WO2019207274A1 - Gene replacement in plants - Google Patents

Abstract

The present invention relates to a method of performing genome editing in any eukaryotic organism using homology directed repair (HDR) and RNA as the repair template. Also described are ribonucleoprotein complexes (RBC) and nucleic acid constructs encoding the RBCs as well as the use of the RBCs and nucleic acid constructs in genome editing and to perform HDR in any eukaryotic organism.

Description

Gene replacement in plants

FIELD OF THE INVENTION

The present invention relates to a method of performing genome editing in any eukaryotic organism using homology directed repair (HDR) and RNA as the repair template. Also described are ribonucleoprotein complexes (RBC) and nucleic acid constructs encoding the RBCs as well as the use of the RBCs and nucleic acid constructs in genome editing and to perform HDR in any eukaryotic organisms.

BACKGROUND OF THE INVENTION

Targeted precise genome modifications, and in particular replacements of nucleotide sequences or genes in plants and other eukaryotic organisms remains a challenge. The CRISPR/Cas9 system has been frequently used and applied to commercially valuable crops such as rice. However, due to the low frequency of homologous recombination in plants, the use of CRISPR/Cas9-mediated homologous recombination to achieve gene-site replacement or site-directed integration in crops has rarely been reported. Moreover, there have been no reports of the use of the CRISPR/Cpf1 system in gene or nucleotide replacement.

It is known that RNA transcripts may be able to serve as repair templates for homology-directed repair (HDR) of double-stranded DNA breaks (DSBs). The use of RNA transcripts as repair templates - known as RNA transcript-templated HDR (TT- HDR) has been described in yeast and human cells. However, the use of HDR-TT has not been employed in genome editing. This is mainly because DNA repair templates can be efficiently introduced into cells by transformation methods such as electroporation, microinjection or transfection. That said, in plant cells, these transformation methods are not suitable due to the presence of cell walls, especially for some crop varieties such as corn, wheat, rice and other monocots and dicots. Protoplasts can be efficiently transformed but nevertheless, regeneration of plants from protoplasts remains very inefficient (Svitashev et al. 2016). As a result, to date it remains technically very challenging to conduct HDR of double-stranded breaks (DSBs) and by extension, deliver repair templates to plant cells using the CRISPR system. This is mainly because: 1) in a plant cell, non-homologous end joining (NHEJ) of DSBs is the predominant pathway and HDR is naturally rare (Puchta H et al. 1998); and 2) as described above, the delivery of a donor repair template (DRT) into plant cells is difficult to achieve. Both particle bombardment and virus based replicons have been used to increase the availability of donor templates.

There therefore exists a need to carry out precise genome modifications, and in particular replacements of nucleotide sequences or genes in plants and other eukaryotic organisms. The present invention addresses this need.

SUMMARY OF THE INVENTION

As RNA transcripts can be abundantly produced in vivo by transcription, we hypothesised that TT-HDR could be effective for targeted precise gene editing in any eukaryotic organism, and in particular plants, if combined with a programmable nuclease. While it has been reported that a sgRNA might serve as both a guide RNA and a donor repair template (DRT) it was not clear whether the observed HDR events were actually mediated by RNA DRTs (Butt et al. 2017).

In one aspect of the invention there is provided a ribonucleoprotein complex (RBC) for use in genome editing, the complex comprising a nuclease, at least one target-DNA binding molecule and at least one RNA donor repair template. In one example, the nuclease is a CRISPR enzyme, preferably Cpf

In one embodiment, the target-DNA binding molecule is a crRNA molecule In an alternative embodiment, the target-DNA binding molecule is a sgRNA molecule. In another embodiment, the RNA repair template comprises at least one mutation compared to the target sequence.

In one embodiment, the complex is DNA-free. In another embodiment, the RNA donor repair template is single stranded.

In another aspect of the invention there is provided a nucleic acid construct comprising at least one nucleic acid sequence encoding a target-DNA binding molecule, at least one nucleic acid sequence encoding a donor repair template and at least one nucleic acid sequence encoding a ribozyme. In one embodiment, the construct comprises at least one regulatory sequence operably linked to at least one of the nucleic acid sequences encoding the target-DNA binding molecule, the donor repair template and the ribozyme. In another embodiment, the construct comprises a single regulatory element operably linked to all of the nucleic acid sequences encoding the target-DNA binding molecule, the donor repair template and the ribozyme. In one example, the regulatory sequence is a promoter, preferably U3 or T7.

In another embodiment, the construct comprises at least one target-DNA binding molecule that is flanked by a 5’ and 3’ nucleic acid sequence that encodes a ribozyme and a donor repair template that is flanked by a 5’ and 3’ nucleic acid sequence that encodes a ribozyme. In one embodiment, the target-DNA binding molecule is a crRNA molecule.

In one aspect of the invention there is provided a vector comprising the nucleic acid construct described above. In an alternative aspect there is provided a host cell comprising the nucleic acid construct or the vector described above. In one example, the cell is a eukaryotic cell, preferably a plant cell.

In another aspect of the invention there is provided a transgenic organism expressing the nucleic acid construct or the vector described above. In one example, the organism is a plant.

In an alternative aspect of the invention there is provided a method of producing a transgenic organism as defined above, the method comprising:

(a) selecting a part of an organism;

(b) transfecting at least one cell of the part of the organism of part (a) with the nucleic acid construct or the vector described above; and

(c) regenerating at least one organism derived from the transfected cell or cells In one example, the organism is a plant.

In yet another aspect of the invention there is provided a method of performing genome editing in an organism, the method comprising introducing an RBC as defined above, or introducing and expressing a nucleic acid construct or a vector as described above into said organism. In one example the organism is a eukaryote, preferably a plant.

In an alternative aspect of the invention there is provided a method of performing homology directed repair (HDR) in an organism, the method comprising introducing an RBC as defined above, or introducing and expressing a nucleic acid construct or a vector as described above into said organism. In one example the organism is a eukaryote, preferably a plant.

In another aspect of the invention there is provided the use of the RBC as described above or the nucleic acid construct described above in homology-directed repair. In an alternative aspect there is provided the use of the RBC as described above or the nucleic acid construct described above in genome editing.

DESCRIPTION OF THE FIGURES

The invention is further described in the following non-limiting figures:

