide RNA suitable for targeting a first target site ("PS-1"), and "sgRNA2" refers to a sequence encoding a guide RNA suitable for targeting a second target site ("PS-2"). "Cas12a" in this figure refers to a Cas12a enzyme with nuclease activity, "nCas12a" in this figure refers to a Cas12a enzyme with nickase activity, and "dCas12a" in this figure refers to a Cas12a enzyme that is inactive, i.e., has no nickase or nuclease activity. Only the idealized situation is presented; Cas12a variants may also exhibit a combination of nickase and nuclease activity and / or reduced nickase and / or nuclease activity. [Figure 4] (Figure 4) shows the results of GFP / RFP detection for selected Cas12a variants: WT: wild type LbCas12a, dLbCas12a: LbCas12a D832A / E925A (mutations with respect to reference sequence SEQ ID NO: 1); LbCas12a R1138A, LbCas12a K932G / N933G and LbCas12a S934A / R935G: mutations with respect to reference sequence SEQ ID NO: 1; LbCas12a K932G / N933G / S934A / R935G: quadruple lid mutant (SEQ ID NO: 14); RuvCL-neg: negative RuvC lid mutant (LbCas12a F931E / K932E / R935D / K937D / K940D, mutations with respect to reference sequence SEQ ID NO: 1). The Y-axis shows the relative fluorescence intensity, i.e., the fluorescence intensity (as determined by the optical density (OD600) of the E. coli culture) relative to the amount of E. coli cells measured. The grey-white bars show the fluorescence due to GFP and the dark grey bars show the fluorescence due to RFP. [Figure 5-1] (Figure 5A) shows the RuvC lid amino acid sequences of the Cas12a variants shown in Figure 5B. The Cas12a proteins shown are LbCas12a WT (SEQ ID NO: 1), pRV26002 (SEQ ID NO: 23), pRV26004 (SEQ ID NO: 16), pRV26006 (SEQ ID NO: 20), pRV26008 (SEQ ID NO: 21), pRV26010 (SEQ ID NO: 19), pRV26180 (SEQ ID NO: 22), pRV26182 (SEQ ID NO: 18), pRV26184 (SEQ ID NO: 17). The displayed sequences are included in the order shown as SEQ ID NOs: 135 to 143, respectively. (Figure 5B) shows the results of GFP / RFP detection for selected Cas12a variants. The Cas12a proteins shown are WT (SEQ ID NO: 1), dLbCas12a (LbCas12a D832A / E925A, mutations relative to reference sequence SEQ ID NO: 1), pRV26002 (SEQ ID NO: 23), pRV26004 (SEQ ID NO: 16), pRV26006 (SEQ ID NO: 20), pRV26008 (SEQ ID NO: 21), pRV26010 (SEQ ID NO: 19), pRV26180 (SEQ ID NO: 22), pRV26182 (SEQ ID NO: 18), pRV26184 (SEQ ID NO: 17). The grey-white bars indicate the fluorescence derived from GFP and the dark grey bars indicate the fluorescence derived from RFP. [Figure 5-2] (Figure 5C) shows the results of GFP / RFP detection for selected Cas12a variants. The Cas12a proteins shown are WT (SEQ ID NO: 1), dLbCas12a (LbCas12a D832A / E925A, mutations relative to reference sequence SEQ ID NO: 1), Lid1.2 (SEQ ID NO: 24), Lid2.3 (SEQ ID NO: 25), Lid2.4 (SEQ ID NO: 26). The grey-white bars show the fluorescence derived from GFP, and the dark grey bars show the fluorescence derived from RFP. (Figure 5D) shows the amino acid sequences within the mutagenized RuvC lid region of selected LbCas12a nickase variants, with the column "Sequence" showing the amino acids at positions 930 to 933 of the respective SEQ ID NO. Furthermore, the respective partial sequences are provided in SEQ ID NOs (107 to 113). [Figure 5-3](FIG. 5E) shows the results of GFP / RFP detection for selected Cas12a variants. The Cas12a proteins shown are LbCas12a wt (SEQ ID NO: 1), LbCas12a inactive (LbCas12a D832A / E925A, mutations relative to reference sequence SEQ ID NO: 1), Lid2.3 (SEQ ID NO: 15), Lid4.1 (SEQ ID NO: 100), Lid4.2 (SEQ ID NO: 101), Lid4.3 (SEQ ID NO: 102), Lid4.4 (SEQ ID NO: 103), Lid4.5 (SEQ ID NO: 104), Lid4.6 (SEQ ID NO: 105), Lid4.7 (SEQ ID NO: 106). The grey-white bars show the fluorescence derived from GFP, and the dark grey bars show the fluorescence derived from RFP. [Figure 6-1] (FIG. 6A) shows the RuvC lid amino acid sequences of the Cas12a variants shown in FIG. 6B. The sequences shown are included in the order shown as SEQ ID NO: 135, 144 and 145, respectively. (FIG. 6B) shows the results of an in vitro plasmid cleavage assay. The Cas12a proteins shown are LbCas12a WT (SEQ ID NO: 1), dLbCas12a (LbCas12a D832A / E925A, mutations relative to reference sequence SEQ ID NO: 1), pRV26004 (SEQ ID NO: 16), RuVCL del1 (lid deletion variant 1, SEQ ID NO: 15). pT: target plasmid, a plasmid containing a target site for the cRNA used; pUC19: a control plasmid without a target site for the cRNA used; EcoRI and NB.BvCl refer to the corresponding restriction endonucleases and nickases, respectively; N: nicked; L: linear; S: supercoiled. [Figure 6-2](FIG. 6C) shows a method for analysis of nicked target DNA by Sanger run-off sequencing. Nicked substrates obtained from in vitro digestion of target plasmids are extracted from agarose gels, purified, and subjected to Sanger sequencing using primers targeting either the top or bottom strand. The Cas12a proteins shown are LbCas12a WT (SEQ ID NO: 1), LbCas12a inactive (LbCas12a D832A / E925A, mutations with respect to reference sequence SEQ ID NO: 1), FnCas12a K969P / D970P (mutations with respect to reference sequence SEQ ID NO: 3), LbCas12a R1138A (mutations with respect to reference sequence SEQ ID NO: 1), RuvCL-del1 (lid deletion variant 1, SEQ ID NO: 15). pT: target plasmid, a plasmid containing a target site for the cRNA used; pUC19: a control plasmid without a target site for the cRNA used; EcoRI and Nt.BbvCl refer to the corresponding restriction endonucleases and nickases, respectively; N: nicked; L: linear; S: supercoiled. [Figure 6-3] (FIG. 6D) shows a schematic model of the dsDNA substrate used in the in vitro fluorescent nickase activity assay. The DNA substrate is labeled with Cy5 on the target strand and Cy3 on the non-target strand. A shift in the position of the fluorescent DNA band indicates that a strand has been cleaved. [Figure 6-4](FIG. 6E) shows the results of an in vitro fluorescent nickase assay. The Cas12a proteins shown are LbCas12a WT (SEQ ID NO: 1), dLbCas12a (LbCas12a D832A / E925A, mutations relative to reference sequence SEQ ID NO: 1), RuVCL del1 (lid deletion variant 1, SEQ ID NO: 15), RuvCL-del1 C931E (lid deletion variant 1+C931E, SEQ ID NO: 56). No digestion: control reaction containing only fluorescently labeled DNA substrate; EcoRI and Nt.BvCl refer to the corresponding restriction endonucleases and nickases, respectively; "-" and "+" indicate the absence and presence of the selected Cas12a proteins in the nicking reaction. Different incubation times were tested for the nicking reaction with the RuvCL-del1 C931E mutant, all other reactions were incubated at 37° C. for 1 h. [Figure 7](FIG. 7A) shows an example set for paired nicking with a +5bp offset. sgRNA3 and sgRNA9 refer to two different guide RNAs. The italicized letters indicate the nucleic acid sequences that have complementarity with the respective guide RNAs, i.e., the guide RNA binding sites on the respective target strands. The bolded letters indicate the nucleic acid sequences (on the respective non-target strands) that correspond to the sequences in the targeting region of the respective guide RNAs. The grey boxes indicate the target sites in this exemplary paired nickase configuration. This configuration was used in the exemplary paired nickase assay shown in FIG. 7B. This exemplary configuration is designed for Cas9-mediated nicking and therefore includes a PAM suitable for the Cas9 protein. For the Cas12a paired nickase strategy, a PAM suitable for the respective Cas12a protein must be selected. Top DNA strand: SEQ ID NO: 34; bottom DNA strand: SEQ ID NO: 35. (Figure 7B) shows an exemplary result of an in vitro paired nicking assay of TXTL with two different guide RNAs (see Figure 7A) targeting sequences encoding Cas9 D10A nickase and GFP. GFP fluorescence over time is shown in gray-white for each sample and in dark gray for a control where the sequence encoding GFP is not targeted. Cas9-sg3: Cas9 nuclease with first guide RNA (sg3:sgRNA3); nCas9 D10A-sg3: Cas9 D10A nickase with first guide RNA; nCas9 D10A-sg9: Cas9 D10A nickase with second guide RNA (sg:sgRNA9); nCas9 D10A-sg3+sg9: Cas9 D10A nickase with first and second guide RNAs. [Figure 8-1](FIG. 8A) shows an analysis of editing results at OsAAT target sites in rice protoplasts transfected with Cas12a nickase candidates. The Y-axis shows the percentage of sequencing reads with indels. The Cas12a proteins shown are LbCas12a (SEQ ID NO: 1), LbCas12a R1138A (mutations with respect to reference sequence SEQ ID NO: 1), LbCas12a K932G / N933G (mutations with respect to reference sequence SEQ ID NO: 1), LbCas12a K932G / N933G / S934A / R935G: quadruple lid mutant (SEQ ID NO: 14). (FIG. 8B) shows an analysis of editing results at OsAAT target sites in rice protoplasts transfected with Cas12a nickase candidates. The Y-axis shows the percentage of sequencing reads with base substitutions. The Cas12a proteins shown are LbCas12a (SEQ ID NO: 1), LbCas12a R1138A (mutations relative to reference sequence SEQ ID NO: 1), LbCas12a K932G / N933G (mutations relative to reference sequence SEQ ID NO: 1), LbCas12a K932G / N933G / S934A / R935G: quadruple lid mutant (SEQ ID NO: 14). [Figure 8-2] (Figure 8C) shows a comparative representation of the data shown in Figures 8A and 8B. Column I shows nuclease activity in percent of wild type LbCas12a (WT), column II shows the percentage of edited reads with indels, and column III shows the percentage of edited reads with base substitutions. [Figure 9](Figure 9A) shows the concept of nicking assay of GFP / dsRed pair. The sequence encoding GFP is targeted by two guide RNAs, while the sequence encoding dsRED is targeted by one. "Cas12a" in this figure means Cas12a enzyme with nuclease activity, "nCas12a" in this figure means Cas12a enzyme with nickase activity, and "dCas12a" in this figure means Cas12a enzyme is inactive Cas12a, i.e., has neither nickase nor nuclease activity. Only the ideal state is shown, and Cas12a variants may also have a combination of nickase and nuclease activity and / or reduced nickase and / or nuclease activity. (Figure 9B) shows an exemplary fluorescence microscope image of nicking analysis of planta GFP / dsRed pair. Rice protoplasts were transfected with either no Cas protein (control); wild-type LbCas12a (SEQ ID NO: 1), inactive LbCas12a D893A (mutations relative to the reference sequence of SEQ ID NO: 1); or LbCas12a K932G / N933G / S934A / R935G (SEQ ID NO: 14). [Figure 10-1] (FIG. 10A) shows the results of different LbCas12a base editor constructs at the OsAAT target site in rice protoplasts. The Y-axis shows the percentage of reads with base edits. LbCas12a-D832A and LbCas12a-K932G / N933G: mutations relative to reference sequence SEQ ID NO: 1. LbCas12a K932G / N933G / S934A / R935G: SEQ ID NO: 14. [Figure 10-2] (FIG. 10B) shows the results of different LbCas12a base editor constructs at the OsAAT target site in rice protoplasts. The Y-axis shows the percentage of reads with indels. LbCas12a-D832A and LbCas12a-K932G / N933G: mutations relative to reference sequence SEQ ID NO: 1. LbCas12a K932G / N933G / S934A / R935G: SEQ ID NO: 14. [Figure 11](FIG. 11) shows an analysis of editing results at OsAAT target sites in rice protoplasts transfected with Cas12a nickase candidates. The Y-axis shows the percentage of sequencing reads with indels. The Cas12a proteins shown are LbCas12a (SEQ ID NO: 1), LbCas12a-RuvC lid deletion (SEQ ID NO: 15) and LbCas12a-RuvC lid deletion / C931E (SEQ ID NO: 56). [Figure 12] (FIG. 12A) shows the results of different LbCas12a base editor constructs at the OsAAT target site in rice protoplasts. The base editors contain either LbCas12a-D832A (mutations relative to reference sequence SEQ ID NO: 1), LbCas12a-RuvC lid deletion (SEQ ID NO: 15), or LbCas12a-RuvC lid deletion / C931E (SEQ ID NO: 56) as the Cas part. The Y-axis shows the base editing efficiency expressed relative to that shown by the LbCas12a-D832A editor. (FIG. 12B) shows the results of different LbCas12a base editor constructs at the BnFAD2 target site in oilseed rape protoplasts. The base editors contain either LbCas12a-D832A (mutations relative to reference sequence SEQ ID NO: 1), LbCas12a-RuvC lid deletion (SEQ ID NO: 15) or LbCas12a-RuvC lid deletion / C931E (SEQ ID NO: 56) as the Cas moiety. The Y-axis shows the base editing efficiency expressed relative to that exhibited by the LbCas12a-D832A editor. [Figure 13] (FIG. 13) shows the effect of target sequence and guide offset on the level of indel formation at the OsDEP1 target site in rice protoplasts co-transfected with paired gRNA and LbCas12a-RuvC lid deletion nickase variant (SEQ ID NO: 15). Guide offset is defined as the distance between the PAM-distal (3') ends of the guide for a given gRNA repair. [Figure 14](Figure 14) shows the indel frequency in rice protoplasts induced by double nicking with selected Cas12a-nickase variants compared to those induced by single nickase or WT LbCas12a. [Figure 15] (Figure 15) shows a schematic diagram of the transient expression vectors used for paired nicking experiments in HEK293 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Based on multiple iterative rounds of in silico analysis, rational protein design and semi-random saturation mutagenesis approaches, and subsequent functional testing, we identified several variants of Cas12a, including Lachnospiraceae Cas12a (LbCas12a), that exhibit efficient nicking both in vitro and in vivo, and the performance of different candidate variants could be tested using several activity assays in different organisms, including E. coli, plants and yeast, as well as mammalian cell culture systems.

[0047] Regarding Cas12a, on the one hand, structural and mechanistic insights are available (e.g., Stella et al., Cell, 2018), which shows that Cas12a contains a so-called "lid" protein compartment that contains the catalytic E1006 (FnCas12a, SEQ ID NO: 3; corresponding to E925 in LbCas12a, SEQ ID NO: 1) and other residues in a loop that closes the catalytic pocket in the apo structure. During hybridization of the crRNA guide region and the target DNA strand in Cas12a, certain key motifs such as the fingers, helix-loop-helix (HLH), and REC linker from the REC lobe and the lid motif in the RuvC domain function in concert to conformationally activate the DNase activity of Cas12a (Stella et al., 2018; Zhang et al., 2021).

[0048] So far, the conformationally mobile part of the lid domain after the catalytically active residue E925 (LbCas12a; SEQ ID NO: 1) has not yet been studied in detail to create an efficient Cas12a-based nickase, since it is highly conserved in all Cas12a orthologs. Therefore, this motif, referred to herein as the "core lid domain" (see SEQ ID NO: 13 for the overall consensus sequence), was specifically analyzed as a target structure for rational protein design to establish a highly functional Cas12a-nickase with an unchanged catalytically active site, but modulating and fine-tuning the nicking activity of only one strand by modifying the mobility of the lid. The core lid domain of LbCas12a as a reference sequence (see SEQ ID NO: 1 and FIG. 1) comprises the core lid domain as defined herein, starting at position L927 and ending at position V924. Homologous positions in conserved Cas12a homologs / orthologs known to those of skill in the art and disclosed herein (e.g., SEQ ID NOs: 1-12) can be determined by those of skill in the art based on the information provided herein.

[0049] SEQ ID NO:13, as detailed in Example 2 below, was identified as a core lid domain and thus a novel sub-motif within Cas12a. This core lid domain corresponds to 927-942 according to SEQ ID NO:1 (LbCas12a) as a reference sequence, which is shown to represent a suitable consensus sequence or motif for characterizing and identifying Cas12 variants. Thus, one skilled in the art can easily identify Cas12a proteins having a core lid domain based on the disclosure presented herein. Based on the in silico analysis detailed in Example 2, position X in SEQ ID NO:13 may correspond to a sequence in the Cas12a wild-type enzyme in various aspects and embodiments disclosed herein below. Xaa at position 2 of SEQ ID NO:13 may be N or S or an amino acid of similar polarity, Xaa at position 3 of SEQ ID NO:13 may be F, H, or Y or an amino acid of similar polarity, Xaa at position 7 of SEQ ID NO:13 may be S, A, K, R, N or an amino acid of similar polarity, Xaa at position 8 of SEQ ID NO:13 may be K or G or an amino acid of similar polarity, Xaa at position 10 of SEQ ID NO:13 may be T, S, F, V, Q or an amino acid of similar polarity, and Xaa at position 11 of SEQ ID NO:13 may be G or K or an amino acid of similar polarity. Xaa at position 12 of SEQ ID NO:13 may be I or V, or an amino acid of similar polarity; Xaa at position 13 of SEQ ID NO:13 may be present or absent and, if present, it may be A, or an amino acid of similar polarity; Xaa at position 15 of SEQ ID NO:13 may be K, R, S, or an amino acid of similar polarity; Xaa at position 16 of SEQ ID NO:13 may be A, G, S, or an amino acid of similar polarity; and Xaa at position 17 of SEQ ID NO:13 may be V or I, or an amino acid of similar polarity.

[0050] All wild-type Cas12a enzymes previously disclosed and provided in the prior art as suitable for genome editing may be eligible as sources for Cas12a nickases as disclosed herein. Orthologs, such as closely related FnCas12a, ErCas12a sequences, may also be eligible, even if they are not included in an independent claim.

[0051] Other sources of species include Francisella tularensis, Prevotella albensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella sp., Acidaminococcus sp., Candidatus Methanoplasma termitum, Eubacterium eligens, Eubacterium lectare, and others. rectale, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens and Porphyromonas macacae, Succinivibrio dextrinosolvens, Prevotella disiens, Flavobacterium sp., Flavobacterium branchiophilum, Helcococcus kunzii, Eubacterium sp.), Microgenomates(Roizmanbacteria)bacterium, Prevotella brevis, Moraxella caprae, Bacteroidetes oral, Porphyromonas cansulci, Synergistes jonesii, Prevotella bryantii, Anaerovibrio sp., Butyrivibrio fibrisolvens, Candidatus Methanomethylophilus, Butyrivibrio sp. or any Cas12a variant or any Cas12 ortholog selected from the group consisting of Oribacterium sp., Pseudobutyrivibrio ruminis and Proteocatella sphenisci., Acidibacillus spp. including Acidibacillus sulfuroxidans, Deltaproteobacteria spp., Planctomycetes spp..

[0052] In a first aspect according to the present invention there is provided an engineered Cas12a enzyme (nCas12a) having nickase activity, or a catalytically active fragment thereof, wherein the engineered Cas12a enzyme may comprise at least one mutation in its core lid domain, the mutation in the core lid domain being selected from the group consisting of (i) at least three point mutations at three consecutive positions in the core lid domain; or (ii) a deletion at least two consecutive positions in the core lid domain; or (iii) a combination of at least one first point mutation at at least one position in the core lid domain comprising two or more point mutations at consecutive positions, and (iiia) at least one deletion at at least one position comprising two or more deletions at consecutive positions in the core lid domain and / or (iiib) a deletion at at least one position comprising two or more deletions at consecutive positions in the core lid domain. at least one, preferably at least two, at least three, or at least four additional point mutations, including two or more point mutations at consecutive positions at different positions compared to the first point mutation in the core lid domain (the positions of the additional point mutations are not in consecutive order with the positions of the at least one first point mutation); (iv) one point mutation at a position in the core lid domain (wherein the at least one mutation in the core lid domain confers broad-spectrum nickase activity, and the reference sequence of the core lid domain comprises a sequence as defined in SEQ ID NO: 13, and optionally the complex further comprises at least one compatible guide RNA, or a sequence encoding same, and is complexed with a cognate engineered Cas12a enzyme having nickase activity, or a catalytically active fragment thereof).

[0053] In one embodiment, at least one mutation in the core lid domain is within positions 5-15 with respect to SEQ ID NO:13.

[0054] Position X or Xaa as defined in SEQ ID NO: 13 may be present in another wild-type Cas12a ortholog or homolog with similar polarity. In this context, "similar polarity" as used herein means a polarity that follows the standard polarity (i.e., charge distribution) of the side chain of the amino acid, and similar polarity means that the amino acid residue at a given position can be exchanged for an amino acid within the same polar group, the polar group being selected from group I, which includes non-polar amino acids selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; group II, which includes polar, uncharged amino acids selected from amino acids serine, cysteine, threonine, tyrosine, asparagine, and glutamine; group III, which includes acidic amino acids selected from aspartic acid and glutamic acid; and group IV, which includes basic amino acids selected from arginine, histidine, and lysine.

[0055] In one embodiment according to various aspects as disclosed herein, 1, 2, 3, 4, 5, 6, 7 or all 8 positions 6-13 with respect to SEQ ID NO: 13 may be deleted or may have point mutations or combinations thereof.

[0056] In one embodiment according to various aspects as disclosed herein, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all 11 positions 5-15 with respect to SEQ ID NO: 13 may be deleted or they may have point mutations or combinations thereof.

[0057] In one embodiment according to various aspects as disclosed herein, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or all 17 positions of the core lid domain with respect to SEQ ID NO: 13 are deleted or have point mutations or combinations thereof.

[0058] In certain embodiments, the at least one point mutation in the core lid domain according to the invention may comprise or consist of at least three point mutations at three positions in the core lid domain, preferably the mutation comprises or consists of (a) a first point mutation at a first position or a first stretch of at least two point mutations at consecutive positions, (b) a second point mutation at a second position or a second stretch of at least two point mutations at consecutive positions, (c) a third point mutation at a third position or a third stretch of at least two point mutations at consecutive positions, and optionally (d) at least one further point mutation at at least one further position or at least one further stretch of at least two point mutations at consecutive positions, wherein the first position or first stretch of positions, the second position or second stretch of positions, the third position or third stretch of positions, and optionally the at least one further position or at least one further stretch of positions are not present in consecutive order with respect to each other.

[0059] In one embodiment according to various aspects as disclosed herein, the at least one point mutation in the core lid domain according to the invention may comprise or consist of a deletion at a first position or at least two deletions of a first section of consecutive positions, and a second deletion at a second position or a second section of consecutive deletions, and optionally at least one further deletion at at least one further position or at least one further section of consecutive deletions, wherein the position of the second deletion or the second section of deletion is not in consecutive order with the first deletion or the first section of consecutive deletions, and optionally the position of the at least one further deletion or the at least one further section of deletion is not in consecutive order with the first section of the first position or consecutive positions and the second section of the second position or consecutive deletions.