Figure 1 shows RNA transcripts-templated homologous directed repair (TT-HDR) of DSBs generated by the LbCpfl nuclease (a) A schematic description of precise gene replacement in rice using TT-HDR. Part of the wild type ALS gene is removed by LbCpfl and a couple of crRNAs. The LbCpfl target sequences are underlined and the PAM sites are shown in red. The wild type ALS fragment is replaced by a mutant version that introduces changes of two discrete amino acid residuals. The PAM sites and an EcoRV site are changed to prevent the replacement from further cleavage by LbCpfl/crRNAs and to facilitate detection of gene replacement events, respectively. ALStestF/ALStestF is the primer set designed to amplify the wild type ALS fragment, whereas ALStestF/T2MR primer set is designed for allelic-specific amplification of the edited ALS gene (b) The features and sequence of the donor repair template (DRT). DRT consists of the left arm (blue), right arm (blue), and the mutated ALS fragment. Note that two point mutations of“t” which are shown in lower case in red are designed targeted substitutions that lead to W548L and S627I changes at protein level, respectively. Both the two triplet codons and the encoded two mutated amino acids are marked in green. EcoRV site is abolished by replacing the“T” with a“C”, but this mutation does not change the protein sequence. Both LbCpfl targets are mutated synonymously so that LbCpfl and the crRNAs can no longer cut the targets. The synonymous mutations in DRT are shown in lower case in red as well (c) Production of crRNAs and the DRT RNA transcripts in vitro. An array of ribozyme-flanked units under the control of the T7 promoter. Upon transcription in vitro by using T7 RNA polymerase, the two crRNAs and RNA transcripts donor repair template are released by self cleavage of the ribozyme units automatically from the primary transcripts. HH and HDV are ribozyme units. HH: Hammerhead ribozyme. HDV: hepatitis delta virus ribozyme. (d) CRISPR/Cpf1-RNP-mediated TT-HDR in rice calli. The in vitro transcription products are digested with DNase I to remove any DNA templates and further purified by column. Recombinant LbCpfl proteins and DNA-free crRNAs and RNA DRT transcripts are pre-assembled to form DNA-free RNP-RNA DRT complex in vitro. These active RNP-RNA DRT complexes are delivered via bombardment to rice calli. PCR products are amplified by allele-specific primer set ALStestF/T2MR as described in Figure 1a. PCR products are then sequenced to determine whether the delivered DNA-free RNP-RNA DRT complex can use RNA transcripts as template for repair of DSBs generated by CRISPR/Cpfl in vivo. Sequence results of the representative TT- HDR events indicate that the callus RDR35 had undergone precise HDR, whereas the callus RDR41 has undergone partial HDR. The sequences shadowed in yellow and blue represent the same bases as that of wild type and the designed DRT, respectively. Specifically, the sequences shadowed in red indicate the designed targeted substitutions.

Figure 2 shows a comparison of RNP-mediated HDR efficiencies of various sources of donor repair templates through ddPCR. (a) RNP-mediated HDR using various sources of donor repair templates (DRTs). In some of the experiments, the DRT fragments were digested by DNase I, RNase H, RNase A, or a combination of both enzymes. Seven different sets of RNP experiments were performed to determine HDR efficiencies of various DRTs. DRTs used were RNA transcripts only, both DNA template and RNA transcripts, single-strands DNA (ssDNA), and single-strands (ssRNA). (b) and (c) Two-dimensional plots of droplets measured for fluorescence signal (amplitude indicated on y-axis) emitted from edited OsALS gene (FAM™ labeled; positive droplets are in blue) or the wild type OsALS gene (HEX™ labeled; positive droplets are in green) in target 1. Negative droplets are shown in black. Droplets (heterozygous events) containing both fluorescent probes are orange (b) Single-stranded (ssRNA) serves as repair template. In the experiment IV, bombardment of the RNP complex using the transcribed RCR1-RCR2 treated with DNase I and ssRNAs leads to HDR events, (c) Single stranded DNA (ssDNA) is an efficient HDR repair template (Experiment VI). (d) and (e) Box-and-whisker plots present the HDR efficiencies of different sources of DRTs at target 1 and target 2, respectively. The graph consists of median values, lower and upper quartile, and outer standard deviations. The Roman numerals under the X-axis correspond to the experiments described in (a). Digestions with both RNase and DNase (III, V, VII) abolish HDR. Either DNA or RNA can serve as repair template for HDR (I, II, IV, and VI). All experiments were performed with nine biological replicates. T1 : target 1. T2: Target 2. **p<0.01.

Figure 3 shows the generation of stable precisely edited rice plants through TT-HDR (a) Ribozyme-based strategy for producing crRNAs and DRT for TT-HDR in rice. (a-1) A schematic presentation of the RDR vector. The two ribozyme-crRNA-ribozyme (RCR) units and DRT ribozyme unit are placed in a tandem array, which is under the control of the OsU3 promoter and NOS terminator. Transcripts undergo self-cleavage to release the mature crRNAs and DRT. (a-2) PCR and restriction enzyme digestion (PCR/RE) analyses of the different genotypes. PCR products amplified by primers ALStestF/R were digested with EcoRV (GACATC). M: DL2000; WT refers to wild-type without EcoRV digestion. WT/+: the PCR products of wild-type were digested with EcoRV, resulting in 481 bp and 322 bp fragments. Successful TT-HDR leads to EcoRV-resistant bands (a-3) Sequence analyses of the TT-HDR events. The line 288- 6 has one allele with precise TT-HDR, while the other allele is wild-type. The line 289-4 and 293-1 have one allele with partial HDR, while the other is wild-type. The sequences shadowed in yellow and blue represent the same bases with that of the wild-type and designed donor repair template, respectively. Specifically, the sequences shadowed in red indicate the expected targeted substitution (b) Generation of stable precisely edited rice lines using the TDT vector through CRISPR/Cpf1 -mediated TT- HDR. (b-1) A schematic show of the TDT vector for TT-HDR-mediated gene replacement. The RCR units and target-DRT-target (TDT) in a tandem array are under the control of the OsU3 promoter and terminated by the NOS terminator. The two crRNA transcripts undergo self-cleavage to release the mature crRNAs. LbCpf-crRNAs can release the RNA transcripts of DRT. (b-2) PCR/RE analyses of the different genotypes. PCR products amplified by primers ALStestF/R were digested with EcoRV (GACATC). M: DL2000; WT: wild-type. WT/+: EcoRV cuts the PCR products of wild- type, resulting in 481 bp and 322 bp fragments. EcoRV failed to digest the PCR products if HDR was successful (b-3) Representative sequences of the different genotypes. The lines 183-2, 185-5 and 278-4 have one allele with precise HDR, while the other is a wild-type. The line 198-1 has one allele with precise HDR and the other has partial HDR. The line 193 has one allele with partial HDR followed by a 28 bp deletion. The sequences shadowed in yellow and blue represent the same bases with that of the wild-type and designed donor repair template, respectively. Specifically, the sequences shadowed in red indicate the expected targeted substitution. In d#, the # refers to the number of bp deleted from the target sites. Different numbers of lines indicate independent lines developed from resistant calli.

Figure 4 shows the detection of HDR events in rice calli by allelic-specific PCR.

Figure 5 shows a schematic of the positions of the primer sets used in ddPCR analysis.

Figure 6 shows a one-dimensional plot of droplets measured for fluorescence signal around target 1.

Figure 7 shows maps of the vectors used in this study.

Figure 8 shows sequence chromatograms of stable rice lines with edited ALS in TO generation.

Figure 9 shows the raw data of ddPCR analyses around target 1 and target 2.

Figure 10 shows the characterization and genotyping of CRISPR/LbCpfl -mediated HDR plants in TO generation.

Figure 11 shows the characterization and genotyping of regenerated plants from Agrobacterium-mediated transformation in TO generation.

Figure 12 shows the segregation analyses of HDR events in T1 seedlings.

Figure 13 shows an analysis of potential off-target effects. Figure 14 shows a schematic of an example of a linearized control construct of the invention. LbCpfl was inserted downstream of the ZmUbi promoter. A Nos terminator was placed at the end of the ORF of LbCpfJ HH and HDV are ribozyme units. The two crRNAs ribozyme units are under the control of OsU3 promoter. Upon transcription, the two crRNAs can be released through ribozyme-catalyzed self-cleavage. Armed donor was designed as a template for HDR and when it is transcribed, it can be released by LbCpfl/crRNAs.

Figure 15 shows the primer sequences used herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, recombinant DNA technology, and bioinformatics which are within the skill of the art. Such techniques are explained fully in the literature.

As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or“gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.

The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

The present invention relates to a method of performing genome editing in any eukaryotic organism using homology directed repair (HDR) and RNA as the repair template. Genome editing is a form of genetic engineering in which DNA is inserted, deleted or replaced in a plant’s genome using engineered nucleases or site-specific nucleases (SSN) to create site-specific double-strand breaks (DSB) in the genome, that are then repaired using homologous recombination (HDR) or non-homologous end-joining (NHEJ) to form targeted mutations.

To achieve effective genome editing via introduction of site-specific DNA DSBs, four major classes of customisable DNA binding proteins can be used: meganucleases derived from microbial mobile genetic elements, ZFN nucleases based on eukaryotic transcription factors, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, and the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats).

A preferred genome editing method that can be used according to the various aspects of the invention is CRISPR. The use of this technology in genome editing is well described in the art, for example in US 8,697,359 and references cited herein. In short, CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR- associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA). Three types (I- III) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers). The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct a CRISPR enzyme, such as a Cas or Cpf1 nucleases to the target site (protospacer). The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson- Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double- stranded break within the protospacer.

One major advantage of the CRISPR-Cas9 system, as compared to conventional gene targeting and other programmable endonucleases is the ease of multiplexing, where multiple genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different gene. In addition, where two sgRNAs are used flanking a genomic region, the intervening section can be deleted or inverted (Wiles et al. , 2015).

Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used.

Alternatively, Cpf1 , which is another Cas protein, can be used as the endonuclease. Cpf1 differs from Cas9 in several ways; Cpf1 requires a T-rich PAM sequence (TTTV) for target DNA recognition, Cpf1 does not require a tracrRNA, and as such only crRNA is required and unlike Cas9, the Cpf1 -cleavage site is located distal and downstream relative to the PAM sequence in the protospacer sequence (Li et al. 2017). Furthermore, after identification of the PAM motif, Cpf1 introduces a sticky-end-like DNA double-stranded break with several nucleotides of overhang. As such, the CRISPR/Cpfl system consists of a Cpf1 enzyme and a guide RNA (or crRNA).

The single guide RNA (sgRNA) is the second component of the CRISPR/Cas system that forms a complex with the nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20 bp. In plants, sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3.

Cpf1 and Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.

It is an object of the present invention to provide a CRISPR system (preferably a CRISPR/Cpf)-mediated homologous recombination method using an RNA transcript as a repair template.

Accordingly, in one aspect of the invention there is provided a ribonucleoprotein complex (RBC) for use in genome editing, the complex comprising a nuclease, at least one target-DNA binding molecule and at least one RNA donor repair template.

In one embodiment of the invention, the RBC does not comprise any DNA - i.e. it is DNA-free. This may be achieved by treating the transcribed target-DNA binding molecule (crRNA) and RNA donor repair template with a deoxyribonuclease, such as DNasel before forming the RBC. Using DNA-free ribonucleoproteins in gene replacement whereby the RNA transcripts serve as templates for DNA repair can be particularly important for gene therapy in human cells as the method does not require the insertion or integration of any foreign gene or gene fragments into a genome. Similarly, for crop species, it allows gene replacement without any transgenes or fragments being integrated into the genome thereby avoiding any biosafety concerns over edited crop plants. Alternatively, the RBC may additionally comprise a DNA donor- repair template (i.e. a DNA DRT). More preferably the DNA DRT may be single- stranded. By“target DNA-binding molecule” is meant any molecule, preferably an RNA molecule, that can bind at least one target nucleotide, preferably a target nucleotide sequence. In one embodiment, the target DNA-binding molecule comprises or consists of a crRNA nucleic acid or a crRNA molecule, most preferably a crRNA molecule. Alternatively, the target DNA-binding molecule may be a sgRNA as described herein.

By “crRNA” or CRISPR RNA is meant the sequence of RNA that contains the protospacer element. By“protospacer element” is meant the portion of crRNA that is complementary to the genomic DNA target sequence, usually around 20 nucleotides. In one embodiment, the crRNA. In a preferred embodiment the RBC comprises more than one crRNA molecule, preferably two crRNA molecules. Two or more crRNA molecules may be useful (i.e. a first and second crRNA molecule as referred to herein), for example, where it is desirable to introduce a fragment of nucleic acid sequence or more than one mutation (e.g. a double mutation) into a target sequence or mutations into two target sequences.

In a further embodiment, the crRNA may include additional nucleotides that are complementary to the tracrRNA. By“tracrRNA” (transactivating RNA) is meant the sequence of RNA that hybridises to the crRNA and binds a CRISPR enzyme thereby activating the nuclease complex to introduce double-stranded breaks at specific sites within the genomic sequence. This may also be known as a spacer or targeting sequence. In this embodiment, where the crRNA includes additional nucleotides, the nuclease is a CRIPSR enzyme, and more specifically is a Cas protein, as discussed below.

By“sgRNA” (single-guide RNA) is meant the combination of tracrRNA and crRNA in a single RNA molecule, preferably also including a linker loop (that links the tracrRNA and crRNA into a single molecule).“sgRNA” may also be referred to as“gRNA" and in the present context, the terms are interchangeable. The sgRNA or gRNA provide both targeting specificity and scaffolding/binding ability for a Cas or Cpf1 nuclease. A gRNA may refer to a dual RNA molecule comprising a crRNA molecule and a tracrRNA molecule. Accordingly, in an alternative embodiment, the target-DNA binding molecule may be a sgRNA molecule. By“RNA donor repair template” or RNA DRT is meant an RNA molecule comprising one or more, preferably a plurality of ribonucleotides. The RNA molecule may correspond to a gene or a part of a gene (a gene fragment) and may or may not comprise one or more mutations (such as additions, deletions or substitutions of one or more base) compared to the equivalent wild-type or control target sequence. This sequence may be referred to herein as a“repair template”. Preferably the RNA DRT comprises all the necessary elements to introduce a specific mutation or sequence into any target sequence, preferably using homology-directed repair or HDR. In one embodiment, the DRT is additionally flanked by at least one, but preferably a left and right arm each that are identical or substantially identical to a sequence within the target sequence. The RNA DRT may be double or single-stranded, but preferably single stranded. In a preferred embodiment, the nucleotide sequence encoding the RNA DRT does not comprise a PAM (protospacer-adjacent) motif (that is, the nucleotide sequence comprises one or more mutations compared to the wild-type or control sequence in at least one PAM motif such that once genome modification is complete, the newly inserted fragment or sequence cannot be targeted by the crRNAs or sgRNAs). The PAM motif may be NGG (where the CRISPR enzyme is a Cas protein) or TTTV (where the CRISPR enzyme is Cpf1). Other PAM motifs would be well known to the skilled person.

Where the RBC also comprise a DNA donor-repair template this may also comprise one or more mutations as described above compared to the (genomic) target sequence.

In a further embodiment, the nucleotide sequence encoding the RNA DRT comprises one or more mutations in a restriction site or a restriction recognition site (i.e. sites recognised by a restriction enzyme), whereby the mutation is any mutation that prevents the binding and cleavage of the DNA strand by a restriction enzyme. In one example the restriction enzyme is EcoRV and the restriction site is GATATC, although the sequence of other restriction sites would be well known to the skilled person.