[0060] In certain embodiments, the at least one point mutation in the core-lid domain is a single deletion at one position, a deletion of two, three, four, five, six, seven, eight, or nine deletions of a stretch of contiguous positions, or in certain embodiments, ten or more deletions (preferably, the position or stretch of positions is within positions 5-15 with respect to SEQ ID NO: 13, optionally in combination with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 point mutations, and some or all positions of the point mutations may be in consecutive order, optionally in consecutive order with the position or stretch of positions of the deletion); or (b) a first deletion at a first position or a consecutive deletion of a first stretch of 2, 3, 4, or 5 positions (preferably, the first position or first stretch of positions is within SEQ ID NO: 13, optionally in combination with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 point mutations, and some or all positions of the point mutations may be in consecutive order, optionally in consecutive order with the position or stretch of positions of the deletion); 13) and a second deletion at a second position, preferably at least one second section of 2, 3, 4, or 5 contiguous deletions (in total) of at least one second section of positions (preferably the second position or at least one second section of positions is within positions 5-15 with respect to SEQ ID NO: 13, and optionally the second deletion or at least one second section of contiguous deletions is not in contiguous order with the first deletion or first section of contiguous deletions, optionally in combination with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 point mutations, some or all of the positions of the point mutations may be in contiguous order and may optionally be in contiguous order with any of the positions of the deletion or sections of the positions of the deletion).

[0061] In one embodiment according to various aspects as disclosed herein, the engineered Cas12a enzyme may be based on a wild-type Cas12a sequence according to any one of SEQ ID NOs: 1-12, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with the corresponding wild-type sequence as a reference sequence, or an ortholog or homolog of a sequence according to any one of SEQ ID NOs: 1-12 having at least 95%, 96%, 97%, 98% or at least 99% sequence identity with the corresponding ortholog or homolog sequence as a reference sequence.

[0062] In another embodiment according to various aspects as disclosed herein, the at least three point mutations in three consecutive amino acids may be located within positions 2-16 with respect to SEQ ID NO: 13, and / or the deletion is a deletion of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 consecutive positions within the core lid domain.

[0063] In another embodiment according to various aspects as disclosed herein, the mutations may be a deletion of at least 4, at least 5, at least 6, at least 7, or at least all 8 of positions 6-13 with respect to SEQ ID NO:13, and / or the mutations are at least one of three point mutations at three consecutive positions within positions 6-13 with respect to SEQ ID NO:13.

[0064] In another embodiment according to various aspects as disclosed herein, the engineered Cas12a enzyme or catalytically active fragment thereof has target strand (TS) nickase activity or non-target strand (NTS) nickase activity, preferably, the engineered Cas12a enzyme or catalytically active fragment thereof has non-target strand (NTS) nickase activity.

[0065] In another embodiment according to various aspects as disclosed herein, the engineered Cas12a enzyme can include an amino acid sequence according to SEQ ID NO: 14-21 or 56, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the corresponding reference sequence. The engineered Cas12a enzyme may or may not comprise at least a core lid domain of any one of SEQ ID NOs: 14-21 or 56 beginning at position 927, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the corresponding core lid domain.

[0066] In another embodiment according to various aspects as disclosed herein, the Cas12a enzyme having nickase activity can comprise at least one additional mutation, wherein the at least one additional modification alters the PAM specificity and / or thermotolerance of the engineered Cas12a enzyme.

[0067] Most wild-type Cas12a proteins have relatively strict requirements for the TTTV PAM sequence, with some variation between different Cas12a orthologs.

[0068] Suitable PAM variants that expand the constraints of the PAM have been described for various Cas12a orthologs (see, e.g., WO2018195545, WO2020033774, WO2018022634).

[0069] According to various aspects and embodiments disclosed herein, at least one mutation resulting in a PAM variant with altered PAM specificity, preferably to extend the PAM constraints of the respective wild-type Cas12a enzyme, may be combined with the nCas12a enzyme as disclosed herein.

[0070] Examples of mutants that modify PAM specificity and / or thermotolerance include LbCas12a-RR(G532R / K595R), LbCas12a-RVR(G532R / K538V / Y542R), LbCas12a-RVRR(G532R / K538V / Y542R / K595R), enLbCas12a(D156R / G532R / K538R), ttLbCas12a(D156R), FnCas12a-RR(N607R / N617R), FnCas12a-RVR(N607R / K613V / N617R), FnCas12a-RVRR(N607R / K613V / N617 R / K671R), AsCas12a-RR(S542R / N552R), AsCas12a-RVR(S542R / K548V / N552R), AsCas12a-RVRR(S542R / K548V / N552R / K607R), enAsCas12a-HF(E174R / N282A / S542R / K548R), MbCas12a-RR(N576R / N582R), MbCas12a-RVR(N576R / K578V / N58 2R), MbCas12a-RVRR(N576R / K578V / N582R / K634R), Mb2Cas12a-RVR(Mb2Cas12a N563R / K569V / N573R), Mb2Cas12a-RVRR (Mb2Cas12a N563R / K569V / N573R / K625R), BsCas12a-3Rv (K155R / N512R / K518R), PrCas12a-3Rv (E162R / N519R / K525R), Mb3Cas12a-3Rv (D180R / N581R / K587R) (International Publication No. 2018195545, International Publication No. 2020033774, International Publication No. 201822634).

[0071] In some embodiments according to various aspects as disclosed herein, at least one mutation in the core lid domain according to the invention may be present in a Cas12a variant having one of the following amino acid reference sequences: SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33.

[0072] In one embodiment, at least one mutation, preferably exactly one, introduced into the core lid domain motif may insert a Cys residue in place of the wild type amino acid, and the at least one inserted Cys residue, preferably exactly one inserted Cys residue, may be introduced in combination with one or more other point mutations and / or deletions according to the invention. Without wishing to be bound by theory, it is postulated that the introduction of an additional cysteine ​​residue may advantageously alter the dynamic lid domain reassembly upon DNA target site binding such that nickase activity is promoted.

[0073] In certain embodiments, nCas12a, or an active fragment thereof, does not contain a point mutation at position 6 (with respect to SEQ ID NO: 13) resulting in a glycine residue in combination with a point mutation at position 7 (with respect to SEQ ID NO: 13) resulting in a glycine residue, and does not contain at least one additional point mutation and / or deletion within the core lid domain (SEQ ID NO: 13).

[0074] In certain embodiments, the Cas12a enzyme as disclosed herein having nickase activity and containing a mobile lid domain can also be selected from orthologs of Cas12a that have the same overall functionality as class 2 type V CRISPR nucleases in their natural environment and have the same overall folding and mechanistic behavior as Cas12a. In particular, such orthologs will have a lid domain that dynamically opens and closes upon substrate binding just like Cas12a (Stella et al., 2017), and as a result, the lid domain of these Cas12a ortholog nickase effectors can also be modified and used as disclosed herein. As shown by Zhang et al. (2020; see Supplementary Data Figure 8) for the Cas12a ortholog Cas12i, the lid domain appears to be conserved in Cas12a orthologs of class 2 type V CRISPR effectors, such that the findings herein can be extended to sub-motifs within the core lid domain as defined herein.

[0075] In one embodiment, nCas12a orthologous enzymes may include Cas12e (also referred to as CasX), including DpbCas12e and PlmCas12e (Selkova et al. RNA Biol. (2020); 17(10): 1472-1479; doi: 10.1080 / 15476286.2020.1777378).

[0076] In another embodiment, the nCas12a orthologous enzyme includes Cas12f1 (Cas14a and V-U3 types), and may include Cas12f variants including AsCas12f1 and Un1Cas12f1, Cas12f2 (Cas14b) and Cas12f3 (Cas14c, V-U2 and U4 types) (Kim et al. Nat Biotechnol. (2022); 40(1): 94-102; doi: 10.1038 / s41587-021-01009-z; Karvalis et al. Nucleic Acids Res. (2020); 48(9): 5016-5023. doi: 10.1093 / nar / gkaa208).

[0077] In a second aspect, there is provided a nucleic acid sequence or nucleic acid molecule (used interchangeably herein in the context of Cas12a enzyme or a catalytically active fragment or variant thereof) encoding a Cas12a enzyme or a catalytically active fragment or variant thereof according to the first aspect of the invention, optionally wherein the nucleic acid sequence is a codon optimized sequence and / or comprises a nucleic acid sequence encoding at least one guide RNA.

[0078] In some embodiments, the nucleic acid sequence is codon-optimized for fungal, prokaryotic or archaeal cells, including yeast cells, particularly for fungal, prokaryotic or archaeal cells disclosed herein. In one embodiment, the nucleic acid molecule comprises or consists of a fungal or prokaryotic optimized sequence according to SEQ ID NOs: 80-87, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%. SEQ ID NOs: 80-87 are sequences encoding LbCas12a-RuvC lid deletions codon-optimized for Bacillus subtilis, Rhodococcus spp., Yarrowia lipolytica, Escherichia coli K12, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Corynebacterium glutamicum, and Pseudozyma tsukubaensis, respectively. The sequences have been adapted by adaptation according to the proportions of the codon usage table of the selected organism, and the removal of repeats of the same codons is removed to avoid translation termination.

[0079] In some embodiments, the nucleic acid sequence is codon-optimized for the disclosed plant cells, particularly the plant cells disclosed herein. In one embodiment, the nucleic acid molecule comprises or consists of a plant-optimized sequence according to SEQ ID NOs: 88-93, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%. SEQ ID NOs: 88-93 are sequences encoding codon-optimized LbCas12a-RuvC lid deletions for soybean (Glycine max), maize (Zea mays), oilseed rape (Brassica napus), cotton species (Gossypium spp), rice (Oryza sativa) and wheat (Triticum aestivum), respectively. The sequences are codon-optimized by using GeneOptimizer, a BASF proprietary adaptation method, according to the proportions of the codon usage table of the selected organism.

[0080] In some embodiments, the nucleic acid sequence is codon-optimized for animal cells, including human cells, particularly for animal cells, including human cells, disclosed herein. In one embodiment, the nucleic acid molecule comprises or consists of an animal-optimized sequence according to SEQ ID NOs: 94-99, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%. SEQ ID NOs: 94-99 are sequences encoding codon-optimized LbCas12a-RuvC lid deletions for Homo sapiens, Rattus norvegicus, Bos taurus, Mus musculus, Sus scrofa, and Gallus gallu, respectively. The sequences have been adapted by using the CLC Genomics Workbench reverse translation tool based on frequency distribution.

[0081] The nucleic acid sequence may be operably linked to promoter and / or terminator sequences suitable for the desired target cell in which the provided nucleic acid sequence may be expressed.

[0082] In a third aspect, there is provided an expression construct or vector comprising at least one nucleic acid sequence according to the second aspect.

[0083] Suitable expression constructs or vectors for a large number of different target cells and means and methods for designing such expression constructs or vectors, including a wide variety of suitable markers, are well known to those of skill in the art.

[0084] Non-limiting examples of expression constructs and classes of vectors include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, bacteriophages, artificial chromosomes, minicircles, or Agrobacterium binary vectors, in double-stranded or single-stranded, linear or circular forms that may or may not be autoinfective or mobilizable. In some embodiments, viral vectors may include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated, or herpes simplex viral vectors.

[0085] In a fourth aspect, there is provided a cell comprising at least one nucleic acid sequence according to the second aspect, or at least one expression construct or vector according to the third aspect.

[0086] In one embodiment, the cell may be a eukaryotic or prokaryotic cell, including a bacterial or archaeal cell.

[0087] In particular, cells with respect to a multicellular organism, as used herein, are preferably isolated and / or cultured cells which can be analyzed and modified.

[0088] In an embodiment according to various aspects as disclosed herein, the cell may be a plant cell, including an algae cell, and preferably the cell is selected from the group consisting of Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp., Artocarpus spp., and the like. spp.), asparagus (Asparagus officinalis), Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.sativa, Avena hybrida), star fruit (Averrhoa carambola), Bambusa sp., Benincasa hispida, Brazil nut (Bertholletia excelsea), sugar beet (Beta vulgaris), Brassica spp. (e.g. Brassica napus, Brassica rapa subsp.), rapa ssp. (canola, rapeseed, turnip rape), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp.), Carex elata, Papaya (Carica papaya), Carissa macrocarpa, Pecan (Carya spp.), Safflower (Carthamus tinctorius), Chestnut (Castanea spp.), Kapok (Ceiba pentandra), Endive (Cichorium endivia), Cinnamomum spp., Watermelon (Citrullus lanatus), Citrus spp., Cocos spp., Coffea spp., Taro (Colocasia esculenta), Cola spp., Corchorus sp., Coriander (Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis spp. (e.g. Elaeis guineensis, Elaeis oleracea), Elaeis oleracea (e.g. ... oleifera), finger millet (Eleusine coracana), teff (Eragrostis tef), Erianthus sp., loquat (Eriobotrya japonica), Eucalyptus sp., pitanga (Eugenia uniflora), buckwheat (Fagopyrum spp.), beech (Fagus spp.), fescue (Festuca arundinacea), fig (Ficus carica), fortunella spp.), Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), cotton (Gossypium hirsutum), Helianthus spp. (e.g. Helianthus annuus, Hemerocallis fulva), Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), sweet potato (Ipomoea batatas), Juglans spp., lettuce (Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mango (Mangifera indica), Cassava spp., Sapodilla (Manilkara zapota), Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp.), Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Parsley (Petroselinum crispum, Reed canary grass (Phalaris arundinacea), Phaseolus spp., Timothy grass (Phleum pratense), Date palm (Phoenix spp.), Common reed (Phragmites australis), Physalis spp., Pinus spp., Pistachio (Pistacia vera), Pea spp., Poa spp., Populus spp., Prosopis spp., Cherry spp., Psidium spp., Pomegranate (Punica granatum), Pear (Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix spp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis spp., Solanum spp.) (e.g. potato (Solanum tuberosum), Solanum integrifolium or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach (Spinacia spp.), myrtaceae (Syzygium spp.), Tagetes spp., tamarind (Tamarindus indica), cacao (Theobroma cacao), Trifolium spp., gamagrass (Tripsacum dactyloides), Triticosecale rimpaui, wheat (Triticum spp.) (e.g. wheat (Triticum aestivum), durum wheat (Triticum durum), riveted wheat (Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare, Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, or Ziziphus spp. The cell may be selected from cells derived from plants belonging to the superfamily of the subkingdom of Chlorophyta, in particular monocotyledonous and dicotyledonous plants, including but not limited to fodder or forage legumes, ornamental plants, food grains, trees or shrubs, selected from the list including:

[0089] Preferred plants include, independently, Abelmoschus spp., Allium spp., celery (Apium graveolens), asparagus (Asparagus officinalis), Avena spp. (e.g., Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), sugar beet (Beta vulgaris), Brassica spp. (e.g., Brassica napus, Brassica rapa subsp. ... spp. (canola, rapeseed, turnip rape)], Capsicum spp., Citrullus lanatus, Cucumis spp., Cynara spp., Daucus carota, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hordeum spp. (e.g. Hordeum vulgare), Lactuca sativa, Medicago sativa, Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Pennisetum sp., Saccharum spp., Secale cereale, Solanum spp.) (for example potato (Solanum tuberosum), Solanum integrifolium or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach species (Spinacia spp.), wheat species (Triticum spp.) (for example Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), or maize (Zea mays).

[0090] Other preferred plants are Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. (canola, rapeseed, turnip rape)]), Capsicum spp., Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Solanum spp. spp. (e.g. potato (Solanum tuberosum), Solanum integrifolium or tomato (Solanum lycopersicum)), Triticum spp. (e.g. wheat (Triticum aestivum), Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), or corn (Zea mays).

[0091] The term "plant" as used herein includes whole plants, ancestors and descendants of plants, and plant parts including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs. The term "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristem regions, gametophytes, sporophytes, pollen, and microspores.

[0092] Plant cells, tissues, organs, materials, or whole organisms, as used herein, include algal cells, tissues, organs, materials, or whole organisms, respectively.

[0093] In another embodiment according to various aspects as disclosed herein, the cell may be an animal cell, including an insect, poultry, fish or crustacean cell, or a mammalian cell, preferably the cell is a mammalian cell; optionally selected from a cell derived from a non-human primate, cow, pig, rodent, including rat or mouse, or a human cell.

[0094] Animal cells, tissues, organs, or materials, as used herein, include human cells, tissues, organs, or materials, respectively.

[0095] In another embodiment according to various aspects as disclosed herein, the cell may be a fungal cell, including a yeast cell, and preferably the fungal cell, including the yeast cell, is selected from the group consisting of Saccharomyces spec., such as Saccharomyces cerevisiae, Hansenula spec., such as Hansenula polymorpha, Schizosaccharomyces spec., such as Schizosaccharomyces pombe, Kluyveromyces spec., such as Kluyveromyces lactis and Kluyveromyces marxianus, Yarrowia lipolytica, and the like. Yarrowia species such as Yarrowia lipolytica, Pichia species such as Pichia methanolica, Pichia stipites, and Pichia pastoris, Zygosaccharomyces species such as Zygosaccharomyces rouxii and Zygosaccharomyces bailii, Candida boidinii, Candida utilis, Candida freyschussii, Candida glabrata, and Candida sonorensis. Candida spec. such as Schwanniomyces occidentalis, Arxula adeninivorans, Candida spec. such as Schwanniomyces occidentalis, Schwanniomyces ...adeninivorans, Ogataea spec., Ogataea minuta, Aspergillus spec., Aspergillus niger, or Myceliophthora thermophila.

[0096] In yet another embodiment according to various aspects as disclosed herein, the cell may be a prokaryotic cell, including gram-positive, gram-negative and gram-variant bacterial cells, preferably gram-negative bacterial cells, or archaeal cells, preferably the prokaryotic cell is selected from the group consisting of Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, and the like. faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, Brevibacterium divaricatum, Brevibacterium lactofermentum lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscumfuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucanthum, fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanensesewanense, Flavobacterium breve, Flavobacterium meningosepticum, Klebsiella spec. such as Klebsiella pneumonia, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas scinxantha synxantha, Pseudomonas azotoformans, Pseudomonas jluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus species ATCC 15592 (Rhodococcus sp. ATCC 15592), Rhodococcus sp. ATCC 19070 (Rhodococcus sp. ATCC 19070), Sporosarcina ureaeureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaaminolyticus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescensmarcescens, Salmonella typhimurium, Salmonella schottmulleri, Xanthomonas citri, Synechocystis sp., Synechococcus elongatus, Thermosynechococcus elongatus, Microcystis aeruginosa, Nostoc sp., N. commune, N. sphaericum, Nostoc punctiforme, Spirulina platensis platensis, Lyngbya majuscula, L. lagerheimii, Phormidium tenue, Anabaena sp., or Leptolyngbya sp.

[0097] In preferred embodiments according to various aspects as disclosed herein, the cell may be a eukaryotic cell or a prokaryotic cell, and the cell may be selected from the group consisting of Rhodococcus rhodochrous, Aerococcus sp., Ashbya gossypii, Aspergillus sp., Bacillus pumilus, Bacillus subtilis, Bacteroides thetaiotaomicron, Clostridium algidicarnis, Corynebacterium efficiens, Corynebacterium glutamicum, and the like. glutamicum, Escherichia coli, Haloferax volcanii, Lactobacillus casei, Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, Myceliophthora thermophila, Pichia pastoris, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas jluorescens, Pseudomonas ovalis ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolensmucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Pseudozyma tsukubaensis, Ralstonia eutropha, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, Shigella boydii, Sinorhizobium meliloti, Streptomyces antibioticus, Streptomyces avermitilis avermitilis, Streptomyces cacaoi, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lavendulae, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces viridochromogenes, Thermoplasma acidophilum The cell is preferably selected from the group consisting of cells derived from Bacillus subtilis, Bacillus acidophilum, Vibrio natrigens or Yarrowia lipolytica.subtilis, Corynebacterium glutamicum, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, or Yarrowia lipolytica.

[0098] In another embodiment, the cell may be a eukaryotic cell or a prokaryotic cell, and the cell may be selected from the group consisting of Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, and Yarrowia lipolytica, Phakopsora species, such as soybean rust, Phakopsora spp. pachyrhizi, Zymoseptoria spec., e.g. Zymoseptoria tritici, Septoria, Mycosphaerella, Phythopthora spec., e.g. Phytopthora infestans, infestans, Puccinia, Sphaerotheca, Blumeria, Erysiphe, Alternaria, Botrytis, Ustilago, Venturia, Verticillium, Pyricularia, Magnaporthe, Plasmopara, Pythium, Sclerotinia, Colletotrichum, Penicillium, Neurospora, Aspergillus, or Ashbya.

[0099] In a fifth aspect, there is provided at least one nucleic acid sequence encoding a complex, or a component thereof, comprising at least one engineered Cas12a enzyme with nickase activity or a catalytically active fragment thereof according to the first aspect of the invention, and at least one compatible guide RNA, and optionally at least one further polypeptide covalently and / or non-covalently linked to the at least one engineered Cas12a enzyme with nickase activity or a catalytically active fragment thereof within the complex, wherein the at least one further polypeptide is selected from an organelle localization sequence comprising a nuclear localization signal (NLS), a mitochondrial localization signal, or a chloroplast localization signal, and / or the at least one further polypeptide is preferably a cell penetrating polypeptide, when the at least one further polypeptide is covalently linked to the at least one engineered Cas12a enzyme with nickase activity or a catalytically active fragment thereof, and wherein the at least one further polypeptide is covalently linked to the N-terminus and / or the C-terminus of the at least one engineered Cas12a enzyme with nickase activity.

[0100] In a sixth aspect, there is provided a fusion protein or at least one nucleic acid sequence encoding same, comprising at least one engineered Cas12a enzyme with nickase activity according to the first aspect of the invention or a catalytically active fragment thereof covalently and / or non-covalently linked to at least one further polypeptide domain having an activity selected from an enzymatic activity, a binding activity or a targeting activity, and optionally comprising at least one guide RNA compatible with the engineered Cas12a enzyme with nickase activity, wherein the at least one compatible guide RNA covalently and / or non-covalently interacts with the at least one engineered Cas12a enzyme with nickase activity or a catalytically active fragment thereof.

[0101] The nCas12a fusion proteins of the invention can be operably linked chimeric nCas12a proteins fused to a polypeptide sequence comprising at least one heterologous polypeptide having an enzymatic activity that modifies at least one target nucleic acid (e.g., a nuclease activity, e.g., an exonuclease activity, a methyltransferase activity, a demethylase activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer formation activity, a helicase activity (e.g., SF1 / 2, SF3, SF4), an integrase activity, a telomerase activity, a topoisomerase activity, e.g., a gyrase activity, a transposase activity, a transcriptase or a reverse transcriptase activity, a recombinase activity, a polymerase activity, e.g., an RNA polymerase activity or a DNA polymerase activity, e.g., a Pol theta activity, a ligase activity, a photolyase activity or a glycosylase activity).

[0102] In some cases, a chimeric nCas12a fusion protein can include at least one heterologous polypeptide having an enzymatic activity that modifies at least one protein and / or polypeptide (e.g., a histone) associated with at least one target nucleic acid.Examples of enzymatic activities that modify at least one protein and / or polypeptide associated with at least one target nucleic acid that may be provided by a fusion partner include histone methyltransferases (HMTs) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1 or KMT1A), euchromatin histone lysine methyltransferase 2 (G9A, KMT1C, EHMT2), SUV39H2, ESET / SETDB 1, such as methyltransferase activity provided by SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2), histone demethylase activity provided by histone demethylases (e.g., lysine demethylase 1A (KDM1A, also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, UTX, JMJD3, etc.), histone acetylase transferases (e.g., human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP6, etc.), 0 / PLIP, MOZ / MYST3, MORF / MYST4, HB01 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK, etc.), deacetylase activity such as that provided by histone deacetylases (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

[0103] In some embodiments, the fusion partner may have an enzymatic activity that modifies at least one target nucleic acid. Examples of enzymatic activities include nuclease activity such as that provided by restriction enzymes (e.g., Fokl nuclease, Clo051 nuclease, homing endonucleases), DNA repair activity, DNA damage activity, deamination activity such as that provided by deaminases (e.g., cytosine deaminases such as rat APO-BEC1 or adenine deaminase), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity such as that provided by integrases and / or resolvases (e.g., hyperactive mutants of Gin integrase, Gin integrase such as GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase, etc.), transposase activity, recombinase activity such as that provided by recombinases (e.g., Gin recombinase, Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RadA, the catalytic domain of Rad51), polymerase activity (e.g., RNA polymerase activity, DNA polymerase activity), ligase activity, helicase activity, photolyase activity, or glycosylase activity.

[0104] In some cases, the nCas12a fusion protein may include at least one detectable label. Suitable detectable labels and / or moieties capable of providing a detectable signal may include, but are not limited to, enzymes, radioisotopes, members of specific binding pairs, fluorophores, fluorescent proteins, quantum dots, and the like.

[0105] Suitable fluorescent proteins include green fluorescent protein (GFP) or variants thereof, blue fluorescent variants of GFP (BFP), cyan fluorescent variants of GFP (CFP), yellow fluorescent variants of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2 (12), mRFPl, pocilloporin, Renilla GFP, Monster. Examples of fluorescent proteins include, but are not limited to, GFP, paGFP, Kaede proteins and kindling proteins, phycobiliproteins and phycobiliprotein conjugates including B-phycoerythrin, R-phycoerythrin and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. 2005), and the like.

[0106] Suitable enzymes that may serve as detectable labels include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosarninidase, f3-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), and the like.

[0107] Further suitable fusion partners include, but are not limited to, proteins (or fragments thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that confer peripheral recruitment (e.g., Lamin A, Lamin B, etc.), protein docking elements (e.g., FKBP / FRB, Pill / Abyl, etc.).

[0108] In certain embodiments, at least one nucleic acid sequence encoding the fusion protein is codon optimized.

[0109] In a seventh aspect of the invention there is provided an adenine or cytidine base editor, or a base editor complex, or at least one nucleic acid sequence encoding same, wherein the base editor or base editor complex comprises at least one catalytically active portion of at least one engineered Cas12a enzyme with nickase activity according to the first aspect of the invention.

[0110] "Base editor" as used herein refers to a protein or catalytically active fragment thereof that induces targeted base modification, i.e., conversion of at least one base to at least one different base, together with a compatible guide RNA, resulting in one or more point mutations. "Base editor complex" refers to a system that includes at least two non-covalently linked components that can function together as a base editor. Base editors are frequently used in the form of a base editor complex. Base editors, such as CBEs (cytosine base editors that mediate C to T conversions) and ABEs (adenine base editors that mediate A to G conversions), are powerful tools for introducing direct mutations that do not require DSB induction (Komor et al., Nature, 2016, 533(7603), 420-424; Gaudelli et al., Nature, 2017, 551, 464-471). A base editor or base editor complex is composed of at least one DNA targeting module, such as a Cas protein or a functional fragment thereof, with at least one suitable guide RNA, and at least one catalytic deaminase module that deaminates cytidine and / or adenine. All four transition mutations of DNA (C GT AA TG C) are possible depending on the choice of deaminase and their possible combinations. CBE and ABE have been optimized and applied in various cellular systems, including mammalian cells and plants (Fan et al., Communications Biology (2021), 4 (1): 882, doi: 10.1038 / s42003-021-02406-5; Zong et al., Nature Biotechnology, vol. 25, no. 5, 2017, 438-440; Yan et al., Molecular Plant, vol. 11, 4, 2018, 631-634; Hua et al., Molecular Plant, vol. 11, 4, 2018, 627-630).

[0111] The terms "cytosine base editor (complex)" and "cytidine base editor (complex)" are used interchangeably herein. Similarly, "cytosine deaminase" and "cytidine deaminase" are used interchangeably herein.

[0112] The terms "adenosine base editor (complex)" and "adenine base editor (complex)" are used interchangeably herein. Similarly, "adenosine deaminase" and "adenine deaminase" are used interchangeably herein.

[0113] In one embodiment of the invention, at least one deaminase module is covalently fused to nCas12a or a catalytically active fragment thereof, optionally the complex further comprises at least one compatible guide RNA, the deaminase module may be fused C-terminally or N-terminally or internally to nCas12a or a catalytically active fragment thereof, each module may be separated from the other modules by suitable linker or spacer regions, as these are known to those skilled in the art. Covalent fusion of different modules of base editors is usually achieved by cloning nucleic acid sequences encoding the desired modules and (optionally) linker sequences.

[0114] In another embodiment, at least one deaminase module can be non-covalently bound to nCas12a or a catalytically active fragment thereof, optionally in a complex further comprising at least one compatible guide RNA. Methods of non-covalent binding, such as protein binding domains, are well known to those skilled in the art.

[0115] In certain embodiments, at least one deaminase module can be covalently or non-covalently bound to at least one compatible guide RNA capable of forming a complex with at least one nCas12a or a catalytically active fragment thereof.

[0116] In certain embodiments, at least one additional polypeptide may be covalently and / or non-covalently linked to at least one base editor or base editor complex, where the at least one additional polypeptide comprises a glycosylase inhibitor activity, such as a uracil glycosylase inhibitor (UGI), a glycosylase activity, such as a uracil DNA glycosylase (UDG), including uracil-n-glycosylase (UNG), an organelle localization sequence comprising a nuclear localization signal (NLS), a mitochondrial localization signal, or a chloroplast localization signal, or a cell penetrating polypeptide, or any combination thereof, including a combination of two or more polypeptide sequences of the same type, including a combination of two or more identical polypeptide sequences, and the covalently linked additional polypeptide or additional polypeptides are linked to the base editor or base editor complex at the N-terminus, the C-terminus, or internally, and each functional module and / or domain may be separated from one or more other functional modules and / or domains by at least one linker region. In embodiments relating to base editor complexes, all protein components of the base editor complex may each be bound (covalently and / or non-covalently) to the same type or identical organelle-localized sequence.

[0117] Various adenine and cytosine deaminases are known to those skilled in the art (e.g., Fan et al., Communications Biology (2021), 4(1): 882, doi: 10.1038 / s42003-021-02406-5; Jeong et al., Molecular Therapy (2020), 28(9): 1938-1952, doi: 10.1016 / j.ymthe.2020.07.021; Yan et al., Molecular Plant (2021), 14(5): 722-731, doi: 10.1016 / j.molp.2021.02.007). Any adenine deaminase and / or cytosine deaminase, including variants of known deaminases, can be used in a base editor or base editor complex using any nCas12a of the invention.

[0118] In one embodiment, the at least one deaminase module comprises at least one adenine deaminase or domain thereof. In another embodiment, the at least one deaminase module comprises at least one cytosine deaminase or domain thereof. In yet another embodiment, the at least one deaminase module comprises at least one adenine deaminase or domain thereof and at least one cytosine deaminase or domain thereof.

[0119] In some embodiments, the adenine deaminase can be a tRNA-specific adenosine deaminase such as TadA (Gaudelli et al., Nature (2017), 551(7681):464-471, doi:10.1038 / nature24644), or adenosine deaminase 1 (ADA1), ADA2; adenosine deaminase 1 acting on RNA (ADAR1), ADAR2, ADAR3 (e.g., Savva et al., Genome Biol. 2012 Dec 28;13(12):252); or adenosine deaminase 1 acting on tRNA (ADAT1), ADAT2, ADAT3, or variants thereof.

[0120] In some embodiments, TadA may be from E. coli. In some embodiments, TadA may be modified and / or truncated. In certain embodiments, TadA does not include an N-terminal methionine. A TadA deaminase that may be used as part of a base editor or base editor complex according to the invention may be, for example, TadA8, TadA8e, TadA8s, TadA7.9 TadA7.10, TadA7.10d, TadA8.17, TadA8.20, TadA9, or a variant thereof.

[0121] In some embodiments, the cytosine deaminase can be an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase can be APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, APOBEC4 deaminase, activation-induced deaminase (AID), such as hAID or AICDA, rAPOBEC1, PpAPOBEC1, AmAPOBEC1, SsAPOBEC3B, RrA3F, FERNY, a cytosine deaminase, such as CDA1, CDA2, pmCDA1, or atCDA1, or a cytosine deaminase acting on rRNA (CDAT), or a variant thereof.

[0122] In one embodiment, at least one nucleic acid sequence encoding a base editor or base editor complex may be codon optimized and may further comprise a nucleic acid sequence encoding at least one compatible guide RNA.

[0123] In an eighth aspect, there is provided a prime editor or prime editor complex, or at least one nucleic acid sequence encoding same, wherein the prime editor or prime editor complex comprises at least one catalytically active portion of at least one engineered Cas12a enzyme having nickase activity according to the first aspect of the invention.

[0124] Prime editing allows the introduction of indels and all 12 base pair base changes without the need to introduce DSBs. For prime editing, so-called prime editing guide RNAs (pegRNAs) are used. The pegRNAs usually contain a primer binding site (PBS) and a reverse transcriptase (RT) template sequence that will be introduced into the gene to be targeted. The PBS region is complementary to the non-targeted strand and will create a primer for the RT that will be linked to the Cas protein. The sequence of the RT template sequence is then copied from the pegRNA into the target DNA sequence. The generation of three prime editors has been used in different target cells: PE1, PE2 and PE3. PE1 is based on Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT). PE2 (called pPE2 in plants) is based on the M-MLV RT D200N / L603W / T330P / T306K / W313F variant. PE3 (called pPE3 in plants) uses an additional guide RNA that specifically targets the sequence to be edited (Marzec et al. 2020; Xu et al. 2020; Lin et al. 2020). It has also been shown that the M-MLV RT can be replaced by different RTs, such as the Cauliflower Mosaic Virus (CaMV) RT, or retron-derived RT (Lin et al. 2020).

[0125] In one embodiment according to various aspects disclosed herein, at least one reverse transcriptase may be fused to at least one nCas12a, optionally as a complex further comprising at least one compatible pegRNA, to form a prime editor, and the at least one reverse transcriptase may be fused N-terminally, C-terminally, or internally to nCas12a, and the at least one reverse transcriptase may be linked to nCas12a via a linker region.

[0126] In another embodiment, at least one reverse transcriptase can be non-covalently bound to at least one nCas12a variant of the invention, optionally as a complex further comprising at least one compatible pegRNA. Methods of non-covalent binding, such as protein binding domains, are well known to those skilled in the art.

[0127] In certain embodiments, at least one reverse transcriptase can be covalently or non-covalently bound to at least one compatible pegRNA capable of forming a complex with at least one nCas12a or a catalytically active fragment thereof.

[0128] In another embodiment, at least one nCas12a or an active fragment thereof and / or at least one reverse transcriptase may comprise at least one further polypeptide covalently and / or non-covalently bound to at least one nCas12a or an active fragment thereof and / or at least one reverse transcriptase, wherein the at least one further polypeptide is selected from an organelle localization sequence including a nuclear localization signal (NLS), a mitochondrial localization signal, or a chloroplast localization signal, and / or the at least one further polypeptide is preferably a cell-permeable polypeptide, when the at least one further polypeptide is covalently bound to at least one nCas12a or an active fragment thereof and / or at least one reverse transcriptase, wherein the at least one further polypeptide is covalently bound to at least one nCas12a or an active fragment thereof and / or at least one reverse transcriptase at the N-terminus and / or the C-terminus and / or internally. In embodiments relating to a prime editor complex, all protein components of the prime editor complex may each be bound (covalently and / or non-covalently) to the same type or identical organelle-localization sequence.

[0129] In certain embodiments, at least one nucleic acid sequence encoding a prime editor or a prime editor complex may be codon optimized and may further comprise a sequence encoding at least one compatible pegRNA, and may further comprise a sequence encoding an additional guide RNA that targets the sequence to be edited.

[0130] In a ninth aspect, the present invention provides a method for the preparation of a nucleic acid sequence comprising the steps of: (i) an engineered Cas12a enzyme with nickase activity (nCas12a) as defined in the first aspect of the invention, or a catalytically active fragment thereof, or an expression construct or vector as defined in the third aspect of the invention, or a complex as defined in the fifth aspect of the invention, or at least one sequence encoding same, or a fusion protein as defined in the sixth aspect of the invention, or at least one sequence encoding same, or an adenine or cytidine base editor or base editor complex as defined in the seventh aspect of the invention, or at least one nucleic acid sequence encoding same, or There is provided a kit comprising a prime editor or a prime editor complex as defined in the eighth aspect of the invention, or at least one nucleic acid sequence encoding same; (ii) at least one compatible guide RNA, or a set of compatible guide RNAs, each guide RNA being complementary to a target sequence of interest; and (iii) a set of reagents; and (iv) optionally a particle, vesicle, or at least one viral vector, or Agrobacterium vector to aid delivery, wherein the particle comprises lipids, including lipid nanoparticles, sugars, metals or polypeptides, or a combination thereof, or the vesicle comprises an exosome or liposome.

[0131] In a tenth aspect, there is provided a method for modifying a genomic locus of interest of at least one cell or construct at or near at least one target site, comprising: (a) providing at least one cell or construct comprising the genomic locus to be modified; (b) providing (i) at least one engineered Cas12a enzyme (nCas12a) having nickase activity as defined in the first aspect of the invention, or a catalytically active fragment thereof, or at least one nucleic acid sequence encoding same; or (ii) at least one expression construct or vector as defined in the third aspect of the invention; or (iii) at least one complex as defined in the fifth aspect of the invention, or at least one nucleic acid sequence encoding same, or at least one fusion protein as defined in the sixth aspect of the invention; or (iv) at least one adenine or cytidine base editor as defined in the seventh aspect of the invention, or at least one base or (v) providing and / or introducing at least one prime editor or at least one prime editor complex as defined in the eighth aspect of the invention, or at least one nucleic acid sequence encoding same, into / into at least one cell or construct; (c) providing and / or introducing at least one compatible guide RNA as defined in the first aspect of the invention, or a sequence encoding same; (d) allowing complex formation of at least one engineered Cas12a enzyme having nickase activity of (a), or a catalytically active fragment thereof, and at least a compatible guide RNA as defined in the first aspect of the invention (b), thus allowing insertion of at least one nick at or near at least one target site at a genomic locus of interest of at least one cell or construct; (e) optionally providing at least one donor repair template, or at least one nucleic acid sequence encoding same;and (f) obtaining at least one edited cell or construct comprising an alteration of a genomic locus of interest at or near the target site (the method also excludes processes for altering the genetic identity of the human germline, the use of human embryos for industrial or commercial purposes and processes for altering the genetic identity of animals that may cause suffering without any substantial medical benefit to humans or animals, as well as animals resulting from such processes, the method comprising the steps of): (g) regenerating at least one population of edited cells, tissues, organs, materials or whole organisms from the at least one edited cell or construct;

[0132] In certain embodiments, at least one nCas12a or active fragment thereof according to the first or fifth aspect, or at least one fusion protein according to the sixth aspect, or at least one base editor or base editor complex according to the seventh aspect, or at least one prime editor or prime editor complex according to the eighth aspect may be provided / introduced to / into at least one cell or construct in a complex with at least one compatible guide RNA or as at least one nucleic acid encoding said complex, wherein at least one nucleic acid encoding said complex may be part of at least one vector, and wherein at least one compatible guide RNA may be a pegRNA.

[0133] In certain embodiments, at least one nCas12a or active fragment thereof according to the first or fifth aspect, or at least one fusion protein according to the sixth aspect, or at least one base editor or base editor complex according to the seventh aspect, or at least one prime editor or prime editor complex according to the eighth aspect, is provided / introduced to at least one cell or construct as a nucleic acid encoding same, said nucleic acid further encoding at least one compatible guide RNA according to the first or fifth aspect, and the at least one nucleic acid may be part of at least one vector, and the at least one compatible guide RNA may be a pegRNA. Alternatively, nCas12a, the fusion protein, the base editor or base editor complex, or the prime editor or prime editor complex, and the at least one compatible guide RNA may be encoded by two separate nucleic acids and provided / introduced to the cell or construct simultaneously or separately.

[0134] Step (c) of providing and / or introducing at least a compatible guide RNA or a sequence encoding same may already be fulfilled by providing and / or introducing at least one complex in step (b) containing at least one compatible guide RNA (including pegRNA) or a nucleic acid encoding same, such that providing and / or introducing at least one (additional) compatible guide RNA or a sequence encoding same may not be necessary.

[0135] In yet another embodiment of providing / introducing a prime editor or prime editor complex, the at least one compatible guide RNA is a pegRNA comprising a PBS region and / or a RT template region, and optionally an additional guide RNA targeting the edited strand is further provided and / or introduced, wherein the at least one prime editor or prime editor complex, the at least one pegRNA and optionally the at least one additional guide RNA may be provided and / or introduced as at least one nucleic acid encoding same, which at least one nucleic acid may be part of at least one vector.

[0136] In certain embodiments, the method of the tenth aspect of the invention does not result in the introduction of DSBs at the genomic locus of interest, which is achieved by the pronounced specific nickase activity (and lack of wild-type DSB activity) of nCas12a variants as disclosed herein.

[0137] In one embodiment, the method is performed in vitro or in vivo and / or ex vivo.

[0138] In certain embodiments, the methods do not include treatment of the human or animal body with therapy.

[0139] In another embodiment, the cell or construct is derived from a prokaryotic cell, including a bacterial or archaeal cell, or a eukaryotic cell.

[0140] In certain embodiments, the cell may be a plant cell, including an algal cell, and preferably the cell is a plant cell, including an algal cell, selected from the group consisting of Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp., Artocarpus spp., and the like. spp.), asparagus (Asparagus officinalis), Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.sativa, Avena hybrida), star fruit (Averrhoa carambola), Bambusa sp., Benincasa hispida, Brazil nut (Bertholletia excelsea), sugar beet (Beta vulgaris), Brassica spp. (e.g. Brassica napus, Brassica rapa subsp.), rapa ssp. (canola, rapeseed, turnip rape), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp.), Carex elata, Papaya (Carica papaya), Carissa macrocarpa, Pecan (Carya spp.), Safflower (Carthamus tinctorius), Chestnut (Castanea spp.), Kapok (Ceiba pentandra), Endive (Cichorium endivia), Cinnamomum spp., Watermelon (Citrullus lanatus), Citrus spp., Cocos spp., Coffea spp., Taro (Colocasia esculenta), Cola spp., Corchorus sp., Coriander (Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis spp. (e.g. Elaeis guineensis, Elaeis oleracea), Elaeis oleracea (e.g. ... oleifera), finger millet (Eleusine coracana), teff (Eragrostis tef), Erianthus sp., loquat (Eriobotrya japonica), Eucalyptus sp., pitanga (Eugenia uniflora), buckwheat (Fagopyrum spp.), beech (Fagus spp.), fescue (Festuca arundinacea), fig (Ficus carica), fortunella spp.), Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), cotton (Gossypium hirsutum), Helianthus spp. (e.g. Helianthus annuus, Hemerocallis fulva), Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), sweet potato (Ipomoea batatas), Juglans spp., lettuce (Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mango (Mangifera indica), Cassava spp., Sapodilla (Manilkara zapota), Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp.), Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Parsley (Petroselinum crispum, Reed canary grass (Phalaris arundinacea), Phaseolus spp., Timothy grass (Phleum pratense), Date palm (Phoenix spp.), Common reed (Phragmites australis), Physalis spp., Pinus spp., Pistachio (Pistacia vera), Pea spp., Poa spp., Populus spp., Prosopis spp., Cherry spp., Psidium spp., Pomegranate (Punica granatum), Pear (Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix spp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis spp., Solanum spp.) (e.g. potato (Solanum tuberosum), Solanum integrifolium or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach (Spinacia spp.), myrtaceae (Syzygium spp.), Tagetes spp., tamarind (Tamarindus indica), cacao (Theobroma cacao), Trifolium spp., gamagrass (Tripsacum dactyloides), Triticosecale rimpaui, wheat (Triticum spp.) (e.g. wheat (Triticum aestivum), durum wheat (Triticum durum), riveted wheat (Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare, Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, or Ziziphus spp. spp.), particularly from plants belonging to the superfamily of the subkingdom Chlorophyta, including but not limited to monocotyledonous and dicotyledonous plants, ornamental plants, food grains, trees or shrubs, including forage or feed legumes.

[0141] Preferred plants are Abelmoschus spp., Allium spp., celery (Apium graveolens), asparagus (Asparagus officinalis), Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), sugar beet (Beta vulgaris), Brassica spp. (e.g. Brassica napus, Brassica rapa subsp. spp. (canola, rapeseed, turnip rape)], Capsicum spp., Citrullus lanatus, Cucumis spp., Cynara spp., Daucus carota, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hordeum spp. (e.g. Hordeum vulgare), Lactuca sativa, Medicago sativa, Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Pennisetum sp., Saccharum spp., Secale cereale, Solanum spp.) (for example potato (Solanum tuberosum), Solanum integrifolium or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach species (Spinacia spp.), wheat species (Triticum spp.) (for example Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), or maize (Zea mays).

[0142] Other preferred plants are Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. (canola, rapeseed, turnip rape)]), Capsicum spp., Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Solanum spp. spp. (e.g. potato (Solanum tuberosum), Solanum integrifolium or tomato (Solanum lycopersicum)), Triticum spp. (e.g. wheat (Triticum aestivum), Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), or corn (Zea mays).

[0143] In other embodiments, the cell may be a fungal cell, including a yeast cell, and preferably the fungal cell, including the yeast cell, is selected from the group consisting of Saccharomyces spec., such as Saccharomyces cerevisiae, Hansenula spec., such as Hansenula polymorpha, Schizosaccharomyces spec., such as Schizosaccharomyces pombe, Kluyveromyces spec., such as Kluyveromyces lactis and Kluyveromyces marxianus, Yarrowia spec., such as Yarrowia lipolytica, Pichia species, such as Pichia methanolica, Pichia stipites, and Pichia pastoris; Zygosaccharomyces species, such as Zygosaccharomyces rouxii and Zygosaccharomyces bailii; Candida species, such as Candida boidinii, Candida utilis, Candida freyschussii, Candida glabrata, and Candida sonorensis; Schwanniomyces species such as Schwanniomyces occidentalis, Arxula species such as Arxula adeninivorans,spec), Ashbya spec, such as Ashbya gossypii, Ogataea spec, such as Ogataea minuta, Aspergillus spec, such as Aspergillus niger, or Myceliophthora thermophila.