In another embodiment, the nucleotide sequence encoding the RNA DRT and the RNA sequence of the DRT may comprise one or more target crRNA sequences. As explained above, a crRNA molecule binds to a target sequence in, for example a genomic sequence. In one embodiment the DRT sequence may additionally comprise one or more of these target sequences. In particular where the RBC comprises a first and second crRNA molecule, the DRT sequence may comprise the target sequence of the first and/or second crRNA molecule. More preferably these target sequences are 5’ and/or 3’ of the repair sequence. As explained below, where the nuclease is a CRISPR enzyme such as Cpf1 the addition of these crRNA target sequences to the RNA DRT sequence permits the self-processing of the RNA sequence by the CRISPR enzyme. As used herein, an RNA DRT comprising crRNA target sequences may be referred to as a“TDT” sequence.

By“nuclease” is meant any enzyme that comprises a DNA cleavage domain. In other words, an enzyme that can cleave at least one DNA strand. In one embodiment, the nuclease is an endonuclease, more preferably a CRISPR enzyme. In one embodiment the nuclease preferably creates cohesive ends or“sticky ends” (5’ and/or 3’ overhangs of one or more nucleotides). The nuclease may be a polypeptide or a nucleic acid or vector that encodes for the nuclease. If the latter, by“nuclease” may also be meant a nucleic acid construct comprising a nucleic acid encoding for a nuclease, wherein preferably the nucleic acid is operably linked to a regulatory sequence. A regulatory sequence is described herein.

In one embodiment, the nuclease is a CRISPR enzyme.

By“CRISPR enzyme” is meant an RNA-guided DNA endonuclease that can associate with the CRISPR system. Specifically, such an enzyme binds to the crRNA sequence. In a preferred embodiment, the CRISPR enzyme is Cpf1 (CRISPR-associated endonuclease in Prevotella and Francisella 1). In one example the Cpf1 may be from Lacnospiraceae bacterium ND2006, and in a particularly preferred embodiment the nucleic acid sequence may encode a sequence as defined in SEQ ID NO: 7 or a variant thereof. In another embodiment, the protein sequence may comprise SEQ ID NO: 8 or a variant thereof. In another example the Cpf1 may be from Acidaminococcus (AsCpfl). In a preferred embodiment, the AsCpfl nucleic acid sequence encodes a protein as defined in SEQ ID NO: 14 or a variant thereof. More preferably the nucleic acid sequence comprises a sequence as defined in SEQ ID NO: 13 or a variant thereof. In a further alternative embodiment, the Cpf1 may be from Francisella novicida (FnCpfl). Preferably, the FnCpfl nucleic acid sequence encodes a protein as defined in SEQ ID NO: 12 or a variant thereof. More preferably the nucleic acid sequence comprises a sequence as defined in SEQ ID NO: 11 or a variant thereof. In one embodiment, the Cpf1 protein may be modified to increase activity. In one particular embodiment, the Cpf1 crRNA may be modified to increase the gene editing efficiency of Cpf1 (see Park et al, 2018); preferably where the Cpf1 crRNA is modified by extending the length of the crRNA at the 5’ end by one or more nucleotides, more preferably where the 5’ end of the crRNA is extended by 59 nucleotides. In a particularly preferred embodiment, the extended 5’ end may also be chemically modified to enhance serum stability, where possible chemical modifications include the introduction of 2’ O-methyl modifications, phosphorothioate linkages, and deoxynucleotide ribose groups. In a further embodiment, the Cpf1 protein may be codon optimised specific for the target organism in question.

Accordingly, the Cpf1 protein may be selected from LbCpfl , AsCpfl and FnCpfl as described above.

In one embodiment, where the CRISPR enzyme is a Cpf1 protein the target DNA- binding molecule is a crRNA molecule (and more preferably does not include the additional nucleotides described above).

In an alternative embodiment, the CRIPSR enzyme is a Cas protein (“CRISPR associated protein), preferably Cas9. Again, the Cas9 protein may also be modified to improve activity. For example, the Cas9 protein may comprise the D10A amino acid substitution, this nickase cleaves only the DNA strand that is complementary to and recognized by the crRNA. In an alternative embodiment, the Cas9 protein may alternatively or additionally comprise the H840A amino acid substitution, this nickase cleaves only the DNA strand that does not interact with the sRNA. In this embodiment, Cas9 may be used with a pair (i.e. two) sgRNA molecules (or a construct expressing such a pair) and as a result can cleave the target region on the opposite DNA strand, with the possibility of improving specificity by 100-1500 fold. In a further embodiment, the Cas9 protein may comprise a D1135E substitution. The Cas 9 protein may also be the VQR variant. Alternatively, the Cas9 protein may be xCas9 (a Streptococcus pyogenes variant that can recognise a broad range of PAM sequences including NG, GAA and GAT). In another alternative, the Cas9 variant is SpCas9-NG (with a relaxed preference to the third nucleotide of the PAM motif, such that the variant can recognise sequences where the PAM motif is NGN rather than NGG). In one embodiment, where the CRISPR enzyme is a Cas protein the target DNA- binding molecule is a sgRNA molecule.

Other examples of CRISPR enzymes include C2c1 , C2c2 and C2c3 or variants thereof. In a further preferred embodiment, the CRISPR enzyme comprises a nuclear localisation signal or NLS to ensure the sequence remains in the nucleus. In particular, the sequence may be a classical or non-classical NLS. An example of a classical NLS is PKKKRKV (SEC ID NO: 17), although the skilled person would be aware of other suitable NLS sequences.

In a further embodiment, the RBC does not comprise a reverse transcriptase enzyme.

In another aspect of the invention there is provided a nucleic acid construct comprising at least one nucleic acid sequence encoding a target-DNA binding molecule as defined above and/or at least one nucleic acid sequence encoding a donor repair template as defined above. In a preferred embodiment, the nucleic acid construct comprises at least one, preferably two or at least two nucleic acid sequences encoding a target-DNA binding molecule and at least one nucleic acid sequence encoding a donor repair template. In a further preferred embodiment, the nucleic acid construct further comprises at least one nucleic acid sequence encoding a CRISPR enzyme as described above.

Preferably, the construct also comprises at least one regulatory sequence operably linked to at least one of the nucleic acid sequences encoding the target-DNA binding molecule and the donor repair template. In a further embodiment, where the nucleic acid construct comprises a CRISPR enzyme, the regulatory sequence may be additionally operably linked to the CRISPR enzyme. Alternatively, the CRISPR enzyme may be operably linked to a second regulatory sequence. The term "operably linked" as used throughout refers to a functional linkage between the promoter sequence and one or more nucleotide sequences of interest, such that the promoter sequence is able to initiate transcription of the nucleotide sequence(s).

In one embodiment, the construct comprises a single regulatory sequence such that all sequences on the construct are operably linked to the single regulatory sequence - such as all of the nucleic acid sequences encoding the target-DNA binding molecule, the donor repair template and the CRISPR enzyme.

In one embodiment, the regulatory sequence is a promoter. According to all aspects of the invention, the term "regulatory sequence" is used interchangeably herein with "promoter" and all terms are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term "regulatory sequence" also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.

The term "promoter" typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene or nucleotide sequence and which is involved in the binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue- specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences.

In a preferred embodiment, the promoter is a constitutive promoter, strong promoter or tissue-specific promoter.