[0144] In certain embodiments, the cell is a eukaryotic cell or a prokaryotic cell, and the cell is selected from the group consisting of Rhodococcus rhodochrous, Aerococcus sp., Aspergillus sp., Bacillus pumilus, Bacillus subtilis, Bacteroides thetaiotaomicron, Clostridium algidicarnis, Corynebacterium efficiens, Corynebacterium glutamicum, Escherichia coli, Haloferax volcanii, and the like. volcanii, Lactobacillus casei, Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, Myceliophthora thermophila, Pichia pastoris, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas jluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovorans acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosaaeruginosa, Pseudozyma tsukubaensis, Ralstonia eutropha, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, Shigella boydii, Sinorhizobium meliloti, Streptomyces antibioticus, Streptomyces avermitilis, Streptomyces cacaoi, Streptomyces coelicolor coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lavendulae, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces viridochromogenes, Thermoplasma acidophilum, Vibrio natrigens or Yarrowia lipolytica lipolytica, and the cell is preferably selected from cells derived from Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli,coli, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, or Yarrowia lipolytica.

[0145] In certain embodiments, the cell is a eukaryotic cell or a prokaryotic cell, and the cell is selected from the group consisting of Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, and Yarrowia lipolytica, Phakopsora species, such as soybean rust, Phakopsora spp. pachyrhizi, Zymoseptoria spec., e.g. Zymoseptoria tritici, Septoria, Mycosphaerella, Phythopthora spec., e.g. Phytopthora infestans, infestans, Puccinia, Sphaerotheca, Blumeria, Erysiphe, Alternaria, Botrytis, Ustilago, Venturia, Verticillium, Pyricularia, Magnaporthe, Plasmopara, Pythium, Sclerotinia, Colletotrichum, Penicillium, Neurospora, Aspergillus, or Ashbya.

[0146] Throughout the various embodiments, the introduction into cells according to step (b) of the tenth aspect can be accomplished by any suitable method known in the art. Those skilled in the art will appreciate that a variety of different transformation or transfection (used interchangeably herein) techniques are available depending on the desired target cells. The introduction can include methods such as, but not limited to, calcium phosphate-mediated transfection, cationic polymer-mediated transfection, liposome-mediated transfection, PEG-mediated transfection, dendrimer transfection, heat shock transfection, magnetofection, electroporation, particles including nanoparticles, uptake or bombardment, or microinjection.

[0147] In embodiments where the cell is a plant cell, introduction into the plant cell may be by methods such as, but not limited to, microprojectile bombardment, particle uptake, whisker-mediated transformation, Agrobacterium transformation including Agrobacterium-mediated introduction of a virus-based vector, PEG-mediated transformation, liposome-mediated transformation, electroporation, cell-penetrating peptides, microinjection or virus-vector-mediated introduction. The skilled artisan will be well aware of some introduction techniques, for example, PEG-mediated transformation, liposome-mediated transformation, electroporation or cell-penetrating peptides, so that the plant cell wall may be removed to create a protoplast prior to introduction. In embodiments involving introduction into at least one protoplast, step (g) of the method of the tenth aspect may include regeneration from at least one protoplast.

[0148] In embodiments where the cell is a fungal cell, including a yeast cell, introduction into the fungal cell, including a yeast cell, may involve partial or complete digestion of the cell wall and / or may involve protoplast transformation.

[0149] In some embodiments, the introduction comprises nuclear transformation, hi some embodiments, the introduction comprises nucleoplastid transformation, such as chloroplast or mitochondrial transformation.

[0150] In one embodiment of the various aspects disclosed herein, the modification can be at least one insertion, at least one deletion, or at least one point mutation.

[0151] In one embodiment of the tenth aspect, at least one additional effector, or a nucleic acid sequence encoding same, may be provided during steps (a)-(c), wherein the additional effector promotes DNA repair and cell regeneration, or another activity, before, during or upon insertion of the at least one nick in the genomic locus of interest at or near the at least one target site. The additional effector may be selected from, but is not limited to, at least one additional effector having an enzymatic activity that modifies at least one target nucleic acid (e.g., a nuclease activity, e.g., an exonuclease activity, a methyltransferase activity, a demethylase activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer formation activity, a helicase activity (e.g., SF1 / 2, SF3, SF4), an integrase activity, a telomerase activity, a topoisomerase activity, e.g., a gyrase activity, a transposase activity, a transcriptase or a reverse transcriptase activity, a recombinase activity, a polymerase activity, e.g., an RNA polymerase activity or a DNA polymerase activity, e.g., a Pol theta activity, a ligase activity, a photolyase activity or a glycosylase activity).

[0152] In one embodiment of the tenth aspect, the method may be a coordinated double nicking method, wherein at least two Cas enzymes with nickase activity (nCas), or catalytically active fragments thereof, or at least one nucleic acid sequence encoding same, are provided in step (b); and in step (c) at least two compatible guide RNAs are provided, wherein the at least two compatible guide RNAs are designed to enable coordinated action of the at least two Cas enzymes with nickase activity, such that the at least two Cas enzymes with nickase activity introduce two individual nicks at at least one target site.

[0153] In one embodiment, the two Cas enzymes having nickase activity, or catalytically active fragments thereof, may be the same or different, and at least one of the at least two Cas enzymes having nickase activity, or catalytically active fragments thereof, is an engineered Cas12a (nCas12a) having nickase activity as defined in any one of claims 1 to 6, or a catalytically active fragment thereof, or a sequence encoding same, and the nCas12a may be the same nCas12a or a different nCas12a.

[0154] In certain embodiments, the two individual nicks are close enough together to result in a DSB. In other embodiments, the two individual nicks do not result in a DSB (see WO2021122080A1).

[0155] In one embodiment, two individual nicks may be introduced into opposite strands within a genomic locus of interest of at least one cell or construct at or near at least one target site, with an offset that is positive, negative, or zero, preferably the offset is between approximately -100bp and +100bp.

[0156] In certain embodiments, the offset may be negative, preferably the offset is between -40bp and -30bp, or between -30bp and -20bp, or between -20bp and -10bp, or between -10bp and -1bp.

[0157] In other embodiments, the offset may be positive, preferably the offset is 1 bp to 10 bp, or 10 bp to 20 bp, or 20 bp to 30 bp, or 30 bp to 40 bp, or 40 bp to 50 bp, or 50 bp to 60 bp, or 60 bp to 70 bp, or 70 bp to 80 bp, or 80 bp to 90 bp, or 90 bp to 100 bp, more preferably the offset is 20 bp to 40 bp, and most preferably the offset is 25 bp to 35 bp.

[0158] In one embodiment, the two Cas enzymes with nickase activity and / or the at least two compatible guide RNAs are provided separately in the form of at least one expression construct or vector, or in the form of at least one complex, or in the form of at least one nucleic acid sequence encoding same, or in the form of at least one fusion protein or at least one nucleic acid sequence encoding same.

[0159] In one embodiment, at least one cell or construct is derived from a prokaryotic cell, including a bacterial or archaeal cell, or a eukaryotic cell.

[0160] In certain embodiments, the cell is a plant cell, including an algal cell, preferably the cell is a plant cell, including an algal cell, such as a cell of Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Celery (Apium graveolens), Arachis spp., Artocarpus spp., Asparagus spp., or the like. officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.sativa, Avena hybrida), star fruit (Averrhoa carambola), Bambusa sp., Benincasa hispida, Brazil nut (Bertholletia excelsea), sugar beet (Beta vulgaris), Brassica spp. (e.g. Brassica napus, Brassica rapa subsp. ssp.) [canola, rapeseed, turnip rape], Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp.), Carex elata, Papaya (Carica papaya), Carissa macrocarpa, Pecan (Carya spp.), Safflower (Carthamus tinctorius), Chestnut (Castanea spp.), Kapok (Ceiba pentandra), Endive (Cichorium endivia), Cinnamomum spp., Watermelon (Citrullus lanatus), Citrus spp., Cocos spp., Coffea spp., Taro (Colocasia esculenta), Cola spp., Corchorus sp., Coriander (Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis spp. (e.g. Elaeis guineensis, Elaeis oleracea), Elaeis oleracea (e.g. ... oleifera), finger millet (Eleusine coracana), teff (Eragrostis tef), Erianthus sp., loquat (Eriobotrya japonica), Eucalyptus sp., pitanga (Eugenia uniflora), buckwheat (Fagopyrum spp.), beech (Fagus spp.), fescue (Festuca arundinacea), fig (Ficus carica), fortunella spp.), Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), cotton (Gossypium hirsutum), Helianthus spp. (e.g. Helianthus annuus, Hemerocallis fulva), Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), sweet potato (Ipomoea batatas), Juglans spp., lettuce (Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mango (Mangifera indica), Cassava spp., Sapodilla (Manilkara zapota), Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp.), Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Parsley (Petroselinum crispum, Reed canary grass (Phalaris arundinacea), Phaseolus spp., Timothy grass (Phleum pratense), Date palm (Phoenix spp.), Common reed (Phragmites australis), Physalis spp., Pinus spp., Pistachio (Pistacia vera), Pea spp., Poa spp., Populus spp., Prosopis spp., Cherry spp., Psidium spp., Pomegranate (Punica granatum), Pear (Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix spp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis spp., Solanum spp.) (e.g. potato (Solanum tuberosum), Solanum integrifolium or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach (Spinacia spp.), myrtaceae (Syzygium spp.), Tagetes spp., tamarind (Tamarindus indica), cacao (Theobroma cacao), Trifolium spp., gamagrass (Tripsacum dactyloides), Triticosecale rimpaui, wheat (Triticum spp.) (e.g. wheat (Triticum aestivum), durum wheat (Triticum durum), riveted wheat (Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare, Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, or Ziziphus spp. The cell may be selected from cells derived from plants belonging to the superfamily of the subkingdom of Chlorophyta, in particular monocotyledonous and dicotyledonous plants, including but not limited to fodder or forage legumes, ornamental plants, food grains, trees or shrubs, selected from the list including:

[0161] Preferred plants are Abelmoschus spp., Allium spp., celery (Apium graveolens), asparagus (Asparagus officinalis), Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), sugar beet (Beta vulgaris), Brassica spp. (e.g. Brassica napus, Brassica rapa subsp. spp. (canola, rapeseed, turnip rape)], Capsicum spp., Citrullus lanatus, Cucumis spp., Cynara spp., Daucus carota, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hordeum spp. (e.g. Hordeum vulgare), Lactuca sativa, Medicago sativa, Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Pennisetum sp., Saccharum spp., Secale cereale, Solanum spp.) (e.g. potato (Solanum tuberosum), Solanum integrifolium or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach species (Spinacia spp.), wheat species (Triticum spp.) (e.g. wheat (Triticum aestivum), durum (Triticum durum), riveted wheat (Triticum turgidum), Triticum hybernum, Mach wheat (Triticum macha), Triticum sativum, einkorn (Triticum monococcum) or Triticum vulgare), or maize (Zea mays).

[0162] Preferred plants also include, in certain embodiments, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp. (canola, rapeseed, turnip rape)), Capsicum spp., Glycine spp. (e.g., Glycine max, Soja hispida, or Soja max), Gossypium hirsutum, Helianthus spp. (e.g., Helianthus annuus), Oryza spp. (e.g., Oryza sativa, Oryza latifolia), latifolia), Solanum spp. (e.g. potato (Solanum tuberosum), Solanum integrifolium or tomato (Solanum lycopersicum)), Triticum spp. (e.g. wheat (Triticum aestivum), Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), or corn (Zea mays).

[0163] In another embodiment, the cell is a fungal cell, including a yeast cell, preferably the fungal cell, including the yeast cell, is selected from the group consisting of Saccharomyces spec., such as Saccharomyces cerevisiae, Hansenula spec., such as Hansenula polymorpha, Schizosaccharomyces spec., such as Schizosaccharomyces pombe, Kluyveromyces spec., such as Kluyveromyces lactis and Kluyveromyces marxianus, Yarrowia spec., such as Yarrowia lipolytica, Pichia species, such as Pichia methanolica, Pichia stipites, and Pichia pastoris; Zygosaccharomyces species, such as Zygosaccharomyces rouxii and Zygosaccharomyces bailii; Candida species, such as Candida boidinii, Candida utilis, Candida freyschussii, Candida glabrata, and Candida sonorensis; Schwanniomyces species such as Schwanniomyces occidentalis, Arxula species such as Arxula adeninivorans,spec), Ogataea spec, such as Ogataea minuta, Ashbya spec, such as Ashbya gossypii, Aspergillus spec, such as Aspergillus niger, or Myceliophthora thermophila.

[0164] In a preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell, and the cell is selected from the group consisting of Rhodococcus rhodochrous, Aerococcus sp., Aspergillus sp., Bacillus pumilus, Bacillus subtilis, Bacteroides thetaiotaomicron, Clostridium algidicarnis, Corynebacterium efficiens, Corynebacterium glutamicum, Escherichia coli, Haloferax volcanii, and the like. volcanii, Lactobacillus casei, Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, Myceliophthora thermophila, Pichia pastoris, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas jluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovorans acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosaaeruginosa, Pseudozyma tsukubaensis, Ralstonia eutropha, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, Shigella boydii, Sinorhizobium meliloti, Streptomyces antibioticus, Streptomyces avermitilis, Streptomyces cacaoi, Streptomyces coelicolor coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lavendulae, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces viridochromogenes, Thermoplasma acidophilum, Vibrio natrigens or Yarrowia lipolytica lipolytica, the cell is preferably selected from the group consisting of Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli,coli, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, and Yarrowia lipolytica.

[0165] In certain embodiments, the cell is a eukaryotic cell or a prokaryotic cell, and the cell is selected from the group consisting of Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, and Yarrowia lipolytica, Phakopsora species, such as soybean rust, Phakopsora spp. pachyrhizi, Zymoseptoria spec., e.g. Zymoseptoria tritici, Septoria, Mycosphaerella, Phythopthora spec., e.g. Phytopthora infestans, infestans, Puccinia, Sphaerotheca, Blumeria, Erysiphe, Alternaria, Botrytis, Ustilago, Venturia, Verticillium, Pyricularia, Magnaporthe, Plasmopara, Pythium, Sclerotinia, Colletotrichum, Penicillium, Neurospora, Aspergillus, or Ashbya.

[0166] In certain embodiments according to various aspects herein, mismatches between the guide RNA and the target strand, e.g., 1, 2, 3 or 4 mismatches, may favor a nicking event. Without being bound by theory, it is hypothesized that reduced mobility mutants, e.g., achieved by substitution with proline, with target DNA mismatches are sufficient to limit conformational changes and block target strand cleavage.

[0167] In an eleventh aspect, there is provided an edited cell, tissue, organ, material or whole organism obtained or obtainable by a method according to the tenth aspect as disclosed.

[0168] In certain embodiments, the edited cell, tissue, organ, material or whole organism is not an edited cell, tissue, organ, material or whole organism of a plant or animal that is obtained exclusively by essentially biological processes.

[0169] A twelfth aspect relates to a compound selected from (i) to (vi): (i) at least one engineered Cas12a enzyme (nCas12a) having nickase activity as defined in the first aspect of the invention, or a catalytically active fragment thereof, or at least one nucleic acid sequence encoding same, for introducing a nucleotide deletion or insertion or modification in a nucleic acid molecule, preferentially a genome; (ii) at least one expression construct or vector as defined in the third aspect of the invention; or (iii) at least one complex as defined in the fifth aspect of the invention, or at least one nucleic acid sequence encoding same, or a fusion protein as defined in the sixth aspect of the invention, or at least one nucleic acid sequence encoding same; or (iv) at least one expression construct or vector as defined in the seventh aspect of the invention, for introducing a nucleotide deletion or insertion or modification in a nucleic acid molecule, preferentially a genome; or (v) at least one prime editor or at least one prime editor complex as defined in the eighth aspect of the invention, or at least one nucleic acid sequence encoding same; or (vi) a kit as defined in the ninth aspect of the invention; comprising use for optimizing or modifying traits in plants, including modifying yield related traits, or disease resistance related traits, and / or for metabolic engineering in prokaryotic or eukaryotic cells, preferably cells comprising plant cells, algal cells, fungal cells, including yeast cells, or archaeal cells.

[0170] Optimizing or modifying traits in plants can involve, for example, the use of endogenous or transgenes conferring herbicide resistance, such as the bar or pat genes conferring resistance to glufosinate ammonium (Liberty®, Basta® or Ignite®; EP 0242236 and EP 0242246); or any modified EPSPS gene, such as the 2mEPSPS gene from maize (EP 0508909 and EP 0507698), or glyphosate acetyltransferase, or glyphosate oxidoreductase, conferring resistance to glyphosate (RoundupReady®), or CP4 or a glyphosate-resistant EPSPS such as an N-acetyltransferase (gat) gene, or a bromoxynitrile nitrilase that confers bromoxynitrile tolerance, or any modified AHAS gene that confers tolerance to sulfonylureas, imidazolinones, sulfonylaminocarbonyltriazolinones, triazolopyrimidines, or pyrimidyl (oxy / thio)benzoates such as the oilseed oil imidazolinone-resistant mutants PM1 and PM2 currently marketed as Clearfield® canola; and / or a 12:0 to obtain high laurate. The genetic modifications may include endogenous or transgenes that confer increased oil content or improved oil composition, such as ACP thioesterase inclease, and confer pollination control, such as barnase under the control of an anther specific promoter to obtain male sterility, or barstar under the control of an anther specific promoter to restore male sterility, or a nuclear restorer of Ogura-type cytoplasmic male sterility and fertility; and / or endogenous or transgenes that confer resistance to glufosinate ammonium (Liberty®, Basta®, or Ignite®); and / or a compendium of genes encoding phosphinothricin-N-acetyltransferase (PAT) enzymes, such as the coding sequence for the bialaphos resistance gene (bar) of Streptomyces hygroscopicus.Such plants may, for example, comprise the elite event MS-BN1 and / or RF-BN1 as described in WO 01 / 41558, or the elite event MS-B2 as described in WO 01 / 31042, or any combination of these events.

[0171] Examples of technically induced mutants in Brassica napus as a result of trait modification optimization are mutants in the FATB gene as described in WO2009007091 or the FAD3 gene as described in WO2011 / 060946, or may be podshatter resistant mutants such as the mutants described in WO2009068313 or WO2010006732, or mutations conferring herbicide tolerance such as the PM1 and PM2 mutations conferring imidazolinone tolerance (Tan et al. 2005; US5545821).

[0172] In an embodiment of the twelfth aspect, the use comprises a paired nickase strategy as defined in the second aspect disclosed herein.

[0173] In a thirteenth aspect, there is provided a method of treating or preventing a disease, comprising administering to a subject in need thereof at least one modification in a genomic locus of interest at or near a target site associated with at least one disease state, the method comprising administering to said subject at least one cell in said cell said disease state at least one modified genomic locus of interest at or near said ... or (iv) at least one adenine or cytidine base editor, or at least one base editor complex, as defined in the seventh aspect of the invention, or at least one nucleic acid sequence encoding same; or (v) at least one prime editor, or at least one prime editor complex, as defined in the eighth aspect of the invention, or at least one nucleic acid sequence encoding same; or (vi) a kit as defined in the ninth aspect of the invention; or (vii) a cell as defined in the fourth aspect of the invention; or (viii) a method comprising using an edited cell, tissue, organ, material or whole organism as defined in the eleventh aspect of the invention.

[0174] In one embodiment, the method may include ex vivo modification of a genomic locus, wherein at least one cell of the subject is provided to perform the ex vivo modification of the genomic locus to obtain at least one edited cell.

[0175] In a fourteenth aspect, there is provided a method for treating or preventing a disease in a patient comprising (i) at least one engineered Cas12a enzyme having nickase activity (nCas12a), or a catalytically active fragment thereof, or at least one nucleic acid sequence encoding same, as defined in the first aspect of the invention; (ii) at least one expression construct or vector as defined in the third aspect of the invention; or (iii) at least one complex as defined in the fifth aspect of the invention, or at least one nucleic acid sequence encoding same, or a fusion protein as defined in the sixth aspect of the invention, or at least one nucleic acid sequence encoding same; or (i) or (v) at least one adenine or cytidine base editor, or at least one base editor complex, as defined in the seventh aspect of the invention, or at least one nucleic acid sequence encoding same; or (v) at least one prime editor, or at least one prime editor complex, as defined in the eighth aspect of the invention, or at least one nucleic acid sequence encoding same; or (vi) a kit as defined in the ninth aspect of the invention; or (vii) a cell as defined in the fourth aspect of the invention; or (viii) an edited cell, tissue, organ, material or whole organism as defined in the eleventh aspect of the invention.

[0176] A fifteenth aspect relates to a method for the manufacture of a medicament for treating or preventing a disease in a patient, comprising: (i) at least one engineered Cas12a enzyme having nickase activity (nCas12a), or a catalytically active fragment thereof, or at least one nucleic acid sequence encoding same, as defined in the first aspect of the invention; (ii) at least one expression construct or vector as defined in the third aspect of the invention; or (iii) at least one complex as defined in the fifth aspect of the invention, or at least one nucleic acid sequence encoding same, as defined in the sixth aspect of the invention; or (iv) at least one adenine or cytidine base editor, or at least one base editor complex, as defined in the seventh aspect of the invention, or at least one nucleic acid sequence encoding same; or (v) at least one prime editor, or at least one prime editor complex, as defined in the eighth aspect of the invention, or at least one nucleic acid sequence encoding same; or (vi) a kit as defined in the ninth aspect of the invention; or (vii) a cell as defined in the fourth aspect of the invention; or (viii) an edited cell, tissue, organ, material or whole organism as defined in the eleventh aspect of the invention.

[0177] All methods disclosed herein exclude processes for modifying the genetic identity of the germline of a human, the use of human embryos for industrial or commercial purposes and processes for modifying the genetic identity of animals that may cause suffering without any substantial medical benefit to humans or animals, as well as animals resulting from such processes, and optionally the methods include the following step: (g) regenerating at least one population of edited cells, tissues, organs, materials or whole organisms from the at least one edited cell or construct.

[0178] (i) at least one engineered Cas12a enzyme (nCas12a) having nickase activity as defined in the first aspect of the invention, or a catalytically active fragment thereof, or at least one nucleic acid sequence encoding same; (ii) at least one expression construct or vector as defined in the third aspect of the invention; or (iii) at least one complex as defined in the fifth aspect of the invention, or at least one nucleic acid sequence encoding same, or a fusion protein as defined in the sixth aspect of the invention; or (iv) at least one adenine or cytidine base editor as defined in the seventh aspect of the invention, or at least one or at least one nucleic acid sequence encoding same; or (v) at least one prime editor or at least one prime editor complex as defined in the eighth aspect of the invention, or at least one nucleic acid sequence encoding same; or (vi) a kit as defined in the ninth aspect of the invention; or (vii) a cell as defined in the fourth aspect of the invention; or (viii) an edited cell, tissue, organ, material or whole organism as defined in the eleventh aspect of the invention, the compound is provided in a functional form, including stabilizing factors, cofactors, means for introducing it into, for example, a target cell or tissue. EXAMPLES

[0179] Example 1: Rational Protein Design One major approach in generating Cas12a mutants with in vivo nickase activity has been rational protein design. This approach is based in part on data available in the literature describing Cas12a mutants with at least partial and / or at least in vitro nickase activity. The mutants used as the basis for rational protein design were LbCas12a R1338A (Yamano et al., 2017; ^=FnCas12a R1218A) and FnCas12a K1013G / R1014G (WO 2019 / 233990; ^=LbCas12a K932G / N933G).

[0180] Second, rational protein design is based on crystal structure information of Cas12a and available mechanistic insights of the cleavage event. In contrast to Cas9, where the RuvC and HNH domains cleave one strand each, the RuvC domain of Cas12a cleaves both the non-target strand (NTS) and the target strand (TS) sequentially. In general, rational design approaches have focused on mutating the so-called lid of the RuvC domain, which is located next to the active site of the RuvC domain and has not been of interest so far for the generation of Cas12a nickase mutants. The lid can open and close to provide access to the active site and play a role in the transition to the second cleavage event (after NTS cleavage). This strategy focuses on mutating the core lid domain (see Figure 1) as defined by SEQ ID NO: 13, avoiding mutation of catalytic residue E925 (LbCas12a) so that the catalytic center of the RuvC domain is not completely inactivated. All mutations were introduced by standard cloning methods. See Figure 2 for the Cas12a domain structure.

[0181] Example 2: Targeted in silico analysis A systematic in silico selection and comparison was set up to provide a basis for extending rational protein design and in vitro and in vivo selection to all Cas12a variants described as effective in genome editing and available in databases, as well as those Cas12a sequences that are also available but not yet annotated. The aim was to define suitable consensus motifs applicable to all Cas12a enzymes described and not yet described to rationally expand the scope of nickase design. For this purpose, a BLAST protein search (NCBI; https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE=Proteins; standard parameters) was performed to obtain an overview of Cas12a / Cpf1 enzymes with known functions and closely related Cas12a enzymes with currently unknown functions. Notably, all enzymes showed very high sequence conservation in the region corresponding to the lid domain, as described for example for LbCas12a and AsCas12a. In addition, there was high overall sequence identity / homology among the selected sequences, and it was therefore hypothesized that the knowledge gained about the Cas12a enzymes investigated herein could be easily transferred to other Cas12a enzymes.

[0182] Next, after completing a search by BLAST using a heuristic algorithm, multiple sequence alignment using seeded guide trees and HMM profile-profile techniques to generate alignments between three or more sequences was performed in Clustal Omega (EMBL-EBI; again using standard parameters) by aligning certain sequences of Cas12a enzymes (provided as SEQ ID NOs: 1-12) analyzed herein and disclosed as suitable for genome editing in various settings. As shown in Figure 1, there was a high degree of sequence conservation within the alpha2 / beta6 and alpha3 domains described for AsCas12a and LbCas12a by structural analysis (see Stella et al., 2018, Suppl. Figure S4). Particularly strong sequence conservation was observed in the domain starting at L927 of Cas12a (for LbCas12a / SEQ ID NO: 1 in Figure 1). This position was completely conserved in all sequences analyzed, so this position was defined as the start of the so-called core lid domain as used herein. Most of the Cas12a sequences (except only AsCas12a) had the same length within the core lid domain. Thus, the last position of the core lid domain was defined as position V942 in LbCas12a (SEQ ID NO:1) and V1011 in AsCas12a (SEQ ID NO:2). It should be noted that several Cas12a variants have been described, for example, for Francisella tularensis (and its various subspecies, including novicida, including U112). Variants can be easily identified via NCBI taxonomy browser searches and sequence databases.Since the alignment of five different Cas12a variants performed on the Cas12a enzyme in Francisella revealed that they were completely identical in their core lid domain consensus sequences (e.g., FIG. 1 with SEQ ID NOs: 3 and 4 as two exemplary sequences), it was decided to include only the two Francisella sequences in the further alignment, but rather to include Cas12a variants from different sources in order to obtain reliable results on the degree of conservation of potential lid consensus sequences across various different species. As derived from FIG. 1, a suitable consensus sequence for the core lid domain motif and its location in the Cas12a enzyme can be easily defined. The core lid domain motif was then defined and used as a basis for further targeted protein design studies (see SEQ ID NO: 13), as it was shown that this motif is indeed highly conserved and therefore could serve as an identifier or consensus sequence for highly conserved regions in the Cas12a enzyme.

[0183] Additional analyses were performed to further demonstrate that the core lid domain motif (see SEQ ID NO: 13) was a novel structural motif useful for generalizing findings on LbCas12a, AsCas12a and other variants as investigated to any homologous Cas12a enzymes. For this purpose, Cas12a sequences (herein SEQ ID NOs: 1-12) were aligned using MUSCLE (EMBL-EBI; multiple sequence comparison with logarithmic prediction; default parameters). To substantiate our previous findings, the MUSCLE alignment confirmed that the selected core lid motif (SEQ ID NO: 13) is a suitable identifier to characterize Cas12a variants (homologs, orthologs, paralogs) in many species, since the defined motif is highly conserved among various variants. To finally confirm that the core lid domain was the preferred structural motif to characterize the Cas12a enzyme, further analysis (based on MUSCLE alignment of SEQ ID NOs: 1-12) was performed (using MView; version 1.63; default parameter settings: see https: / / www.ebi.ac.uk / seqdb / confluence / display / JDSAT / MView+Help+and+Documentation) with the overall sequence identity / homology derivable from the database (primary amino acid sequence) and structural features on the three-dimensional level known for a given Cas12a enzyme. MView allowed the calculation of the percentage of coverage (cov) and identity (pid) for 100%, 90%, 80% and 70% consensus sequences using AsCas12a (SEQ ID NO: 2), which has the longest core lid domain, as a reference sequence together with other Cas12a variants (SEQ ID NOs: 1, 3-12). Based on this knowledge, a core lid domain consensus sequence was constructed (currently: SEQ ID NO: 13), which was used iteratively for alignment purposes. First, a BLAST protein search for Cas12a variants was performed, followed by a sub-search for the presence of the core lid domain consensus.Taken together, these analyses confirmed that the core lid domain, as defined during the project, is indeed a highly conserved characteristic motif and represents a useful consensus sequence for identifying and characterizing Cas12a enzymes.

[0184] Interestingly, new insights into the mechanism for target recognition and cleavage by other Cas12 endonucleases have demonstrated that the core lid domain is also structurally conserved in Cas12i, Cas12b, and Cas12e, although the protein sequences of the lid region in these Cas12 orthologs are highly diverse (Zhang et al. 2018, see Extended Data Fig. 8). Due to this structural conservation, the core lid domain may also constitute an intriguing motif that offers new opportunities to improve and extend genome editing applications of class II, type V enzymes other than Cas12a.

[0185] Example 3: In vivo screening assay for Cas12a nickase candidates An in vivo assay for the different forms of Cas nickases has been developed, consisting of a 3-plasmid system: two reporter plasmids are used and the third is a Cas-encoding plasmid. The reporter plasmid consists of a GFP-encoding plasmid encoding guide RNA1 and carrying target-1 flanked by the appropriate PAM motif. The second plasmid is an RFP-encoding plasmid encoding two guide RNAs and carrying overlapping target-1 and target-2, each with the appropriate PAM motif. Upon transformation of the Cas-encoding plasmid into cells hosting the two reporter plasmids (in the absence of antibiotic selection for the two reporter plasmids, but in the presence of a selective antibiotic for the Cas-encoding plasmid), the red / green fluorescent readout results in a characteristic phenotype for the nickase, wild-type or inactive Cas nuclease. Nuclease activity results in the loss of both GFP and RFP, while nickase activity will destroy only RFP due to double nicking on two overlapping target sites, but not GFP, as there is only one target site to be nicked. A catalytically inactive Cas12a variant will result in both RFP and GFP fluorescence (see Figure 3).

[0186] The in vivo screening assay was first established and optimized using Cas9 nuclease, Cas9 DH10A and Cas9 H840A nickases, as well as inactive Cas9 to validate the accurate readout of the assay. Upon establishment and validation of the Cas9 reporter assay, it was used to test LbCas12a candidate nickases either in single-genotype experiments (one at a time) or in a high-throughput format using fluorescence-activated cell sorting (FACS).

[0187] The following plasmids were generated for Cas12a in vivo nicking assay: pGFP (SEQ ID NO:52; pSC101 RepA N99D, KanR; GFP under PlacIQ promoter; target-1; Cas12a guide RNA1 under PJ23119 promoter); pRFP (SEQ ID NO:53; pBR322 AmpR; RFP under Amp(Bla) promoter; target-1 under PJ23119 promoter; target-2, Cas12a guide RNA2); pCas LbCas12a WT (SEQ ID NO:54; p15A(pCB482), CamR; LbCas12a under PJ23108 promoter; SEQ ID NO:1), pCas LbCas12a inactive (SEQ ID NO:55; p15A(pCB482), CamR; LbCas12a inactive under PJ23108 promoter; LbCas12a E925A / D832A (mutations relative to reference sequence SEQ ID NO:1).

[0188] To generate rationally designed Cas12a variants, point mutations were introduced into the pCas LbCas12a WT template (SEQ ID NO: 53). Inverse PCR site-directed mutagenesis was used to introduce mutations using a 5' phosphorylated primer containing the desired mutation at the 5' end of the sequence. Different primer sets were designed depending on the variant to be generated.

[0189] In the first experiment, individual LbCas12a variants were introduced into E. coli GFP / RFP reporter strain (DH10b). After individual (single) transformation of LbCas12a variants (10 ng) by heat shock, transformed cells were allowed to recover in 950 μL of LB medium for 1 h, and then 2 μL of recovered transformants were inoculated into 200 μL of M9TG medium containing chloramphenicol [35 mg / l] and incubated overnight at 37 °C (day 1). The next day (day 2), a 1:10,000-fold dilution was re-inoculated into 200 μL of fresh M9TG medium containing chloramphenicol [35 mg / l] and incubated overnight at 37 °C. After 20 h, the resulting cultures were diluted in 1xPBS (1:10 dilution) and the green and red fluorescence of the samples was measured in a plate reader.

[0190] The results of several selected variants mutated in the RuvC lid are shown in Figure 4. LbCas12a S934A / R935G (mutations with reference to sequence SEQ ID NO: 1) and LbCas12a K932G / N933G (mutations with reference to sequence SEQ ID NO: 1; this double mutant is the LbCas12a homolog of the previously reported FnCas12a K1013G / R1014G mutant, WO 2019 / 233990) showed wild-type-like nuclease activity and appeared to cleave both strands in vivo. In contrast, the LbCas12a quadruple mutant K932G / N933G / S934A / R935G (SEQ ID NO: 14) showed the desired nickase phenotype. The negative RuvC lid mutation (LbCas12a F931E / K932E / R935D / K937D / K940D, mutations relative to reference sequence SEQ ID NO: 1) appears to be inactive Cas12a. Similarly, the previously reported LbCas12a R1138A mutant showed an inactive Cas12a phenotype.

[0191] Example 4: Laboratory evolution - semi-random RuvC lid mutagenesis As mentioned above, the objective of the present invention is to provide a robust nickase variant of LbCas12a. In addition to the rational design described above (Examples 1 and 3), a laboratory evolution approach was carried out in parallel. Laboratory evolution is an exemplary and powerful approach to optimize protein functionality in an unbiased manner. The essential requirement of laboratory evolution is the linkage of genotype (the gene encoding the desired Cas12a variant) to phenotype (the desired Cas12a functionality, in this case: efficient dsDNA nicking). This was achieved by transforming a GFP / RFPP E. coli strain (see Example 3) with a library of Cas12a variants and selecting green fluorescent transformants manually or using fluorescence-activated cell sorting (FACS).

[0192] Because the Cas12a RuvC lid quadruple mutant (LbCas12a K932G / N933G / S934A / R935G, SEQ ID NO: 14) showed reduced GFP signal compared to inactive LbCas12a (see Example 3 and Figure 4), semi-random saturation mutagenesis was performed in an attempt to further improve the nickase activity of this variant. We randomly substituted amino acid residues 931-940 (10 residues, residues refer to positions in SEQ ID NO: 1 and correspond to positions 5-15 in SEQ ID NO: 13, note that SEQ ID NO: 13 has one optimal position not present in LbCas12a) using a degenerate NNK codon (N=A,C,G,T; K=G,T), which encodes 20 different canonical amino acids and a single stop codon. This design aims to replace the entire lid with random amino acids, including the wild type residues.

[0193] pCas Lb12a WT (SEQ ID NO:53) was "opened" at the positions encoding G930 and Q941 using a pair of primers containing a 5'SapI restriction site. The digested PCR product was then ligated (T4 DNA ligase) using two short complementary oligos as inserts that upon annealing form complementary overhangs to the overhangs left by the SapI nuclease. The insert oligos contain degenerate NNK nucleotides that upon correct assembly of the construct will generate a library of plasmids encoding LbCas12a with different coding sequences at the oligo insertion site.

[0194] The resulting RuvC-lid NNK library was then introduced into the E. coli GFP / RFP reporter strain (Examples 3 and 4). Cultures generated after transformation were diluted and plated onto media (chloramphenicol [50 mg / L]) selecting for the plasmid encoding Cas12a. GFP + / RFP - (green) cells are expected in the case of LbCas12a nickase. Single green colonies in the plate were selected for Sanger sequencing to extract the LbCas12a genotype within the green fluorescent phenotype colonies. The extracted single genotype variants were then individually reintroduced into an E. coli GFP / RFP reporter strain to verify nicking activity based on the fluorescent signal readout from each culture / variant (i.e., individual LbCas12a sequences isolated from the population).

[0195] Manual selection of green colonies (i) DH10b chemically competent cells containing the pGFP pRFP reporter plasmid were transformed with 500 ng (approximately 100 fmol) of the RuvC lid NNK library. The transformed cells were allowed to recover in 950 μl of LB medium at 37° C. for 1 hour. After recovery, the recovered transformants were aliquoted into 50 ml of LB medium and incubated overnight (ON) at 37° C. (ii) The next day (day 2), a 1:10,000 dilution was plated onto LB agar + chloramphenicol [50 mg / L] and incubated overnight at 37°C. (iii) The next day (day 3), plates were removed from the refrigerator and placed at 4° C. for approximately 5 hours (fluorophore maturation). Plates were visualized under blue light and screened for green colonies. Single green colonies were transferred (restreaked) to new plates containing Lb agar + chloramphenicol [50 mg / L] and incubated overnight at 37° C. (iv) The next day (day 4), the restreaked plates were replicated onto plates containing LB agar + chloramphenicol [50 mg / L] (each streak was transferred to fresh medium) and incubated overnight at 37°C. (v) The next day (day 5), plates were placed at 4° C. for approximately 5 hours (fluorophore maturation). After restreak incubation, plates were visualized under blue light to select for green fluorescent colonies. Colonies exhibiting the green fluorescent phenotype were independently plated (N=32) in LB medium + chloramphenicol [50 mg / L] and incubated overnight at 37° C. (vi) The next day (day 6), the generated cultures were processed to extract plasmids (minipreps) and Sanger sequencing was used to reveal the sequence of the mutated region in the RuvC lid of each colony. The resulting sequencing information was processed in BenchLing to sequence each colony and classify them based on sequence repeats.

[0196] An exemplary GFP / RFP readout of manually selected RuvC lid variants (see Figure 5A for different core lid mutations) is shown in Figure 5B.

[0197] Selection of green colonies by FACS (i) DH10b chemically competent cells containing pGFP and pRFP reporter plasmids were transformed with 500 ng (approximately 100 fmol) of the RuvC-lid NNK library. Transformed cells were allowed to recover in 950 μl of LB medium at 37° C. for 1 hour. After recovery, the recovered transformants were aliquoted into 10 ml of LB medium and incubated overnight (ON) at 37° C. (ii) The next day (day 2), the culture was diluted 40-fold in sterile 1xPBS. A portion of the culture was analyzed via flow cytometry (day 1, pre-sorting) overnight at 37°C. The resulting samples were sorted by FACS (first round of sorting). Strong GFP + and RFP - Cells exhibiting the phenotype were collected into separate tubes containing 2 ml of LB medium + chloramphenicol [50 mg / L] and incubated overnight at 37°C. (iii) The next day (day 3), the culture was diluted 40-fold in sterile 1xPBS. A portion of the culture was analyzed via flow cytometry (day 2, sorted once). Additionally, a 1:10,000 dilution of the culture was plated (10 plates) on Lb agar + chloramphenicol [50 mg / L] and incubated overnight at 37°C. The resulting samples were FACS sorted (second round of sorting). Strong GFP + and RFP - Cells exhibiting the phenotype were harvested into separate tubes containing 2 ml of LB medium + chloramphenicol [50 mg / L]. The harvested cells were aliquoted into 10 ml of LB medium + chloramphenicol [50 mg / L] and incubated overnight at 37°C. (iv) The following day (day 4), a 1:10,000 dilution of the culture was plated (10 plates) onto Lb agar + chloramphenicol [50 mg / L] and incubated overnight at 37° C. A portion of the culture was analyzed via flow cytometry (day 3, sorted twice). (v) The next day (day 5), plates were placed at 4° C. for approximately 5 hours (fluorophore maturation). Plates were visualized under blue light to select for green fluorescent colonies. Individual green colonies were transferred (restreaked) to new plates containing LB agar + chloramphenicol [50 mg / L] and incubated overnight at 37° C. (vi) The next day (day 6), the restreaked plates were replicated onto plates containing LB agar + chloramphenicol [50 mg / L] (each streak was transferred to fresh medium) and incubated overnight at 37°C. (vii) The next day (day 7), plates were placed at 4° C. for approximately 5 hours (fluorophore maturation). Plates were visualized under blue light to select for green fluorescent colonies. Colonies exhibiting the green fluorescent phenotype were independently grown (N=12, n=6 per biological replicate) in LB medium + chloramphenicol [50 mg / L] and incubated overnight at 37° C. (viii) The next day (day 8), the generated cultures were processed to extract plasmids (minipreps) and Sanger sequencing was used to reveal the sequence of the mutated region in the RuvC lid of each colony. The resulting sequencing information was processed in BenchLing to sequence each colony and classify them based on sequence repeats.

[0198] The GFP / RFP results of exemplary mutants after FACS sorting are shown in Figure 5C.

[0199] Optimization of RuvC lid deletion variant A second round of site-directed saturation mutagenesis was performed to randomly replace both the four amino acid residues (Y930, C931, S932, and S933) that comprise the lid domain of the deletion variant (RuvCL-del1, SEQ ID NO: 15) identified in the first screen, as well as E925, a residue that is part of the highly conserved DED active site of Cas12a.

[0200] Diversity libraries were generated essentially as described above using insert oligos containing degenerate NNK nucleotides. The resulting plasmid populations were Sanger sequenced to confirm correct construction of the constructs and then transformed into an E. coli GFP / RFP reporter strain (see Examples 3 and 4). GFP + / RFP - After FACS sorting to enrich the cells, the sorted population was plated on chloramphenicol-containing medium to select for the Cas12a-encoding plasmid, and single green fluorescent colonies were selected for Sanger sequencing to extract the LbCas12a genotype, and a multiple sequence alignment was generated enumerating all monogenic variants identified in the population (data not shown, all sequences for alignment shown in the attached sequence listing). Interestingly, all variants obtained encoded glutamic acid at position 925, indicating that only cells containing catalytically active LbCas12a variants were sorted during the experiment. Furthermore, significant sequence diversity was observed within the mutagenized lid region, while the original deletion mutant ((RuvC L-del1 , SEQ ID NO:15) was not found among the picked colonies.

[0201] Although only 57 colonies were sequenced, several variants were identified multiple times (see Figure 5D). These enriched variants (SEQ ID NO: 100 to SEQ ID NO: 106) were then individually reintroduced into an E. coli GFP / RFP reporter strain to verify nicking activity based on the fluorescent signal readout from each culture / variant. Plasmids encoding either wild-type LbCas12a (pRV060) or a catalytically inactive variant (pRV061) were used as positive and negative controls, while the original lid deletion variant (Lid2.3; SEQ ID NO: 15) was included to assess the nicking activity of the newly identified variants. Figure 5E shows the normalized relative fluorescence units (fluorescence / OD600, average of three biological replicates). Interestingly, multiple variants showed either enhanced GFP expression or lower RFP signal compared to the original Lid2.3 mutant, suggesting enhanced nickase activity and / or reduced residual DSB activity.

[0202] In vitro validation of variants recovered from the RuvC-lid NNK library Lid variant pRV26004 (SEQ ID NO: 16) and a version of the lid deletion variant (RuvC L-del1 , SEQ ID NO: 15) (see FIG. 6A), as well as wild-type and inactive LbCas12a, were used for in vitro validation. Selected LbCas12a variants were cloned into the pET (pML-1B, KanR.Addgene#29653) vector, which contains a 6x histidine tag at the N-terminus of the protein.

[0203] Vectors encoding the selected variants were introduced into E. coli Rosetta DE3 competent cells (each variant individually). A single colony from each transformed variant was used to inoculate 10 ml LB medium containing chloramphenicol [50 mg / l] and kanamycin [35 mg / l] and incubated overnight at 37°C. The next day, an overnight culture of each variant was used to inoculate 250 ml LB medium containing chloramphenicol [50 mg / l] + kanamycin [35 mg / l] and incubated at 37°C at 180 rpm until OD600 = 0.5, at which point 50 μl of 0.5 M IPTG (0.1 mM final) was added to the culture and incubated at 120 rpm for 18 hours at 18°C. The next day, the resulting culture was centrifuged at 6,000 rpm for 15 min to collect the cells, and the pellet was resuspended in 10 ml of ice-cold lysis buffer I (NaCl 500 mM, Tris 20 mM, and imidazole 10 mM, pH 8 + 1 tablet / 10 ml of cOmplete protease inhibitor). The resuspended pellet was sonicated (30% amplitude, 1 sec on cycle, 2 sec off cycle, repeated for 15 min) and the cell lysate was centrifuged at 30,000 rpm for 45 min. After centrifugation, the supernatant was passed through a 0.22 μm filter to generate a cell-free extract.

[0204] A gravimetric column was loaded with 500 μl of Ni-NTA slurry and the loading solution was eluted. Three column volumes of lysis buffer I were passed through the column for resin equilibration. Cell-free lysate was passed through the column and the flow-through was collected for later SDS-page analysis. The column was washed with 4 column volumes of wash buffer II (NaCl 500 mM, Tris 20 mM and imidazole 20 mM, pH 8) and fractions were collected for SDS-page analysis. After washing, 5 column volumes of elution buffer III (NaCl 500 mM, Tris 20 mM and imidazole 250 mM, pH 8) were applied to the column to release bound proteins and elution fractions were collected for later SDS-PAGE analysis.

[0205] Eluted fractions were pooled together and the concentration was determined using a NanoDrop (Mw: 145.66 kDa extinction coefficient (εmolar concentration (M-1cm-1)) = 169270) and diluted to a final stock solution of 1 μM in SEC buffer (KCl 500 mM, HEPES 20 mM DTT 1 mM).