A "constitutive promoter" refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Examples of constitutive promoters include the cauliflower mosaic virus promoter (CaMV35S or 19S), rice actin promoter, maize ubiquitin promoter, polyubiquitin (UBQ10) promoter, rubisco small subunit, maize or alfalfa H3 histone, OCS, SAD1 or 2, GOS2 or any promoter that gives enhanced expression. A "strong promoter" refers to a promoter that leads to increased or overexpression of the target gene. Examples of strong promoters include, but are not limited to, CaMV- 35S, CaMV-35Somega, Arabidopsis ubiquitin UBQ1 , rice ubiquitin, actin, Maize alcohol dehydrogenase 1 promoter (Adh-1), AtPykIO, BdEFIa, FaRB7, FMDS2, HvPhtl .1 , LjCCaMK, MtCCaMK, MtlPD3, MtPT 1 , MtPT2, OsAPX, OsCd , OsCCaMK, OsCYCLOPS, OsPGDI , OsR1G1 B, OsRCc3, OsRS1 , OsRS2, OsSCPI , OsUBI3, SbCCaMK, SiCCaMK, TobRB7, ZmCCaMK, ZmEFIa, ZmPIP2.1 , ZmRsyn7, ZmTUBIa, ZmTUB2a and ZmUBI.

Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, for example the egg-specific YAO promoter.

For the identification of functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter may be analysed for example by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant. Suitable well-known reporter genes are known to the skilled person and include for example beta-glucuronidase or beta- galactosidase.

In a preferred embodiment, the promoter may be a polymerase promoter, preferably selected from a T7 promoter (preferably as defined in SEQ ID NO: 9 or a variant thereof), a U3, preferably rice U3 (referred to herein as“OsU3”)(preferably as defined in SEQ ID NO: 10 or a variant thereof), a U6 or Pol II promotor such as ubiquitin or actin. In particular, the U3 promotor belongs to the pol III RNA polymerase, which is used to transcribe small RNA molecules. Use of this promoter with the constructs of the invention will mean that the transcripts remain in the nucleus. Where the target organism is an animal cell, the promoter may be a U3 or U6 promoter.

As used herein a“target sequence” may refer to any nucleic acid sequence or gene, genomic or non-genomic that could possibly be and/or would be of value to genetically modify or mutate. In a preferred embodiment the construct comprises at least one sequence that allows self-cleavage/self-processing of the target-DNA binding molecule and/or DRT upon transcription.

In one example the construct comprises at least one nucleic acid sequence that encodes a ribozyme enzyme. Preferably the construct comprises at least one target- DNA binding molecule that is flanked by a 5’ and 3’ nucleic acid sequence that encodes a ribozyme enzyme and a donor repair template that is flanked by a 5’ and 3’ nucleic acid sequence that encodes a ribozyme enzyme, as shown, for example in Figure 1c. As described in the examples, transcription of the construct will result in at least one ribozyme-crRNA-ribozyme unit (referred to herein as a“RCR”), preferably two, and a ribozyme-DRT-ribozyme unit (referred to herein as a “RDR”). Once transcribed, the primary transcripts will undergo self-catalysed cleavage to generate either a crRNA or a RNA DRT. In a further embodiment, where the nucleic acid construct further comprises a CRISPR enzyme, the CRISPR enzyme may be also be flanked by at least one, preferably two (a 5’ and 3’) ribozyme enzyme sequences (also referred to herein as a“ribozyme unit”).

In one example, the ribozyme enzyme may be selected from a Hammerhead (HH) ribozyme unit and/or a hepatitis delta virus (HDV) ribozyme unit.

In one embodiment, the sequence of the HH (Hammerhead) ribozyme is: M¹ M²M³M⁴M⁵M⁶CTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTC (SEQ ID NO: 15) or a variant thereof as described herein in which the first six nucleotides (M¹ M²M³M⁴M⁵M⁶) of the Hammerhead (HH) ribozyme are preferably complementary to the first six nucleotides of the sequence between HH and HDV units. In a further embodiment, the sequence of hepatitis delta virus (HDV) ribozyme is: GGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGG CATGGCGAATGGGAC (SEQ ID NO: 16) or a variant thereof as described herein.

In a preferred embodiment, the HH ribozyme sequence is the 5’ flanking sequence, and the HDV ribozyme sequence is the 3’ flanking sequence, as shown in Figure 1 c. In an alternative embodiment, the HDV sequence is the 5’ flanking sequence and the HH sequence is the 3’ flanking sequence. In an alternative embodiment, a tRNA sequence or ribosomal skipping sequence or direct repeat (DR) sequence may be used in place of a ribozyme sequence. Accordingly, in one example, the nucleic acid construct comprises at least one target- DNA binding molecule that is flanked by a 5’ and 3’ nucleic acid sequence that encodes a tRNA or a ribosomal skipping sequence and a donor repair template that is flanked by a 5’ and 3’ nucleic acid sequence that encodes a tRNA or a ribosomal skipping sequence or a DR sequence. Similarly where the nucleic acid construct also comprises a CRISPR enzyme, the CRISPR enzyme may also be flanked by at least one, preferably two (a 5’ and 3’) tRNA or ribosomal skipping sequence.

To allow two proteins to be expressed as individual proteins from a single mRNA molecule, ribosomal skipping sequences may be added to the 5’ and/or 3’ end of the individual proteins (for example, the target-DNA binding molecule or the DRT). During translation, when the ribosome encounters a ribosomal skipping sequence it is prevented from creating the peptide bond with the last proline in the ribosomal skipping sequence. As a result, translation is stopped, the nascent polypeptide is released and translation is re-initiated to produce a second polypeptide. This results in the addition of a C-terminal ribosomal skipping sequence (or the majority of such a sequence) to the first polypeptide chain, and an N-terminal proline to the next polypeptide. Accordingly, in a further embodiment, the nucleic acid construct comprises at least one ribosomal skipping sequence. In one example, the ribosomal skipping sequence is a 2A-like peptide.

Alternatively, tRNA sequences may be used to allow multiple RNAs to be produced from a single engineered polycistronic gene consisting of tandemly arrayed tRNA-RNA units. After the polycistronic gene is transcribed by endogenous transcriptional machinery, the endonucleases RNAse P and RNAse Z (or RNAse E in bacterium) recognise and specifically cleave the tRNAs at specific sites at the 3’ and 5’ ends, releasing mature RNAs and tRNAs. Advantageously, tRNAs and its processing system are virtually conserved in all living organisms and therefore this method can be used in all known species. In particular, this technology can allow for the production of multiple excised mature gRNAs which can direct Cas9 to multiple targets, wherein the polycistronic gene contains tandemly arrayed tRNA-gRNA units, where each gRNA contains a target-specific spacer and a conserved gRNA scaffold (see Xie et al, 2015). Accordingly, in a further embodiment, the nucleic acid construct comprises at least one tRNA sequence.

In a further alternative to the use of ribozyme sequences, where the target-DNA binding molecule is at least one crRNA molecule and the CRISPR enzyme is Cpf1 , the DRT sequence may be flagged by a 5’ and/or 3’ crRNA target sequence including the PAM motif. As a result, the RNA transcripts of the at least one donor repair template (DRT transcripts) can be released by Cpf1/crRNAs once transcribed. This is possible as Cpf1 has both DNA and RNA cleavage activity.

In a further embodiment, the construct comprises at least one sequence to prevent export of the transcribed sequences (such as the target-DNA binding molecule and the RNA donor repair template) into the cytosol.