[0206] His-tagged proteins were purified on a nickel column using standard protein purification protocols. The purified Cas12a protein was incubated with a guide RNA and a plasmid containing a target site for the guide RNA. The target plasmid (and a control plasmid lacking the target site) were then loaded onto a gel to analyze the presence of nicked, linear (broken double-stranded) or supercoiled (neither nicked nor broken double-stranded) plasmid. Reactions were set up in 1x nuclease buffer (HEPES [20 mM], NaCl [100 mM], MgCl2 [5 mM], EDTA [0.1 mM]) containing purified LbCas12a variant [100 mM], together with synthetic guide RNA [200 nM] and negatively supercoiled pUC19 plasmid substrate [150 fmol], whose sequence contains a target protospacer that perfectly matches the provided guide RNA. First, the LbCas12a variant was incubated with the guide RNA in 1x nuclease buffer at room temperature for 20 minutes. After assembling the RNP, the plasmid DNA substrate was added to the reaction and incubated at 37°C for 1 hour. After incubation, the reaction was stopped by adding NEB purple loading dye and the reaction was loaded onto a 1% agarose gel.

[0207] As a control for plasmid topology, negative controls were made using DNA substrates in 1x nuclease buffer. Linear topology controls were made by digesting DNA substrates with EcoRI-HF restriction enzyme, and nicked topology was regenerated using Nb.BbvCI nickase restriction enzyme. All controls were made using the same input amount of DNA substrate as in the reaction containing LbCas12a variants.

[0208] Surprisingly, pRV26004 (SEQ ID NO: 16), which showed a GFP signal comparable to inactive Cas12a in the in vivo analysis (suggesting nickase activity but no or little nuclease activity), showed nicking and cleavage of target DNA in vitro, at least under selected conditions (see FIG. 6B). However, the lid deletion mutant (SEQ ID NO: 15) showed very strong nicking activity but little residual nuclease activity. To determine which strand the lid deletion mutant was cleaving, the nicked DNA fragments were extracted from the gel and analyzed by Sanger run-off sequencing (see FIG. 6C). RuvC L del1* Three replicates of reverse primer (NTS as template) sequencing of targets digested with showed termination of the sequencing reaction within the target site as depicted in the right sequencing chromatogram diagram in FIG. 6C, while three replicates of forward primer (TS as template) sequencing showed continuous sequencing reaction across the target region as depicted in the left sequencing chromatogram diagram in FIG. 6C. The negative control showed continuous sequencing reaction for both strands, and the positive control with a restriction enzyme cleaving either TS or NTS showed termination of the sequencing reaction for each strand. The results obtained clearly show that the lid deletion mutant generates nicks in the non-target strand that is replaced, indicating that it acts as a non-target strand nickase.

[0209] To further improve the RuvC lid deletion mutant, the cysteine ​​residue at position 931 (Cys / C-931) was replaced by selected alternative residues containing either bulky (Trp / W), positively charged (Lys / K), or negatively charged (Glu / E) amino acids. The resulting LbCas12a variants were cloned into a pET (pML-1B, KanR.Addgene#29653) vector containing a 6x histidine tag at the N-terminus of the protein and expressed in E. coli Rosetta DE3 competent cells as described above. For initial testing of activity, a fluorescent nickase assay was performed (see Figure 6D). In this assay, a 331 bp PCR substrate in which the target strand (complementary to the crRNA used) is labeled with Cy5 and the non-target strand is labeled with Cy3 was incubated with each nickase candidate and separated via denaturing gel electrophoresis.

[0210] Nicking reactions were performed as described above for the plasmid nickase assay, except that a dual Cy3 / Cy5-labeled dsDNA substrate was used. After incubation, the reaction was stopped by digesting the samples with proteinase K for 10 min. TBE-urea sample buffer was then added and the samples were heated at 95°C for 5-10 min to denature the substrate strands. Samples were resolved on a denaturing 10-15% TBE-urea gel at 8-15 mA and fluorescence imaged on an Amersham Typhoon imaging system.

[0211] Fluorescently labeled DNA substrate in 1x nuclease buffer was used as a non-digested control, while nuclease and nickase controls were made by incubating the DNA substrate with EcoRI-HF and Nb.BbvCI restriction enzymes, respectively. All controls were made using the same input amount of labeled DNA substrate as in the reactions containing LbCas12a variants. As shown in Figure 6E, the reaction with the C931E (SEQ ID NO: 56) variant showed a clear band at the expected position of cleavage of the non-target strand, indicating that it preferentially nicks the non-target strand. Interestingly, time series analysis showed that this mutant also nicked the original RuvC deletion mutant (RuvC L-del1 , SEQ ID NO: 15), with only slight levels of target strand cleavage being detected from 150 min onwards. In contrast, the C931W variant showed stronger nicking specificity and reduced background double-strand breaks but no increase in overall activity, while the C931K variant produced increased initial nicking activity but comparable levels of double-strand breaks (data not shown). Taken together, these findings indicate that the C931E variant is a superior nickase variant that exhibits the highest ratio of nickase activity to double-strand break activity among all LbCas12a mutants tested.

[0212] Example 5: Analysis in an in vitro transcription-translation system In addition to the in vivo GFP / RFP detection method, a second analytical approach was used based on an in vitro cleavage system. Genes encoding Cas12a variants, guide RNAs and GFP are co-expressed in one reaction compartment (one well of a 96-well plate) using a cell-free transcription-translation (TXTL) system (Marshall et al., Mol Cell, 2018). In this assay, the expressed guide RNA targets the sequence encoding GFP, while GFP fluorescence is measured in each reaction compartment over time using a plate reader. Control reactions are prepared with guide RNAs that do not target the sequence encoding GFP. GFP fluorescence increases over time in the non-targeting control reaction, while Cas-mediated cleavage strongly suppresses GFP fluorescence.

[0213] A particular interest for using Cas12a nickase is a paired nickase strategy, in which at least two guide RNAs are designed to enable the coordinated action of at least two Cas enzymes, which may be the same or different Cas enzymes with nickase activity, such that at least two Cas enzymes with nickase activity introduce at least two individual nicks at at least one target site, and the at least two individual nicks can result in a DSB.

[0214] The TXTL system has therefore been modified to function as an in vitro double-nicking assay, in which the GFP coding sequence is targeted not by a single guide RNA, but instead by a pair of guide RNAs that create a DSB through the introduction of two nicks.

[0215] First, the system was prepared and optimized using wild-type Cas9 and wild-type LbCas12a to achieve high GFP expression and fluorescence detection in non-targeted control samples and suitable conditions for efficient cleavage by Cas enzyme in targeted samples. Next, double nicking assays were tested and optimized using Cas9 D10A and different pairs of guide RNAs. For illustration, Figure 7B shows an example of the results of an in vitro double nicking assay using Cas9 D10A and a pair of guide RNAs (see Figure 7A). Experiments with Cas12a nickase have been initiated and are ongoing. One purpose of this assay is to further test the ability of Cas12a nickase to introduce DSBs through paired nicks. Experiments are performed with one Cas12a variant and two suitable pairs of guide RNAs suitable for Cas12a targeting and Cas9 targeting, respectively, or one Cas12a variant and two guide RNAs combined with Cas9D10A. In addition to the ability to introduce paired nicks and thus quantify nickase activity, this in vitro assay can be used as an additional means to analyze Cas12a variants for residual nuclease activity; thus providing a rapid and scalable tool for quantitative and time-dependent characterization of Cas12 activity.

[0216] Example 6: Analysis of Cas12a nickase variants in Bacillus subtilis Cas12a variants will be extensively tested in Bacillus subtilis and initial work on these experiments has been carried out. Validation of different Cas12a variants in Bacillus subtilis will be undertaken according to the following protocol.

[0217] The Cas9 gene of plasmid pCC0027 (WO2021175759) is replaced by the coding sequence of the Cas12a nickase variant gene by Gibson assembly (NEBuilder® HiFi DNA Assembly Cloning Kit, New England Biolabs) to obtain plasmid pNCP001.

[0218] The Cas12a nickase-based gene deletion plasmid pNCP002 for deletion of the amyB gene of Bacillus subtilis is constructed as described below.

[0219] A fragment containing the amyB-specific FnCas12a crRNA and the 5' and 3' homology regions of the amyB gene (amyB-HomAB) was PCR amplified from plasmid pcrA3 (Wu Y, Liu Y, Lv X, Li J, Du G, Liu L CAMERS-B: CRISPR / Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis. Biotechnol Bioeng. 2020 Jun;117(6):1817-1825. doi:10.1002 / bit.27322. Epub 2020 Mar 16. PMID:32129468.) with primers carrying flanking BsaI restriction sites. A Cas12a-nickase-based gene deletion plasmid for the amyB gene was then constructed by type II assembly with plasmid pCC027 and the PCR-amplified crRNA-amyB-HomAB region with the restriction endonuclease BsaI as described in (Radeck et al., 2017). The reaction mixture was transformed into E. coli DH10B cells (Life technologies). The transformants were spread on LB-agar plates containing 20 μg / ml kanamycin and incubated overnight at 37° C. Plasmid DNA was isolated from individual clones and analyzed for accuracy by restriction digestion and sequencing. The resulting amyE gene deletion plasmid is designated pNCP002.

[0220] Electrocompetent Bacillus subtilis ATCC 6051a cells are prepared as described by Brigidi et al. (Brigidi, P., Mateuzzi, D. (1991). Biotechnol. Techniques 5, 5) with the following modifications: Upon DNA transformation, cells were harvested in 1 ml of LBSPG buffer and plated on selective LB-agar plates and incubated at 37°C for 60 min (Vehmaanperae J., 1989, FEMS Microbio. Lett., 61:165-170).

[0221] Electrocompetent Bacillus subtilis ATCC6051a cells were plated on LB-agar plates containing 20 μg / ml kanamycin and, after overnight incubation at 37 °C, transformed with 1 μg of the amyE deletion plasmid pNCP002 isolated from E. coli DH10B cells.

[0222] The next day, 20 clones of each transformation reaction were analyzed by colony-PCR for successful Cas12a-nickase-based deletion of the amyE gene with oligonucleotides located 5' and 3' of the homologous region and further transferred onto fresh LB-agar plates without antibiotics after overnight incubation at 48°C for plasmid curing.

[0223] Correct clones with the deletion of the amyE gene and cured of the plasmid pNCP002 are identified and the corresponding B. subtilis ATCC 6051a strain with the deletion of the amyE gene is isolated.

[0224] Similarly, gene integration is performed in the amyE locus of B. subtilis ATCC6051a. A protein expression construct containing the GFP-gene under the control of the aprE gene promoter is placed between the 5' and 3' homologous regions of the amyE gene as described for the Cas9-based construct pCC043 (WO2021175759) using Gibson assembly. The resulting Cas12a-nickase-based gene integration plasmid pNCP003 is transformed into electrocompetent Bacillus subtilis ATCC6051a cells and the gene integration procedure is performed as described for the gene deletion procedure.

[0225] A resulting B. subtilis ATCC6051a strain with a PaprE-GFP expression cassette integrated at the amyE locus is isolated.

[0226] Example 7: Evaluation of DNA nicking activity in plant cells Cloning methods and plasmid construction Unless otherwise indicated, cloning procedures carried out for the purposes of the present invention, including restriction digestion, agarose gel electrophoresis, purification and ligation of nucleic acids, transformation of bacterial cells, selection and culture, are performed as described (Sambrook J, Fritsch EF and Maniatis T (1989)). Sequence analysis of recombinant DNA was performed by LGC Genomics (Berlin, Germany) using the Sanger technique (Sanger et al., 1977). Restriction endonucleases and Gibson assembly reagents used to construct plasmids are from New England Biolabs (Ipswich, MA, USA). Oligonucleotides are synthesized by Integrated DNA Technologies (Coralville, IA, USA). Codon-optimized genes are from Genewiz (South Plainfield, NJ, USA).

[0227] The selected LbCas12a nickase candidates were optimized for expression in plant cells using GeneOptimzer, a BASF proprietary software tool. Different settings were tested with parameter sets for codon usage for wheat highly expressed genes and optional removal of major cryptic splice sites. Alternatively, more stringent parameters were used for codon usage and only the most abundant wheat amino acid codons were selected during optimization, followed by manual removal of major cryptic splice sites.

[0228] Codon-optimized nickase variants were synthesized tagged with the SV40 nuclear localization signal (SEQ ID NO: 36) at the N-terminus and the Xenopus laevis nucleoplasmin C nuclear localization signal (SEQ ID NO: 37) at the C-terminus. The synthesized genes were digested with NcoI and NheI and cloned into a proprietary expression plasmid between the NcoI and NheI sites. The resulting expression vectors contain a maize polyubiquitin (Ubi) promoter (SEQ ID NO: 38) for constitutive expression located upstream of the Cas9 gene and a fragment of the 3' untranslated region of either the Agrobacterium tumefaciens nopaline synthase gene (SEQ ID NO: 39) or the Cauliflower mosaic virus 35S gene (SEQ ID NO: 40) at the 3' end.

[0229] Guide RNA expression cassettes containing Cas12a guide RNAs consisting of a 21-bp direct repeat sequence (SEQ ID NO: 41), a 23-bp protospacer region, and a rice polymerase III terminator sequence (nnnnntttttttt, where n is a, c, g, or t) were arranged as synthetic fragments. Expression of the guide RNA is driven by the polymerase III type promoter of the rice U6 snRNA gene (SEQ ID NO: 43). The synthesized cassettes were cloned into standard E. coli vectors (pUC derivatives) via EcoRV blunt-end ligation.

[0230] All plasmids were transformed into E. coli for propagation and isolated using the ZymoPure II Plasmid GigaPrep Kit (Zymo Research, Irvine, CA, USA) for DNA purification.

[0231] Rice protoplast isolation and transfection Transformation of rice protoplast cells was performed as described by Wang et al. (2014) with minor modifications. Protoplasts were isolated from the leaf sheaths of 3-week-old axenically grown rice seedlings. Healthy stems and leaf sheaths were bundled together in groups of 20 and cut into strips with a sharp razor blade. The strips were then infiltrated with a cell wall lytic enzyme solution (1.5% Cellulase R10 and 0.75% Macerozyme R10 in 10 mM KCl and 0.6 M mannitol, pH 7.5) and incubated overnight in the dark at 24 °C with gentle shaking (40 rpm). After enzymatic digestion, the liberated protoplasts were collected by filtering the mixture through a 40-μm nylon mesh and resuspended in W5 solution. After washing the resuspended protoplasts with W5 solution, the cell pellet was suspended in MMG solution at a density of 2.5 million cells / ml. For transformation, 200 μl of cells (5×105 cells) were mixed with 20 μg of plasmid DNA and 220 μl of freshly prepared polyethylene glycol (PEG) solution. The mixture was incubated for 15-20 min in the dark. After removing the PEG solution, the protoplasts were resuspended in 2 ml of WI solution, transferred into a 6-well plate and incubated at 24° C. for at least 48 h. Finally, the protoplasts were collected by centrifugation at 12,000 rpm at room temperature for 1 min, and the pelleted fraction was stored at −80° C. until further analysis.

[0232] Brassica napus protoplast isolation and transfection Brassica napus protoplasts were isolated from leaves of 4- to 7-week-old axenically grown plants and transfected as described for rice cells. After enzymatic digestion, the liberated protoplasts were collected by filtering the mixture through a 40-μm nylon mesh and resuspended in W5 solution. The resuspended protoplasts were kept on ice for at least 30 min and allowed to settle by gravity, after which the cell pellet was resuspended in MMG. For transformation, 200 μl of cells (2.5x10 5) was mixed with 20 μg of plasmid DNA and 220 μl of freshly prepared polyethylene glycol (PEG) solution. The mixture was incubated in the dark for 15-20 min. After removing the PEG solution, the protoplasts were resuspended in 2 ml of W5 solution, transferred into a 6-well plate, and incubated at 24°C.

[0233] In planta nickase activity assay A convenient in vitro assay for LbCas12a nickase variants is to monitor the processing of negatively supercoiled dsDNA plasmid substrates isolated from E. coli. Exposing the plasmid to Cas12a-derived nuclease variants allows the identification of variants that generate DSBs or nicks by analysis of linear and nicked cleavage products using agarose gel electrophoresis. However, this simple assay cannot be easily performed in plants, as the presence of relaxed circles in extracted DNA is insufficient to infer whether nicking occurred in vivo or during DNA extraction and / or analysis. Therefore, a different assay was designed to evaluate the performance of selected Cas12a nickase candidates in plant cells.

[0234] The first assay takes advantage of new molecular insights into the pathways and factors that regulate the repair of nicks in genomic DNA. As the simplest and most frequent form of DNA damage, nicks are usually repaired either sequentially or by high-fidelity homologous recombination repair. However, recent findings have highlighted the possibility that nicked genomic DNA may undergo mutagenic repair, including the introduction of single nucleotide mutations (Zhang Y, et al. PLoS Genet. 2021 doi:10.1371 / journal.pgen.1009329). Thus, low-level frequency of base substitutions at or near the nick site can be used as a proxy for nickase activity in vivo. In this context, the selected nickase variants were co-transfected with Cas12a guide RNA (SEQ ID NO: 44) targeting the AAT gene (LOC_Os01g55540.1) in rice protoplasts using PEG-mediated transformation as described above. All Cas12a variants were codon-optimized for monocotyledonous plants and transcribed from the maize Ubi promoter. Three days after transfection, protoplasts were harvested by centrifugation and genomic DNA was extracted using the Qiagen DNeasy Plant kit. The AAT target region was amplified by PCR using primers SEQ ID NO: 45 and SEQ ID NO: 46 and subjected to amplicon deep sequencing.

[0235] As shown in Figure 8A, transfection of WT LbCas12a (SEQ ID NO: 1) resulted in a high frequency of indels at the predicted cleavage site (average 22.54%), demonstrating efficient generation of double-strand breaks (DSBs). On-target indels were also frequently observed with the R1138A and K932G / N933G mutants (mutations relative to reference sequence SEQ ID NO: 1). Both variants showed indels in 2.98% and 0.62% of total sequencing reads, respectively, which correspond to 13.21% and 2.73% of the indel-induced activity observed in the Cas12a nuclease control, respectively (Figure 8A). Interestingly, the K932G / N933G / S934A / R935G quadruple mutant (SEQ ID NO: 14) induced much fewer indels (average 0.18%, i.e., less than 1% for WT Cas12a). Also, the K932G / N933G / S934A / R935G quadruple variant supported a higher number of base substitutions at the AAT target site compared to both the R1138A and K32G / N933G variants (up to 1.09% of total sequencing reads) (Figure 8B). Comparison of the number of NGS reads with indels versus those with base conversions further highlighted the differences between the various variants (Figure 8C). Unlike Cas12a-R1138A and Cas12a-K932G / N933G, which yielded levels of indels reaching 99.44% and 49.04%, respectively, Cas12a-K932G / N933G / S934A / R935G predominantly generated base changes (86.19% of edited sequence reads). Although nicked DNA can, in rare cases, be processed through a DSB intermediate resulting in an NHEJ event (Certo et al., 2011 doi:10.1038 / nmeth.1648), the high ratio of base changes observed for both R1138A and K932G / N933G to indels suggests substantial nuclease activity for the latter variant.

[0236] To further evaluate nickase activity in plants, a dual-plasmid reporter system was devised similar to the GFP / RFP system used in E. coli (Example 3). In this system, a plasmid encoding an engineered GFP reporter (SEQ ID NO: 47) with two Cas12a targeting sites located in close proximity on opposite strands within the GFP coding sequence and a plasmid encoding an engineered dsRed reporter (SEQ ID NO: 48) carrying a single Cas12a targeting site are co-transfected into rice protoplast cells with the selected nickase variant and three Cas12a gRNAs targeting the GFP (SEQ ID NO: 49 / SEQ ID NO: 50) and dsRed (SEQ ID NO: 51) reporters, respectively (see FIG. 9A). Three days after transfection, cells transfected with inactive Cas12a will show both GFP and dsRed; cells expressing WT Cas12a will show no or minimal GFP and dsRed; and cells expressing nickase will be positive for dsRed (due to single nicking) but low in GFP (due to double nicking), so the fluorescent signature of transfected cells can be used to distinguish nickase from catalytically active and inactive enzymes.

[0237] Figure 9B shows the results for protoplasts transfected with plasmids encoding either WT LBCas12a (SEQ ID NO: 1), catalytically inactive Cas12a-D832R (mutations relative to reference sequence SEQ ID NO: 1), or Cas12a-K932G / N933G / S934A / R935G (SEQ ID NO: 14) variants. Expression of WT Cas12a caused a strong reduction in the number of GFP- and RFP-positive cells compared to those in cells transfected with the fluorescent reporter alone. In contrast, GFP and dsRed fluorescence with the inactive Cas12a variant was comparable to that of the positive control, while transfecting cells with the Cas12a quadruple variant resulted in a reduction in the GFP signal but not in dsRed.

[0238] In a third activity assay, the base editing results induced by LbCas12a nickase variants were compared to those by WT LbCas12a. In the absence of a suitable variant that nicks the unedited strand, Cas12a base editors routinely use catalytically inactive Cas12a as the Cas moiety. By analogy with the previously characterized Cas9 base editor (Komor et al., 2016; Nishida et al., 2016; Gaudelli et al., 2017), it is reasonable to assume that the use of Cas12a nickase will affect base editing activity. That is, variants that nick the unedited strand (i.e., the target strand) are expected to increase editing levels, while nickase variants that target the edited strand should decrease editing efficiency.

[0239] Taking advantage of this phenomenon, different nickase candidates were introduced into the LbCas12-BE (LbCas12 base editing) construct, and the editing of the AAT target site was measured after 3 days by amplicon deep sequencing. As shown in Figure 10A, Cas12a-mediated base editing by K932G / N933G (mutations with respect to reference sequence SEQ ID NO: 1) and K932G / N933G / S934A / R935G (SEQ ID NO: 14) was approximately 9-fold and 7-fold reduced, respectively, compared to the corresponding D832A (mutations with respect to reference sequence SEQ ID NO: 1) variant. Importantly, as shown in Figure 10B, BE-K932G / N933G also led to high levels of indel formation (average 10.81%), suggesting that the introduction of DSBs and subsequent NHEJ repair, rather than DNA nicking, contributes to the reduction in editing. BE-K932G / N933G / S934A / R935G induced indels at a much lower frequency (<1%) than BE-K932G / N933G, showing a nearly 10-fold reduction in the percentage of reads with indels. The difference in editing outcomes between the Cas12a double and quadruple variants was also evident from alignments of the 20 most abundant sequencing reads (data not shown). While the quadruple mutant-derived base editors edited different bases in the range C5–C22 (counting the distal end relative to the protospacer adjacent motif as position 1), introduction of K932G / N933G almost exclusively resulted in deletions and was rarely accompanied by concomitant base editing. Combined with the relatively high frequency of base changes and low levels of indel formation at individual nick sites, as well as reduced GFP-derived but not dsRed-derived fluorescence in dual-color reporter assays, these findings strongly suggest that the LbCas12a-K932G / N933G / S934A / R935G quadruple variant exhibits significant nickase activity in plant cells and no or at least very low residual nuclease activity.

[0240] A different activity assay was also performed using the RuvC lid deletion mutant (RuvC L-del1, SEQ ID NO: 15) and its C931E variant (SEQ ID NO: 56) were used to evaluate the in planta performance of LbCas12a-K932G / N933G / S934A / R935G-Q variant (K932G / N933G / S934A / R935G-Q variant) and its C931E variant (SEQ ID NO: 56). As shown in FIG. 11, transfection of RuvC lid deletion mutants in rice protoplasts with Cas12a guide RNA targeting the AAT gene resulted in a strong reduction in indel formation compared to WT LbCas12a, while much lower levels of on-target indels were observed in the RuvC lid C931E mutant. Similar to the LbCas12a-K932G / N933G / S934A / R935G quadruple variant, both mutants also induced detectable levels of base substitutions (up to 90% of edited sequence reads) at the AAT target site, which may indicate nickase activity.

[0241] To further evaluate the nickase activity, RuvC lid deletion and C931 mutation were introduced into the LbCas12-BE construct, and editing at the AAT target site was quantified after 3 days by amplicon deep sequencing. The results are shown in Figure 12. When pooled across six independent experiments, the RuvC lid deletion mutation reduced Cas12a base editing by nearly 4.5-fold compared to the corresponding variant LbCas12a-D832A, while the additional C931E mutation resulted in a 1.4-fold reduction in editing efficiency (see Figure 12A). A similar picture emerges when targeting the FAD2 gene (LOC106452409) in canola (Brassica napus) protoplasts. In this case, transfecting a Cas12a base editor with the RuvC lid deletion and C931E mutant along with a FAD2-targeting gRNA (SEQ ID NO:57) reduced base editing by 1.98-fold and 4.43-fold, respectively, compared to the Cas12a D832A BE construct (mutations with respect to reference sequence SEQ ID NO:1) (see FIG. 12B). Given that the RuvC lid deletion mutant preferentially cleaves the non-target strand (see FIG. 6E) and the low levels of residual nuclease activity of both mutants (see FIG. 11 and FIGS. 6B and 6E), it is reasonable to assume that the observed reduction in base editing is due to nicking of the edited strand.

[0242] Finally, the in planta activity of different variants was evaluated in a dual nickase experiment. In this approach, indel formation at target sites is evaluated using nickase candidates directed by either a single guide or a pair of offset guides targeting opposite DNA strands. Single nicks are primarily repaired via high-fidelity base excision repair, while cooperative nicking of opposite DNA strands is expected to generate site-specific double-strand breaks and subsequent indel formation. As previously demonstrated for Cas9 nickase (Ran et al., DOI:10.1016 / j.cell.2013.08.021), different factors can affect cooperative nicking leading to indel formation, including steric hindrance, overhang type, and sequence composition between two adjacent Cas12a RNPs. To assess how offsets between Cas12a gRNA target sequences and guides may affect the generation of indels, a set of gRNA pairs separated by a range of offset distances from +62 to -95 bp targeting the rice OsDEP1 gene (LOC106452409) and creating either 5' or 3' overhangs were designed and tested for their ability to induce on-target indels in rice protoplasts co-transfected with a RuvC lid deletion variant (RuvCL del1, SEQ ID NO:15; gRNAs: SEQ ID NO:57 to SEQ ID NO:73).

[0243] As shown in Figure 13, only gRNA pairs that create 5' overhangs with at least 9bp offset between guides were able to mediate detectable indel formation. Notably, a significant proportion of induced mutations showed large deletions (>50bp) between the two nick sites, likely resulting from cooperative cleavage of opposite DNA strands by sequentially or simultaneously bound nickases. The highest indel frequency (up to 1.49% of sequencing reads) was observed for the gRNA3+gRNA17 pair, which creates a 64-bp 5' overhang. Using the gRNA3 / gRNA17 pair, we next compared the indel frequency induced by the paired nickases to that induced by a single nickase or WT LbCas12a. As expected, transfection of WT LbCas12a with gRNA3 or gRNA17 alone resulted in significant indel formation at the respective target sites (average 3.84% and 3.48%, respectively), whereas few indels were detected when using single guides with either the LbCas12a-K932G / N933G / S934A / R935G quadruple variant, the RuvC lid deletion mutant, or its C931E variant (see Figure 14). Clear differences between WT and Cas12a nickase candidates were also evident when testing paired gRNAs. Indeed, while the indel frequencies induced by WT LbCas12a and co-transfected gRNA3 and gRNA17 were comparable to those generated by WT Cas12a paired with each gRNA alone (3.86% vs. 3.84% and 3.48%, respectively), co-delivery of both gRNA and Cas12a nickase candidates had synergistic effects and strongly enhanced indel formation compared to single nickases. This was particularly evident for the quadruple and C931E mutants, where no or few indels were detected with single nickases, while the guide combinations successfully generated on-target indels at frequencies of 0.65 and 0.91%, respectively.Similarly, dual targeting of RuvC lid deletion variants with the gRNA3 / gRNA17 pair also induced indel formation at a significantly higher frequency than targeting with a single gRNA. Collectively, these findings not only demonstrate the robust performance of different RuvC lid nickase variants in plants, but also indicate that these Cas12a mutant proteins can be exploited to promote targeted DNA double-strand breaks using paired guide RNAs.

[0244] Example 8: Genetic modification in Ashbya gossypii using Cas12a-nickase Construction of CRISPR-Cas12a-Nickase vector The Cas12a-nickase system is constructed in a single vector that contains all the required modules for genome editing. The Ashbya gossypii CRISPR-Cas9 vector is used as the backbone, containing replication origins (yeast 2μm and bacterial ColE1) and resistance markers (AmpR and G418R) (Jimenez A, Munoz-Fernandez G, Ledesma-Amaro R, Buey RM, Revuelta JL. One vector CRISPR-Cas9 genome engineering of the industrial fungus Ashbya gossypii. Microb Biotechnol 2019;12:1293-1301). The donor DNA and modules for expression of Cas12a-nickase and crRNA are constructed as follows: a synthetic codon-optimized ORF of Cas12a-nickase enzyme (LbCas12a-nickase) with SV40 nuclear localization signal is constructed with the promoter and terminator sequences of A. gossypii TSA1 and ENO1 genes, respectively. Expression of the crRNA is driven by the promoter and terminator sequences of A. gossypii SNR52 gene transcribed by RNA polymerase III. A synthetic donor DNA containing the corresponding genome edit is also constructed in the nCas12a-nickase vector. The construction of the fragments is accomplished according to the Golden Gate assembly method as previously described (Ledesma-Amaro R, Jimenez A, Revuelta JL. Pathway grafting for polyunsaturated fatty acids production in A. gossypii through Golden Gate Rapid Assembly. ACS Synth Biol 2018;7:2340-2347). A directional cloning strategy is used by introducing BsaI sites at the ends of the fragments. The BsaI sites are flanked by 4 nucleotide (nt) sticky end sequences.Thus, after BsaI digestion, all modules contain compatible 4-nt sticky ends that facilitate single-step directional assembly of Cas12a-nickase vectors.

[0245] Using the described cloning strategy, a Cas12a-nickase system based on different Cas12a-nickase variants is designed to inactivate the ADE2 gene in A. gossypii. ADE2-deficient mutants show a red color due to the accumulation of intermediates of the purine synthesis pathway. Therefore, the ADE2 gene is a suitable reporter for gene inactivation. The same system was already used to show the applicability of the CRISPR-Cas12a system for A. gossypii (Jimenez A, Hoff B, Revuelta JL. Multiple genome editing in Ashbya gossypii using CRISPR-Cas12a. New Biotechnol 2020;57:29-33). In this experiment, the same crRNA and donor DNA sequences were chosen, the only difference being that they induce single-stranded DNA breaks, which allow the inactivation of the ADE2 gene. Use of Cas12a-nickase to induce DNA repair systems in Ashbya.

[0246] Transformation and Cas12a-nickase-mediated genome editing of A. gossypii 5-10 μg of one of the Cas12a-nickase variants and the above-mentioned plasmids encoding ADE2-specific crRNA and donor DNA sequences are used to transform spores of A. gossypii wild-type strain ATCC10895 as previously described (Jimenez A, Santos MA, Pompejus M, Revuelta JL. Metabolic engineering of the purine pathway for riboflavin production in Ashbya gossypii. Appl Environ Microbiol 2005;71:5743-5751). Heterokaryon transformants are selected on G418-containing MA2 medium, thereby confirming the uptake of the plasmid. G418-resistant colonies are isolated and grown again in G418-MA2 medium at 30° C. for 2 days to promote genome editing events. Loss of the CRISPR-Cas12a-nickase plasmid is performed after sporulation of heterokaryon clones in sporulation medium lacking G418. Homokaryon clones are isolated in MA2 medium lacking G418. The desired genomic inactivation of the ADE2 gene results in red colonies on agar plates. Genomic DNA of the red transformants is isolated and the transformants are analyzed via PCR and sequencing to confirm the desired ADE2 editing.

[0247] The sequencing results of the obtained transformants are expected to show that using Cas12a nickase instead of Cas12a nuclease will result in more clones carrying the desired short ADE2 deletion, while fewer clones should only carry random single point mutations resulting from non-homologous end joining repair. In that regard, nuclease and nickase activity can be distinguished by sequencing. In line with the investigation of Cas9 nickase, it is expected that the efficiency of obtaining specific HDR-mediated genome editing events will be improved using Cas12a-nickase.

[0248] Example 9: In vivo double nicking in yeast cells The ADE2 disruption strategy (see Example 8) will be further used to test for paired nicking in fungal cells in vivo. The selected Cas12a nickase candidates will be tested in vivo for nuclease and nickase activity in yeast cells by targeting the reporter gene ADE2 with either a single guide RNA or a pair of guide RNAs in parallel, similar to the in vivo GFP / RFP (Example 3) or GFP / dsRed (Example 7) assays. Loss of ADE2 leads to a red phenotype in yeast cells due to the accumulation of red intermediates in the adenine synthesis pathway. Yeast cells will be transformed with either a single guide RNA or a suitable pair of guide RNAs targeting the different Cas12a nickase candidates and the ADE2 gene. The nuclease activity of the Cas12a protein should lead to a red phenotype with both a single guide RNA and a pair of guide RNAs, while the nickase activity should lead to a red phenotype only when the guide RNA pair is present. Inactive Cas12a variants should not result in a red phenotype in either scenario.

[0249] Example 10: Analysis of Cas12 nickase variants in mammalian cells Further examples for testing selected nCas12a variants, or their orthologs, are planned in immortalized cell lines such as HEK293, HeLA, A549, or Jurkat cells, primary mouse and human cells, embryos, egg cells, stem cells, etc.

[0250] The target cell of interest can be transfected with the selected nCas12a variant or its orthologue as disclosed herein, using appropriately codon-optimized and cell-compatible NLS and regulatory sequences optimized for the given target cell of interest, and the nCas12 enzyme can be provided with either one guide RNA (single crRNA, or crRNA:.tracrRNA heteroduplex, or chimeric single guide RNA), or a pair of guide RNAs suitable for paired nickase approach. The guide RNA or guide RNA pair can target any chromosomal target or a target on a plasmid, such as a reporter construct, for easier assessment of nickase activity and residual nuclease activity. Transfection and transformation protocols (chemical (nucleofection, lipofection, etc.), viral-mediated, physical (e.g., bombardment, electroporation, microinjection for embryos, oocytes or zygotes), biological, using vectors and plasmids), buffers and equipment are known to the skilled artisan for a given target cell of interest.

[0251] To characterize the nicking activity of LbCas12a-RuvC lid deletion variants in mammalian cells, three different genes are selected (EMX1, DYRK1A and GRIN2BA) to be targeted with different variants of LbCas12a (wild type, nickase and inactive; corresponding gRNAs: SEQ ID NO:74-SEQ ID NO:79). In principle, the generation of a single nick should not induce indel formation at the target site, contrary to paired nicking, which generates double-stranded breaks (DSBs) and leads to non-homologous end joining (NHEJ) and subsequent indel formation. LbCas12a nickase is expected not to generate DSBs when only one locus is targeted (one guide), but should lead to DSB generation when two adjacent loci are targeted simultaneously (two guides). In this manner, the use of paired nicking provides higher on-target cleavage specificity compared to standard double-stranded DNA cleavage-dependent approaches, resulting in a higher frequency of precisely edited cells.

[0252] Cloning and replication of the expression vector is carried out in E. coli DH10b cloning strain. The following modules are integrated in an E. coli plasmid (pBR322, selection marker AmpR under the control of the natural bla / AmpR promoter): (i) a gene encoding one of three LbCas12a variants (wild type (LbCas12a-WT), nickase (e.g., LbCas12a-RucC lid deletion variant) and inactive (LbCas12a-inactive)) downstream of a CMV promoter, (ii) a synthetic CRISPR array downstream of a U6 promoter (allowing to target one of three target genes), and (iii) a gene encoding a GFP marker downstream of an SV40 promoter (see Figure 15). Upon individual transfection of each of these plasmids into human cells (HEK293), Cas12a / CRISPR and gfp genes are transiently expressed and Cas12a / crRNA RNP complexes are formed. Different combinations of LbCas12a variants and guides are required to evaluate pairwise nicking at selected loci. For this purpose, a set of different plasmids is created (3 nucleases x 3 loci x 2 CRISPR arrays (single guide array or two guide arrays)).

[0253] HEK293 cells are transfected using Lipofectamine according to standard procedures and then incubated. Due to variable transfection efficiency and to avoid sequencing of untransfected cells, the resulting bacterial culture is FACS sorted to enrich for GFP-positive cells (indicating successful transfection). After pooling the transfected populations, chromosomal DNA is extracted from each population and PCR reactions are carried out to generate amplicons of the three target sites, followed by calculating the frequency of indel formation in each treatment by amplicon deep sequencing (Illumina). A detailed protocol is described below.

[0254] [Table 1]

[0255] protocol 1. Cloning a. Generate different plasmids with either LbCas12a Lid2.3, LbCas12a inactive or LbCas12a WT and each guide (single guide array or two guide arrays) for a total of 18 plasmids. b. Golden Gate Cloning using BsaI restriction enzyme 2. HEK293 Cell Transfection a. Cells are transfected with the desired plasmid using Lipofectamine 2000 b. The cells are cultured in an incubator at 37° C. for 6 hours. After 6 hours, Opti-MEM medium is replaced with D-MEM to optimize cell growth, and the cells are incubated at 37° C. for at least 48 hours before being sorted. 3.GFP+ sorting a. FACS sorting and pooling only GFP+ cells 4. DNA Extraction and Isolation 5. PCR to Generate Sequencing Amplicons 6. NGS Sequencing 7. Data Analysis

[0256] Example 11: Base editing and prime editing The selected nickase variants will be tested in base editing systems (both single base editors and dual base editors using different configurations with different cytidine and / or adenosine deaminases and different linker regions) and optionally prime editing systems (with different reverse transcriptases, different pegRNA designs, both with and without additional guide RNA targeting the sequence to be edited, i.e., PE2 and PE3). Base editing, and optionally prime editing, will be tested in the most important target systems, including crop plants and optionally fungal systems and human cells. Exemplary first results for base editing in rice protoplasts are shown in Figures 10A and 10B (Example 7). While these results showed a negative impact of the tested Cas12a nickase variants on base editing levels (due to cleavage of the edited strand), it should be noted that these mutants may be employed to improve editing efficiency in a similar manner to the previously described Cas9 PPE3 system (Anzalone et al., 2019). In this approach, the selected NTS-nickase forms a complex with a nicking gRNA, and the resulting RNP is co-delivered with a Cas12a base editor with a catalytically inactive Cas12a. While the Cas12a base editor is directed to the target site by the first gRNA, the nicking gRNA will direct the NTS nickase to cleave the unedited DNA strand, which should promote favorable DNA repair by inducing the cell to use the edited strand as a repair template. Optionally, the nicking gRNA can be designed to specifically target the sequence to be edited, thereby preventing nicking of the unedited strand until after editing has occurred (Anzalone et al., 2019). Because optimal nicking positions may vary depending on the genomic site, various unedited strand nick positions should be tested using gRNAs that are located at 5' or 3' and induce nicks at different distances (e.g., 10-120 bp) from the editing site.

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Claims

1. An engineered Cas12a enzyme (nCasl2a) having nickase activity, or a catalytically active fragment thereof, comprising at least one mutation in its core lid domain, wherein said mutation in said core lid domain: (i) at least three point mutations at three consecutive positions within the core lid domain; or (ii) a deletion of at least two consecutive positions within the core lid domain; or (iii) at least one first point mutation combination at at least one position within the core lid domain; and (iiia) at least one deletion at at least one position within the core lid domain, and / or (iiib) at least one, preferably at least two, at least three, or at least four additional point mutations at different positions relative to the first point mutation within the core lid domain, wherein the positions of the additional point mutations are not in consecutive order with the positions of the at least one first point mutation; (iv) a point mutation at a position within the core lid domain; an engineered Cas12a enzyme or catalytically active fragment thereof, wherein said at least one mutation in said core lid domain confers broad-spectrum nickase activity, and wherein the reference sequence of said core lid domain comprises the sequence as defined in SEQ ID NO: 13, and optionally, the complex further comprises at least one compatible guide RNA, or a sequence encoding same, that forms a complex with said cognate engineered Cas12a enzyme or catalytically active fragment thereof that has nickase activity.

2. wherein said engineered Cas12a enzyme is based on a wild-type Cas12a sequence according to any one of SEQ ID NOs: 1-12, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity with the corresponding wild-type sequence as a reference sequence, or an ortholog or homolog of a sequence according to any one of SEQ ID NOs: 1-12 having at least 95%, 96%, 97%, 98% or at least 99% sequence identity with the corresponding ortholog or homolog sequence as a reference sequence; the at least three point mutations in three consecutive amino acids are located within positions 2-16 with respect to SEQ ID NO: 13, and / or the deletion is a deletion of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 consecutive positions within the core lid domain; the mutations are deletions of at least 4, at least 5, at least 6, at least 7, or at least all 8 of positions 6-13 relative to SEQ ID NO: 13, and / or the mutations are at least one of three point mutations at three consecutive positions within positions 6-13 relative to SEQ ID NO: 13; the engineered Cas12a enzyme or the catalytically active fragment thereof has target strand (TS) nickase activity or non-target strand (NTS) nickase activity, preferably the engineered Cas12a enzyme or the catalytically active fragment thereof has non-target strand (NTS) nickase activity; or wherein the engineered Cas12a enzyme comprises or has an amino acid molecule according to SEQ ID NOs: 14-21 or 56 or 100-106, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the corresponding reference sequence; or the Cas12a enzyme comprises at least the core lid domain of any one of SEQ ID NOs: 14-21 or 56 or 100-106 beginning at position 927, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the corresponding core lid domain; 2. The engineered Cas12a enzyme of claim 1 or a catalytically active fragment thereof.

3. 2. The engineered Cas12a enzyme or catalytically active fragment thereof of claim 1, wherein the Cas12a enzyme having nickase activity comprises at least one additional mutation, wherein at least one additional modification alters the PAM specificity and / or thermotolerance of the engineered Cas12a enzyme.

4. 10. A nucleic acid molecule encoding the Cas12a enzyme or a catalytically active fragment thereof of claim 1, optionally wherein the nucleic acid molecule is codon-optimized, preferably codon-optimized for a fungal cell, including a yeast cell, a prokaryotic cell or an archaeal cell, or codon-optimized for a plant cell or an animal cell (including a human cell), and / or comprises a nucleic acid molecule encoding at least one guide RNA.

5. 5. The nucleic acid molecule of claim 4, wherein the nucleic acid molecule comprises or consists of a sequence according to SEQ ID NOs: 80 to 87 or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to SEQ ID NOs: 80 to 87, respectively.

6. An expression construct or vector comprising at least one nucleic acid molecule according to claim 4.

7. 10. A cell comprising at least one engineered Cas12a enzyme or catalytically active fragment thereof according to any one of claims 1 to 3; and / or at least one nucleic acid molecule according to claim 4 or 5; and / or at least one expression construct or vector according to claim 6.

8. The cell of claim 7 , wherein the cell is a eukaryotic or prokaryotic cell, including a bacterial or archaeal cell.

9. The cell is a fungal cell, including a yeast cell, and preferably the fungal cell, including the yeast cell, is selected from the group consisting of Saccharomyces spec., including Saccharomyces cerevisiae, Hansenula spec., including Hansenula polymorpha, Schizosaccharomyces spec., including Schizosaccharomyces pombe, Kluyveromyces lactis and Kluyveromyces marxanus. marxianus), Yarrowia spec. including Yarrowia lipolytica, Pichia spec. including Pichia methanolica, Pichia stipites, and Pichia pastoris, Zygosaccharomyces rouxii, and Zygosaccharomyces bailiii, Zygosaccharomyces spec. including Candida boidinii, Candida utilis, Candida freyschussii, Candida glabrata, and Candida sonorensis; Schwanniomyces spec. including Schwanniomyces occidentalis; Arxula species, including Arxula adeninivorans8. The cell of claim 7, selected from the group consisting of Ogataea spec, Ogataea spec including Ogataea minuta, Aspergillus spec including Aspergillus niger, and Myceliophthora thermophila.

10. The cell is a prokaryotic cell, including a gram-positive, gram-negative, or gram-variant bacterial cell, preferably a gram-negative bacterial cell, or an archaeal cell, and preferably the prokaryotic cell is selected from the group consisting of Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, and the like. tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumecens, Arthrobacter parafineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, Brevibacterium divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicumBrevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes aerogenes), Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium lennanum rhenanum), Flavobacterium sewanense, Flavobacterium brevebreve), Flavobacterium meningosepticum, Klebsiella species including Klebsiella pneumonia, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas juluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovorans, Pseudomonas musiderens, mucidolens), Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, ureae), Staphylococcus aureus, Vibrio metschnikoffiimetschnikovii), Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces flavellas, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae lavendulae), Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, marcescens, Salmonella typhimuriumtyphimurium), Salmonella schottmulleri, Xanthomonas citri, Synechocystis sp., Synechococcus elongatus, Thermosynechococcus elongatus, Microcystis aeruginosa, Nostoc sp., N. commune, N.

8. The cell of claim 7, wherein the cell is selected from cells derived from N. sphaericum, Nostoc punctiforme, Spirulina platensis, Lyngbya majuscula, L. lagerheimii, Phormidium tenue, Anabaena sp., or Leptolyngbya sp.