In a further embodiment, the construct may further comprises at least one, preferably two terminator sequences, which marks the end of the operon causing transcription to stop. In one example, the nucleic acid construct can be considered to comprise a first and second expression cassette. The first expression cassette comprises a regulatory sequence, at least one DNA-binding sequence (preferably two) and/or a DRT sequence, as defined herein where the first expression cassette is terminated by a first termination sequence. The second expression cassette comprises a regulatory sequence, a CRISPR sequence and/or a DRT sequence wherein the second expression cassette is terminated by a second terminator sequence.

The first and second terminator sequences can be the same or different. A suitable terminator sequence would be well known to the skilled person, and may in one example, be Nos.

In another aspect of the invention, there is provided a vector or expression vector comprising the nucleic acid construct described herein. In one embodiment, the vector backbone is pCXUN.

In another aspect of the invention there is provided a host cell comprising the nucleic acid construct or the vector. The host cell may be a prokaryotic or eukaryotic cell. Preferably the cell is a mammalian, bacterial or plant cell. Most preferably the cell is a plant cell.

In another aspect of the invention there is provided a transgenic organism where the transgenic organism expresses the nucleic acid construct or vector. Again, the organism is any prokaryote or eukaryote, but in a preferred embodiment, the organism is a plant.

In one embodiment, the progeny organism is transiently transformed with the nucleic acid construct or vector. In another embodiment, the progeny organism is stably transformed with the nucleic acid construct described herein and comprises the exogenous polynucleotide which is heritably maintained in at least one cell of the organism. The method may include steps to verify that the construct is stably integrated. Where the organism is a plant, the method may also comprise the additional step of collecting seeds from the selected progeny plant.

In a further aspect of the invention there is provided a method of producing a transgenic organism as described herein. In a different aspect there is provided a method of producing a gene-edited organism. In either aspect the method comprises at least the following steps:

a. selecting a part of the organism;

b. transfecting at least one cell of the part of the organism of part (a) with the nucleic acid construct or the vector; and

c. regenerating at least one organism derived from the transfected cell or cells.

Transformation or transfection methods for generating a transgenic organism of the invention are known in the art. Thus, according to the various aspects of the invention, a nucleic acid construct as defined herein is introduced into an organism and expressed as a transgene. The nucleic acid construct is introduced into said organism through a process called transformation. The term “transfection”, "introduction" or "transformation" as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Such terms can also be used interchangeably in the present context. Where the organism is a plant, tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets in plants include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). Mammalian cell starting materials include but are not limited to mouse connective tissue, mouse fibroblasts, mouse embryonic stem cells, mouse monocytes, mouse macrophages, mouse spleen cells, rat fibroblasts, rat hepatomas, human lymphomas, human keratinocytes, human small cell lung cancer cells and human embryonic kidney cell HEK293 cell lines. The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed cell may then be used to regenerate a transformed organism in a manner known to persons skilled in the art.

Transformation of plants and animals is now a routine technique in many species. Advantageously, any of several transformation methods may be used to introduce the RBC or nucleic acid construct as described herein into a suitable ancestor cell in any eukaryote. In particular, various transfection techniques exist for mammalian cells and the method of choice is a trade-off between a high transfection efficiency, low cell toxicity, minimal physiological effects and a simple reproducible method. The methods described for the transformation of an organism’s cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the target organism, particle gun bombardment, transformation using viruses or pollen and microprojection. Chemicals transformation methods include the use of chemicals with a positive charge to form a nucleic acid/chemical complex for subsequent cell uptake (Cationic polymer, Calcium phosphate and Catioinc lipid). Physical transformation methods include direct injection, particle gun bombardment, electroporation, laser-irradiation and sonoporation. Methods may be also selected from the calcium/polyethylene glycol method for protoplasts, electroporation (of protoplasts where the organism is a plant), microinjection into cell material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.

In one embodiment, the preferred method of transformation is particle bombardment or biolistics. For ribonucleoprotein (RNP) delivery by particle bombardment, a CRISPR enzyme, such as Cas or Cpf1 as described above and the transcribed crRNA(s) or sgRNAs together with at least one DRT are delivered in the form of a single RNP complex into cells.

To select transformed plants, the plant material obtained in the transformation is subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker or expression of a constitutively expressed reporter gene, as described above. Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern blot analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western blot analysis, both techniques being well known to persons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non- transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion). In a further aspect of the invention, there is provided an organism, preferably a plant obtained or obtainable by the methods described herein.

In another aspect of the invention there is provided a method of performing genome editing in an organism, the method comprising introducing an RBC as defined above or introducing and expressing a nucleic acid construct or vector as described above into a target organism.

In yet another aspect of the invention, there is provided a method of performing homology directed repair (HDR) in an organism, the method comprising introducing an RBC as defined above or introducing and expressing a nucleic acid construct or vector as described above into a target organism.

In a preferred embodiment of the above methods, the method may additionally comprise introducing a CRISPR enzyme or introducing and expressing into the target organism a second nucleic acid construct comprising a nucleic acid sequence encoding a CRISPR enzyme, preferably Cpf1 as described herein.

In a further aspect of the invention there is provided the use of any of the RBCs, nucleic acid constructs or vectors described herein for genome editing or modification of a target sequence in a eukaryotic organism or to perform HDR in a eukaryotic organism.

The term “organism” as used herein refers to any eukaryotic organism. Some examples of eukaryotes include a human, a non-human primate / mammal, a livestock animal (e.g. cattle, horse, pig, sheep, goat, chicken, camel, donkey, cat, and dog), a mammalian model organism (mouse, rat, hamster, guinea pig, rabbit or other rodents), an amphibian (e.g., Xenopus), fish, insect (e.g. Drosophila), a nematode (e.g., C. elegans), a plant, an algae, a fungus. Examples of prokaryotes include bacteria (e.g. cyanobacteria) and archaea.

The term“plant” as used herein may refer to any plant. For example, the plant may be a monocot or dicot. Preferably, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In a preferred embodiment, the plant is a cereal. In another embodiment the plant is Arabidopsis or Medicago truncatula. In one example the plant is selected from wheat, barley, rice, soybean, cotton, maize, canola and brassicas. In one embodiment, the rice may be selected from the Japonica cv or Zhonghua 11 rice varieties.

The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned comprise the nucleic acid construct as described herein. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the nucleic acid construct or RBC.

The invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. The aspects of the invention also extend to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins. Another product that may be derived from the harvestable parts of the plant of the invention is biodiesel. The invention also relates to food products and food supplements comprising the plant of the invention or parts thereof. In one embodiment, the food products may be animal feed. In another aspect of the invention, there is provided a product derived from a plant as described herein or from a part thereof.

In a most preferred embodiment, the plant part or harvestable product is a seed or grain. Therefore, in a further aspect of the invention, there is provided a seed produced from a transgenic or genetically altered plant as described herein.

In an alternative embodiment, the plant part is pollen, a propagule or progeny of the genetically altered plant described herein. Accordingly, in a further aspect of the invention there is provided pollen, a propagule or progeny produced from a transgenic or genetically altered plant as described herein.