11. The cell is a plant cell, preferably a plant cell selected from the group consisting of maple spp., Actinidia spp., Abelmoschus spp., sisal (Agave sisalana), wheatgrass spp., bedweed (Agropyron spp.), bedweed (Agrostis stolonifera), Allium spp., Amaranthus spp., Ammophila arenaria, pineapple (Ananas comosus), Annona spp., celery (Apium graveolens), peanut species (Arachis spp.), breadfruit species (Artocarpus spp.), asparagus (Asparagus officinalis), oat species (Avena spp.) (e.g., Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), star fruit (Averrhoa carambola), bamboo species (Bambusa sp.), wax gourd (Benincasa hispida), Brazil nut (Bertholletia excelsea), sugar beet (Beta vulgaris), Brassica spp. (e.g., Brassica napus, Brassica rapa ssp. [canola, rapeseed, turnip rape]), Cadaba farinosa, tea plant (Camellia sinensis), Canna indica, cannabis (Cannabis sativa), sativa), Capsicum spp., Carex elata, papaya (Carica papaya), Carissa macrocarpa, pecan spp.spp.), safflower (Carthamus tinctorius), chestnut spp. (Castanea spp.), kapok (Ceiba pentandra), endive (Cichorium endivia), cinnamon spp. (Cinnamomum spp.), watermelon (Citrullus lanatus), citrus spp., coconut spp. (Cocos spp.), coffee spp. (Coffea spp.), taro (Colocasia esculenta), cola spp., coriander spp., coriander spp., sativum, Hazel (Corylus spp.), Crataegus spp., Saffron (Crocus sativus), Pumpkin (Cucurbita spp.), Cucumis spp., Artichoke (Cynara spp.), Wild carrot (Daucus carota), Desmodium spp., Longan (Dimocarpus longan), Dioscorea spp., Persimmon spp., Echinochloa spp. spp.), Elaeis (e.g., Elaeis guineensis, Elaeis oleifera), finger millet (Eleusine coracana), teff (Eragrostis tef), Erianthus sp., loquat (Eriobotrya japonica), eucalyptus sp., Eugenia uniflora, buckwheat (Fagopyrum spp.), beech (Fagus spp.), fescue (Festuca arundinacea), fig (Ficus carica), Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g., Glycine max, Soja hispida),hispida or Soja max), cotton (Gossypium hirsutum), Helianthus spp. (e.g., sunflower (Helianthus annuus), daylily (Hemerocallis fulva)), Hibiscus spp., Hordeum spp. (e.g., barley (Hordeum vulgare)), sweet potato (Ipomoea batatas), walnut spp., lettuce (Lactuca sativa), Lathyrus spp. spp.), lentil (Lens culinaris), flax (Linum usitatissimum), litchi (Litchi chinensis), lotus spp., Luffa acutangula, lupin spp., Luzula sylvatica, Lycopersicon spp. (e.g., Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), pyriforme), Macrotyloma spp., Malus spp., Acerola (Malpighia emarginata), Mammea americana, Mango (Mangifera indica), Cassava spp., Sapodilla (Manilkara zapota), Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp.), Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp.spp. ), Oryza spp. (e.g., rice (Oryza sativa), Oryza latifolia)), millet (Panicum miliaceum), Panicum virgatum, passionflower (Passiflora edulis), parsnip (Pastinaca sativa), Pennisetum sp., Persea spp., parsley (Petroselinum crispum), reed canary grass (Phalaris arundinacea), Phaseolus spp. spp.), Timothy grass (Phleum pratense), Date palm (Phoenix spp.), Common reed (Phragmites australis), Nightshade (Physalis spp.), Pine spp., Pistachio (Pistacia vera), Pea spp., Poa spp., Populus spp., Prosopis spp., Cherry spp., Psidium spp., Pomegranate (Punica granatum, pear (Pyrus communis), oak (Quercus spp.), radish (Raphanus sativus), rhubarb (Rheum rhabarbarum), currant (Ribes spp.), castor bean (Ricinus communis), rubus spp., sugarcane spp., willow spp., elderberry spp., rye (Secale cereale), sesame spp., Sinapis spp. sp.), Solanum spp. (e.g. potato (Solanum tuberosum), Solanum integrifolium, or tomato (Solanum lycopersicum)), sorghum (Sorghumbicolor), Spinacia spp., Myrtaceae spp., Tagetes spp., Tamarind (Tamarindus indica), Cocoa (Theobroma cacao), Trifolium spp., Gamagrass (Tripsacum dactyloides), Triticosecale rimpaui, Triticum spp. (e.g., Triticum aestivum, Triticum durum, Triticum riveted), turgidum, Triticum hibernum, Triticum macha, Triticum sativum, einkorn (Triticum monococcum) or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis vinifera spp.), maize (Zea mays), wild rice (Zizania palustris), or jujube species (Ziziphus spp.), or cells derived from plants belonging to the superfamily of the subkingdom Chlorophyta, in particular monocotyledons and dicotyledons, ornamentals, food grains, trees or shrubs, including fodder or forage legumes; or the cell is an animal cell, said animal cell being an insect, poultry, fish or crustacean cell, or a mammalian cell, preferably a mammalian cell, optionally selected from a cell derived from a non-human primate, cow, pig, rodent, including rat or mouse, or a human cell; The cell of claim 7.

12. 10. A complex, or at least one nucleic acid molecule encoding a component of said complex, said complex comprising at least one engineered Cas12a enzyme or catalytically active fragment thereof with nickase activity according to claim 1, and at least one compatible guide RNA, and optionally at least one additional polypeptide covalently and / or non-covalently linked to said at least one engineered Cas12a enzyme with nickase activity or said catalytically active fragment thereof within said complex, wherein said at least one additional polypeptide is selected from the group consisting of a nuclear localization signal (NLS), a mitochondrial localization signal (MLS), a mitochondrial targeting signal (MTS), a mitochondrial targeting signal (MTT ... or an organelle localization sequence comprising a chloroplast localization signal, and / or said at least one further polypeptide is preferably a cell-permeable polypeptide when said at least one further polypeptide is covalently linked to said at least one engineered Cas12a enzyme having nickase activity or said catalytically active fragment thereof, wherein said at least one further polypeptide is covalently linked to the N-terminus and / or the C-terminus of said at least one engineered Cas12a enzyme having nickase activity, or said catalytically active fragment thereof.

13. 10. A fusion protein or at least one nucleic acid molecule encoding the same, comprising at least one engineered Cas12a enzyme with nickase activity according to claim 1 or a catalytically active fragment thereof covalently and / or non-covalently linked to at least one further polypeptide domain, wherein the at least one further polypeptide domain has an activity selected from an enzymatic activity, a binding activity, or a targeting activity, and optionally comprises at least one guide RNA compatible with the engineered Cas12a enzyme with nickase activity, wherein the at least one compatible guide RNA interacts covalently and / or non-covalently with the at least one engineered Cas12a enzyme with nickase activity or the catalytically active fragment thereof.

14. 10. An adenine or cytidine base editor or base editor complex, or at least one nucleic acid molecule encoding same, wherein the base editor or base editor complex comprises at least one catalytically active portion of at least one engineered Cas12a enzyme with nickase activity according to claim 1.

15. 10. A prime editor or prime editor complex, or at least one nucleic acid molecule encoding same, wherein the prime editor or prime editor complex comprises at least one catalytically active portion of at least one engineered Cas12a enzyme with nickase activity described in claim 1.

16. (i) an engineered Cas12a enzyme with nickase activity (nCasl2a) as defined in claim 1, or a catalytically active fragment thereof, or an expression construct or vector as defined in claim 6, or a complex as defined in claim 12, or at least one sequence encoding same, or a fusion protein as defined in claim 13, or at least one sequence encoding same, or an adenine or cytidine base editor or base editor complex as defined in claim 14, or at least one nucleic acid molecule encoding same, or a prime editor or prime editor complex as defined in claim 15, or at least one nucleic acid molecule encoding same; (ii) at least one compatible guide RNA, or a set of compatible guide RNAs, each guide RNA being complementary to a target sequence of interest; and (iii) comprising a set of reagents; (iv) Optionally, the kit includes at least one vector comprising a particle, vesicle, or viral vector to aid in delivery, wherein the particle comprises a lipid, including lipid nanoparticles, a sugar, a metal, or a polypeptide, or a combination thereof, or the vesicle comprises an exosome or a liposome.

17. 1. A method for modifying a genomic locus of interest in at least one cell or construct at or near at least one target site, comprising: (a) providing at least one cell or construct comprising a genomic locus to be modified; (b) (i) at least one engineered Cas12a enzyme (nCasl2a) having nickase activity as defined in claim 1, or a catalytically active fragment thereof, or at least one nucleic acid molecule encoding same; or (ii) At least one expression construct or vector as defined in claim 6; or (iii) at least one complex as defined in claim 12 or at least one nucleic acid molecule encoding same; or At least one fusion protein as defined in claim 13 or at least one nucleic acid molecule encoding same; or (iv) at least one adenine or cytidine base editor, or at least one base editor complex, as defined in claim 14, or at least one nucleic acid molecule encoding same; or (v) at least one prime editor or at least one prime editor complex as defined in claim 15, or at least one nucleic acid molecule encoding same; providing and / or introducing into / into said at least one cell or construct; (c) providing and / or introducing at least one compatible guide RNA as defined in claim 1 or a sequence encoding same; (d) allowing complex formation of the at least one engineered Cas12a enzyme having the nickase activity of (a), or the catalytically active fragment thereof, and at least the compatible guide RNA as defined in claim 1, thus allowing insertion of at least one nick at or near at least one target site at a genomic locus of interest of the at least one cell or construct; (e) optionally providing at least one donor repair template, or at least one of said nucleic acid molecules encoding same; and (f) obtaining at least one edited cell or construct comprising an alteration of the genomic locus of interest at or near the target site; The method also excludes processes for altering the genetic identity of the germ line of humans, the use of human embryos for industrial or commercial purposes, and processes for altering the genetic identity of animals that may cause suffering without any substantial medical benefit to humans or animals, as well as animals resulting from such processes; Optionally, the method comprises the steps of: (g) regenerating at least one population of edited cells, tissue, organ, material, or whole organism from said at least one edited cell or construct.

18. 18. The method of claim 17, wherein the method is performed in vitro or in vivo.

19. The cell or construct is derived from a prokaryotic cell, including a bacterial or archaeal cell, or a eukaryotic cell, preferably the cell is (i) fungal cells, including yeast cells (preferably, the fungal cells, including the yeast cells, are selected from the group consisting of Saccharomyces species, including Saccharomyces cerevisiae, Hansenula species, including Hansenula polymorpha, Schizosaccharomyces species, including Schizosaccharomyces pombe, Kluyveromyces lactis and Kluyveromyces marxanus). marxianus), Yarrowia spec. including Yarrowia lipolytica, Pichia spec. including Pichia methanolica, Pichia stipites, and Pichia pastoris, Zygosaccharomyces rouxii, and Zygosaccharomyces bailiii, Zygosaccharomyces spec. including Candida boidinii, Candida utilis, Candida freyschussii, Candida glabrata, and Candida sonorensis; Schwanniomyces spec. including Schwanniomyces occidentalis; Arxula species including Arxula adeninivorans, Ogataea minuta,minuta), Aspergillus spec., including Aspergillus niger, or Myceliophthora thermophile; or (ii) prokaryotic cells, including gram-positive, gram-negative, or gram-variant bacterial cells, preferably gram-negative bacterial cells, or archaeal cells (preferably, the prokaryotic cells are selected from the group consisting of Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, and the like); tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumecens, Arthrobacter parafineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, Brevibacterium divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicumBrevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes aerogenes), Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium lennanum rhenanum), Flavobacterium sewanense, Flavobacterium brevebreve), Flavobacterium meningosepticum, Klebsiella species including Klebsiella pneumonia, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas juluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovorans, Pseudomonas musiderens, mucidolens), Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, ureae), Staphylococcus aureus, Vibrio metschnikoffiimetschnikovii), Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces flavellas, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae lavendulae), Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, marcescens, Salmonella typhimuriumtyphimurium), Salmonella schottmulleri, Xanthomonas citri, Synechocystis sp., Synechococcus elongatus, Thermosynechococcus elongatus, Microcystis aeruginosa, Nostoc sp., N. commune, N.

18. The method of claim 17, wherein the cell is derived from a species selected from the group consisting of N. sphaericum, Nostoc punctiforme, Spirulina platensis, Lyngbya majuscula, L. lagerheimii, Phormidium tenue, Anabaena sp., or Leptolyngbya sp.

20. The cell is a plant cell, preferably a plant cell selected from the group consisting of maple spp., Actinidia spp., Abelmoschus spp., sisal (Agave sisalana), wheatgrass spp., Agropyron spp., bedweed (Agropyron spp.), Allium spp., Amaranthus spp., Ammophila arenaria, pineapple (Ananas comosus), Annona spp., celery (Apium graveolens), peanut species (Arachis spp.), breadfruit species (Artocarpus spp.), asparagus (Asparagus officinalis), oat species (Avena spp.) (e.g., Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), star fruit (Averrhoa carambola), bamboo species (Bambusa sp.), wax gourd (Benincasa hispida), Brazil nut (Bertholletia excelsea), sugar beet (Beta vulgaris), Brassica spp. (e.g., Brassica napus, Brassica rapa ssp. [canola, rapeseed, turnip rape]), Cadaba farinosa, tea plant (Camellia sinensis), Canna indica, cannabis (Cannabis sativa), sativa), Capsicum spp., Carex elata, papaya (Carica papaya), Carissa macrocarpa, pecan spp.spp.), safflower (Carthamus tinctorius), chestnut spp. (Castanea spp.), kapok (Ceiba pentandra), endive (Cichorium endivia), cinnamon spp. (Cinnamomum spp.), watermelon (Citrullus lanatus), citrus spp., coconut spp. (Cocos spp.), coffee spp. (Coffea spp.), taro (Colocasia esculenta), cola spp., coriander spp., coriander spp., sativum, Hazel (Corylus spp.), Crataegus spp., Saffron (Crocus sativus), Pumpkin (Cucurbita spp.), Cucumis spp., Artichoke (Cynara spp.), Wild carrot (Daucus carota), Desmodium spp., Longan (Dimocarpus longan), Dioscorea spp., Persimmon spp., Echinochloa spp. spp.), Elaeis (e.g., Elaeis guineensis, Elaeis oleifera), finger millet (Eleusine coracana), teff (Eragrostis tef), Erianthus sp., loquat (Eriobotrya japonica), eucalyptus sp., Eugenia uniflora, buckwheat (Fagopyrum spp.), beech (Fagus spp.), fescue (Festuca arundinacea), fig (Ficus carica), Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g., Glycine max, Soja hispida),hispida or Soja max), cotton (Gossypium hirsutum), Helianthus spp. (e.g., sunflower (Helianthus annuus), daylily (Hemerocallis fulva)), Hibiscus spp., Hordeum spp. (e.g., barley (Hordeum vulgare)), sweet potato (Ipomoea batatas), walnut spp., lettuce (Lactuca sativa), Lathyrus spp. spp.), lentil (Lens culinaris), flax (Linum usitatissimum), litchi (Litchi chinensis), lotus spp., Luffa acutangula, lupin spp., Luzula sylvatica, Lycopersicon spp. (e.g., Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), pyriforme), Macrotyloma spp., Malus spp., Acerola (Malpighia emarginata), Mammea americana, Mango (Mangifera indica), Cassava spp., Sapodilla (Manilkara zapota), Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp.), Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp.spp. ), Oryza spp. (e.g., rice (Oryza sativa), Oryza latifolia)), millet (Panicum miliaceum), Panicum virgatum, passionflower (Passiflora edulis), parsnip (Pastinaca sativa), Pennisetum sp., Persea spp., parsley (Petroselinum crispum), reed canary grass (Phalaris arundinacea), Phaseolus spp. spp.), Timothy grass (Phleum pratense), Date palm (Phoenix spp.), Common reed (Phragmites australis), Nightshade (Physalis spp.), Pine spp., Pistachio (Pistacia vera), Pea spp., Poa spp., Populus spp., Prosopis spp., Cherry spp., Psidium spp., Pomegranate (Punica granatum, pear (Pyrus communis), oak (Quercus spp.), radish (Raphanus sativus), rhubarb (Rheum rhabarbarum), currant (Ribes spp.), castor bean (Ricinus communis), rubus spp., sugarcane spp., willow spp., elderberry spp., rye (Secale cereale), sesame spp., Sinapis spp. sp.), Solanum spp. (e.g. potato (Solanum tuberosum), Solanum integrifolium, or tomato (Solanum lycopersicum)), sorghum (Sorghumbicolor), Spinacia spp., Myrtaceae spp., Tagetes spp., Tamarind (Tamarindus indica), Cocoa (Theobroma cacao), Trifolium spp., Gamagrass (Tripsacum dactyloides), Triticosecale rimpaui, Triticum spp. (e.g., Triticum aestivum, Triticum durum, Triticum riveted), turgidum, Triticum hibernum, Triticum macha, Triticum sativum, einkorn (Triticum monococcum) or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis vinifera spp.), maize (Zea mays), wild rice (Zizania palustris), or jujube species (Ziziphus spp.), or cells derived from plants belonging to the superfamily of the subkingdom Chlorophyta, in particular monocotyledons and dicotyledons, ornamentals, food grains, trees or shrubs, including fodder or forage legumes; or the cell is an animal cell, said animal cell being an insect, poultry, fish or crustacean cell, or a mammalian cell, preferably a mammalian cell, optionally selected from a cell derived from a non-human primate, cow, pig, rodent, including rat or mouse, or a human cell; 18. The method of claim 17.

21. 18. The method of claim 17, wherein the modification is at least one insertion, at least one deletion, or at least one point mutation.

22. 18. The method of claim 17, wherein at least one additional effector, or a nucleic acid molecule encoding the same, is provided during steps (a) to (c), said additional effector promoting DNA repair and cell regeneration before, during or at the time of insertion of at least one nick in the genomic locus of interest at or near the at least one target site.

23. 18. The method of claim 17, wherein the method is a coordinated double-nicking method, wherein at least two Cas enzymes with nickase activity (nCas), or catalytically active fragments thereof, or at least one nucleic acid molecule encoding same, are provided in step (a); and at least two compatible guide RNAs are provided in step (c), wherein the at least two compatible guide RNAs are designed to enable coordinated action of the at least two Cas enzymes with nickase activity, such that the at least two Cas enzymes with nickase activity introduce two individual nicks at the at least one target site.

24. 24. The method of claim 23, wherein the two Cas enzymes having nickase activity, or the catalytically active fragments thereof, may be the same or different, and wherein at least one of the at least two Cas enzymes having nickase activity, or the catalytically active fragment thereof, is an engineered Cas12a enzyme having nickase activity (nCas12a) as defined in claim 1, or a catalytically active fragment thereof, or a sequence encoding the same, and wherein the nCas12a may be the same nCas12a or a different nCas12a.

25. 24. The method of claim 23, wherein two individual nicks are introduced into opposite strands within a genomic locus of interest of at least one cell or construct at or near said at least one target site, the offset being positive, negative or zero, preferably said offset being between approximately -100 bp and +100 bp.

26. 24. The method of claim 23, wherein the two Cas enzymes with nickase activity and / or the at least two compatible guide RNAs are provided separately in the form of at least one expression construct or vector, or in the form of at least one complex, or in the form of at least one nucleic acid molecule encoding same, or in the form of at least one fusion protein or at least one nucleic acid molecule encoding same.

27. 18. An edited cell, tissue, organ, material or whole organism obtained or obtainable by the method of claim 17.

28. (i) Use of at least one engineered Cas12a enzyme (nCas12a) having nickase activity as defined in claim 1, or a catalytically active fragment thereof, or at least one nucleic acid molecule encoding same, for metabolic engineering in cells comprising prokaryotic or eukaryotic cells, preferably fungal cells including yeast cells, or prokaryotic cells comprising gram-positive, gram-negative or gram-variant bacterial cells, preferably gram-negative bacterial cells, or archaeal cells, and / or for introducing nucleotide deletions or insertions or modifications in nucleic acid molecules, preferably genomes, to optimize or modify traits of plants, including modifying yield-related traits or modifying disease resistance-related traits, Optionally, the use comprises a paired nickase strategy as defined in claim 24. (ii) Use of at least one expression construct or vector as defined in claim 6, for metabolic engineering in cells comprising prokaryotic or eukaryotic cells, preferably fungal cells including yeast cells, or prokaryotic cells comprising gram-positive, gram-negative or gram-variant bacterial cells, preferably gram-negative bacterial cells, or archaeal cells, and / or for introducing nucleotide deletions or insertions or modifications in nucleic acid molecules, preferably genomes, to optimize or modify traits of plants, including modifying yield-related traits or modifying disease resistance-related traits, Optionally, the use comprises a paired nickase strategy as defined in claim 24. (iii) Use of at least one complex as defined in claim 12 or at least one nucleic acid molecule encoding same, for metabolic engineering in cells comprising prokaryotic or eukaryotic cells, preferably fungal cells including yeast cells, or prokaryotic cells comprising gram-positive, gram-negative or gram-variant bacterial cells, preferably gram-negative bacterial cells, or archaeal cells, and / or for introducing nucleotide deletions or insertions or modifications in nucleic acid molecules, preferably genomes, to optimize or modify traits of plants, including modifying yield-related traits or modifying disease resistance-related traits, Optionally, the use comprises a paired nickase strategy as defined in claim 24. (iii) Use of a fusion protein as defined in claim 13 or at least one nucleic acid molecule encoding same, for metabolic engineering in cells comprising prokaryotic or eukaryotic cells, preferably fungal cells including yeast cells, or prokaryotic cells comprising gram-positive, gram-negative or gram-variant bacterial cells, preferably gram-negative bacterial cells, or archaeal cells, and / or for introducing nucleotide deletions or insertions or modifications in nucleic acid molecules, preferably genomes, to optimize or modify traits of plants, including modifying yield-related traits or modifying disease resistance-related traits, Optionally, the use comprises a paired nickase strategy as defined in claim 24. (iv) Use of at least one adenine or cytidine base editor, or at least one base editor complex, as defined in claim 14, or at least one nucleic acid molecule encoding same, comprising: for metabolic engineering in cells comprising prokaryotic or eukaryotic cells, preferably fungal cells including yeast cells, or prokaryotic cells comprising gram-positive, gram-negative or gram-variant bacterial cells, preferably gram-negative bacterial cells, or archaeal cells, and / or for introducing nucleotide deletions or insertions or modifications in nucleic acid molecules, preferably genomes, to optimize or modify traits of plants, including modifying yield-related traits or modifying disease resistance-related traits, Optionally, the use comprises a paired nickase strategy as defined in claim 24. (v) Use of at least one prime editor or at least one prime editor complex as defined in claim 15, or at least one nucleic acid molecule encoding same, for metabolic engineering in cells comprising prokaryotic or eukaryotic cells, preferably fungal cells including yeast cells, or prokaryotic cells comprising gram-positive, gram-negative or gram-variant bacterial cells, preferably gram-negative bacterial cells, or archaeal cells, and / or for introducing nucleotide deletions or insertions or modifications in nucleic acid molecules, preferably genomes, to optimize or modify traits of plants, including modifying yield-related traits or modifying disease resistance-related traits, Optionally, the use comprises a paired nickase strategy as defined in claim 24. (vi) Use of a kit as defined in claim 16, comprising: for metabolic engineering in cells comprising prokaryotic or eukaryotic cells, preferably fungal cells including yeast cells, or prokaryotic cells comprising gram-positive, gram-negative or gram-variant bacterial cells, preferably gram-negative bacterial cells, or archaeal cells, and / or for introducing nucleotide deletions or insertions or modifications in nucleic acid molecules, preferably genomes, to optimize or modify traits of plants, including modifying yield-related traits or modifying disease resistance-related traits, Optionally, the use comprises a paired nickase strategy as defined in claim 24.

35. Use of at least one compound selected from (i) to (viii) for the manufacture of a medicament for treating or preventing a disease in a patient: (i) at least one engineered Cas12a enzyme (nCas12a) having nickase activity according to claim 1, or a catalytically active fragment thereof, or at least one nucleic acid sequence encoding same; (ii) at least one expression construct or vector according to claim 6; (iii) at least one conjugate according to claim 12 or at least one nucleic acid sequence encoding same, or a fusion protein according to claim 13 or at least one nucleic acid sequence encoding same; (iv) at least one adenine or cytidine base editor or at least one base editor complex according to claim 14, or at least one nucleic acid sequence encoding same; (v) at least one prime editor or at least one prime editor complex according to claim 15, or at least one nucleic acid sequence encoding same; (vi) the kit of claim 16; (vii) the cell of claim 7; (viii) A compound selected from the cell, tissue, organ, material or whole organism of claim 27.

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