The term“variant” or“functional variant” as used throughout with reference to any of SEQ ID NOs refers to a variant nucleotide, ribonucleotide or protein sequence that retains the biological function of the full non-variant sequence. A functional variant also comprises a variant that has sequence alterations that do not affect function, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active. Alterations in a nucleic acid sequence or ribonucleic acid sequence that result in the production of a different amino acid at a given site that does not affect the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

As used in any aspect of the invention described throughout a“variant” or a“functional variant” has at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,

51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%,

66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 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% overall sequence identity to the non-variant nucleic acid, ribonucleic acid or amino acid sequence.

In another aspect, the present invention provides an expression cassette A (or nucleic acid construct or vector - these terms can be used interchangeably) comprising a fragment of a target plant genome for substitution in A, and further includes a promoter and terminator, wherein the sequence between the promoter and the terminator include the following three sections: section I, section II and section III, wherein section III is section 111-1 or section MI-2 and wherein the expression cassette comprises

at least one, preferably two coding sequences for a nuclease or ribozyme enzyme in section I and a coding sequence for a first crRNA sequence (crRNAI) located between them; at least one, preferably two coding sequences for a nucleases or ribozyme enzyme in section II and a coding sequence for a second crRNA sequence (crRNA2) located between them;

at least one, preferably two coding sequence for a nucleases or ribozyme enzyme in section ill-1 and a template segment (i.e. the DRT as described above) located between them. Alternatively, there are two target sequences (preferably crRNA target sequences) in section ill-2 and a template segment (i.e. the DRT as described above) between them. More preferably, at one end of the target fragment is the target sequence of crRNAI in section I, and the other end is the target sequence of crRNA2 in section II.

In one embodiment, the template segment comprises an upstream (i.e. 5’) homology arm, a donor fragment sequence and a downstream (i.e. 3’) homology arm. In one embodiment, the donor fragment differs from the target fragment by one or more of the following:

1. one or more nucleotide mutations (for example, a substitution, deletion or addition of one or more nucleotides) compared to the target sequence (that is, the original sequence in the genome that is to be edited by the present method); and/or

2. at least one mutation i.e. substitution, deletion or addition of one or more nucleotides in the PAM motif (e.g. this is TTTN for Cpf1 and NGG for Cas) in the target sequence for crRNAI and/or crRNA 2 in the donor sequence. This means that crRNAI and/or 2 cannot bind to the target sequence and cleave the newly inserted sequence

In a further embodiment, section I comprises a Hammerhead-type nuclease coding sequence (preferably as defined above), a coding sequence for crRNAI , and a coding sequence for hepatitis D virus nuclease (preferably as defined above) from the 5 ' to 3 ' end.

Similarly, section II has a Hammerhead-type nuclease coding sequence (preferably as defined above), a coding sequence for crRNA2, and a coding sequence for hepatitis D virus nuclease (preferably as defined above) from the 5 ' to 3 ' end.

In a further preferred embodiment, in section 111-1 , the coding sequence of the Hammerhead-type nuclease, the upstream homology arm, the donor fragment sequence, the downstream homology arm and the hepatitis D virus nuclease are sequenced or transcribed in this order from the 5 ' to the 3 ' end. Alternatively, in segment ill-2, the target sequence of the crRNM , the upstream homology arm, the donor fragment sequence, the downstream homology arm and the target sequence of crRNA2 are sequenced or transcribed in this order from the 5 ' to the 3 ' end.

Examples of the expression cassettes (or nucleic acid constructs) of the invention are shown in SEQ ID NOs 1 to 3. In this example, the target sequence is a fragment of the ALS gene, as shown in SEQ ID NO: 6.

In a further aspect of the invention, there is provided a recombinant vector comprising the expression construct or nucleic acid construct described above. In one embodiment, the vector may further comprise a second expression cassette - expression cassette B. Preferably expression cassette B comprises a nucleic acid sequence encoding a CRISPR enzyme, such as Cas or Cpf1 , more preferably LbCpf Even more preferably the CRISPR enzyme is operably linked to a regulatory sequence. In one embodiment, the regulatory sequence is a promoter, such as a Ubiquitin promoter. In a further embodiment, the second expression cassette comprises a termination sequence, for example a Nos terminator.

In another aspect of the invention, there is provided a method for homologous recombination of a target gene by using an RNA transcript as a template in the plant comprising introducing and expressing the recombinant vector according to any of the above into a starting plant to achieve homologous recombination of the target gene in the plant. In one example, the target gene is an ALS gene.

In a further aspect of the invention, there is provided a homologous recombination vector using the rice ALS gene as a research object, wherein the vector may be pCXUN-OsU3-RCR1-RCR2-RDR-Nos-Ubi-LbCpf1-Nos or pCXUN-OsU3-RCR1- RCR2-armed donor (with targets)- Nos-Ubi-LbCpfl-Nos as described herein.

In one such embodiment, the RCR1-RCR2-RDR fragment is transcribed in vitro, and the RNA transcript is used as a repair template by the RNP method to achieve homologous recombination repair of the target gene in rice callus, wherein using the gene gun method, the vectors pCXUN-OsU3-RCR1-RCR2-RDR-Nos-Ubi-LbCpf1-Nos , pCXUN-Osll3-RCR1-RCR2-armed donor (with targets)-Nos-Ubi-LbCpf1-Nos and pCXUN -OsU3-RCR1-RCR2- Ubi-LbCpf1-Nos- armed donor (with targets) are introduced into rice callus, and rice plants with ALS gene site-directed modification are obtained, wherein pCXUN-OsU3-RCR1-RCR2- Ubi-LbCpf1-Nos -armed donor (with targets) is the control vector for the DNA repair template. The result of this embodiment demonstrate that using RNA as a repair template can successfully mediate homologous recombination of the target gene, providing a new approach for crop breeding with significant potential in agricultural breeding.

The plasmid pCXUN-Cas9 is described in He et al. , 2017 and Sun et al., 2016, both of which are incorporated herein by reference and is available to the public from the Crop Science Institute of the Chinese Academy of Agricultural Sciences.

The plasmid pRS316-RCR-GFP is described in Zhang et al., 2017, both of which are incorporated herein by reference, and is publicly available from the Crop Science Institute of the Chinese Academy of Agricultural Sciences.

The LbCpf1-OsU6 vector is described in Wang et al., 2017 and is available from the Crop Science Institute of the Chinese Academy of Agricultural Sciences. pCXUN-Cas9-OsU3 is described in Sun et al, 2016; Institute and is available from the Chinese Academy of Agricultural Sciences.

The endonucleases, kits and PCR enzymes used in the following examples were purchased from the reagent company. Other reagents are of domestic analytical purity.

Primers, DNA synthesis and sequencing in the following examples were performed by Huada Company.

While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

The foregoing application, and all documents and sequence accession numbers cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

The invention is now described in the following non-limiting examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. In the quantitative tests in the following examples, three replicate experiments were performed and the results were averaged.

EXAMPLE I

As discussed above, RNA transcripts-templated homology-directed DNA repair (TT- HDR) potentially can overcome the obstacles in delivery of donor repair template (DRT) into plant cells to repair double-stranded DNA breaks (DSBs) through HDR because RNA transcripts can be produced abundantly in vivo. However, primary transcripts are often processed/modified and transported to cytosol, rendering them unavailable for HDR. Herein we coupled LbCpfl with a single array comprising of the CRISPR RNAs (crRNAs) flanked with ribozymes and a DRT flanked with either ribozymes or crRNA targets. The primary transcripts from the arrays underwent self processing to release the crRNAs and DRT inside the nucleus. Using TT-HDR and DNA-free ribonucleoprotein (RNP) complexes, we achieved targeted gene replacement in rice, greatly expanding our ability to improve agriculturally important traits in crops through CRISPR/Cpf1 -mediated HDR technology.

We chose Cpf1 nuclease over the most widely used SpCas9 nuclease to test TT-HDR in plants because Cpf1 is a dual nuclease. It can process the precursor of CRISPR RNA into mature crRNA. It also uses crRNA to guide the cleavage of target DNA ^{16, 17}. Moreover, Cpf1 cleavage produces 5’ protruding sticky ends, which may facilitate HDR ¹⁷. Among several proteins exploited in the Cpf1 family, LbCpfl from Lachnospiraceae bacterium ND 2006 acts more effectively in human cells and plants than other orthologues ^{18 21}. Herein, we demonstrate that TT-HDR coupled with LbCpfl-crRNAs is effective in achieving precise targeted gene replacement in rice. We replaced the rice Acetolactate synthase ( ALS ) gene with a mutant version using a DNA-free ribonucleoprotein (RNP) complex that contains the recombinant LbCpfl protein, crRNAs, and DRT transcripts. We also generated rice stable lines that were introduced two desired discrete mutations in the ALS gene by TT-HDR.

To test whether TT-HDR can be effective in plants, we first developed a RNP system to replace the ALS gene with a mutated version (Fig. 1a). ALS, a major target for herbicides, catalyzes the initial step of the biosynthesis of the branched-chain amino acids ²². The W548L and S627I mutations in ALS render rice plants resistance to ALS- inhibiting herbicides ¹². The two substitutions could not be achieved by base-editing strategy due to a lack of appropriate PAM sites ^{23 25}. We designed two crRNAs that enable LbCpfl to remove a fragment from wild-type ALS (Fig. 1a). We also modified the crRNA target sequences so that the successfully introduced mutant ALS fragment can no longer be released by the same LbCpfl-crRNAs (Fig. 1a). We designed a DRT that contains all of the intended mutations and two homologous arms (Fig. 1 b). We used the ribozyme-based technology ²⁶ to produce two crRNAs and RNA transcripts of DRT in vitro from a single transcript (Fig. 1c). The two ribozyme-crRNA-ribozyme (RCR) units and one ribozyme-DRT-ribozyme (RDR) unit in tandem were transcribed from the T7 promoter. Transcripts underwent self-cleavage to release the mature crRNAs and DRT RNA transcripts (Fig. 1c). To remove any DNA templates from the in vitro transcription mixture, we performed DNase I digestion. We delivered the ribonucleoprotein (RNP) complexes, which contained LbCpfl protein, two crRNAs, and the RNA templates, into rice calli by particle bombardment (Fig. 1c). As shown in Fig. 1 d, by using allele-specific primer set ALSTestF/T2MR (Fig. 15), we precisely replaced the wild-type ALS gene with the mutant version. We also observed partial homologous recombination, by which only one end was replaced (Fig. 1 d), suggesting that there might be template-switching during the repair of DSBs ²⁷.

To demonstrate unambiguously that an RNA template is used for gene targeting, we performed DNase I, RNase H, and RNase A digestions of the intended DRT fragments, respectively. We conducted seven different sets of RNP experiments (Fig. 2a) in rice calli to test if we can achieve HDR events by using various DRT templates including RNA transcripts only, both DNA template and RNA transcripts, single-stranded DNA (ssDNA), and ssRNA, respectively. We performed droplet digital PCR (ddPCR) to detect HDR events and to evaluate the RNP-mediated HDR efficiency using various DRT templates (Fig. 9). It is very clear that ssRNA could serve as DRT for HDR (experiment IV) (Fig. 2b; Fig. 6 and Fig. 9), although the efficacy was much lower than ssDNA (experiment VI) (Fig. 2c). Because ddPCR is only effective when the amplification length is less than 300 bp, we had to evaluate the HDR efficiencies around target 1 and target 2 loci, respectively. As shown in Fig. 2d and 2e, RNA template alone (experiment I and experiment IV, in which DNA was removed by DNase I digestion) was able to achieve HDR. Without DNase I treatment (experiment II), HDR efficiency was much higher, suggesting that the availability of both DNA and RNA DRTs made HDR more effective (Fig. 2d, 2e; Fig. 9). We also digested DRT transcripts with either RNase A or RNase H (Fig. 2a), which degrades RNA non-specifically and removes RNA from the RNA-DNA duplexes, respectively, to determine whether HDR depends on RNA transcripts. Removal of RNA DRTs (experiment III, V and VII) abolished HDR (Fig. 2a, 2d, 2e and Fig. 9).

Encouraged by the results of experiment II (Fig. 2a, 2d, 2e) that uses both DNA and RNA transcripts as DRTs, we tested whether we could conduct CRISPR/Cpf1- mediated HDR in rice by placing all of the essential HDR components in one single expression cassette (Fig. 3). Because RNA transcripts are often modified and transported into cytosol, we developed two systems to ensure that RNA transcripts stay in the nucleus as templates for HDR. First, we put two RCR units and a RDR unit in a tandem array under the control of the OsU3 promoter and terminated by the NOS terminator (Fig. 3a-1 and Fig. 7a). When transcribed, both the mature crRNAs and the repair RNA template are released by self-cleavage of the ribozymes. This RDR strategy enables the production of the desired RNA transcripts even if the 5’ and 3’ end of primary transcripts are modified ²⁶. Secondly, we took advantage of the unique property of Cpf1 , which can also process its own pre-crRNA ¹⁶. We flanked the DRT with two crRNA target sites and coupled with the two RCR units in a single expression cassette (Fig. 3b-1 and Fig. 7b). Therefore, the RNA transcripts of DRT can be released by LbCpfl-crRNAs (hereafter referred to as TDT, target-donor-target). One caveat of TDT design is that the LbCpfl-crRNAs can also release the DRT DNA fragment, making it difficult to distinguish between DNA and RNA DRTs. To clarify the source of DRT, we also constructed a vector that produces two crRNAs from two tandem RCRs and DRT flanked with Cpf1 targets, but lacked a promoter so that the DRT cannot be transcribed. The obtained vector was referred as control, and DRT in this control vector could only be released by LbCpfl/crRNAs at DNA level (Fig. 7c).

To investigate whether the three constructs (RDR, TDT, and control) can achieve TT- HDR-mediated targeted gene replacement in rice without co-bombardment of any additional free DNA donor repair templates, we introduced these vectors into rice (Japonica cv. Zhonghua 11) calli by particle bombardment. For RDR, TDT, and the control vector, a total of 203, 192, and 139 calli were bombarded, respectively. The calli that survived one round of hygromycin selection were transferred onto the induction media with 0.4 mM bispyribac-sodium (BS). Then, the plants recovered from regeneration media with 0.4mM BS were used for PCR and restriction enzyme digestion assay (PCR-RE). PCR primer set ALStestF/R was designed to amplify an ALS fragment from both wild-type ALS locus and the edited ALS, but not from the plasmids (Fig. 3a-2 and Figure 15). All plantlets developed from one callus were treated as a pool. The plantlets in a pool that gave PCR-RE patterns different from that of wild-type were then transferred to soil individually and were further analyzed by PCR-RE and sequencing. No obvious phenotypic variations were observed between the lines and wild-type plants. In total, 58, 87, and 32 plants developed from 19, 20, and 8 BS-resistant calli for the three treatments, respectively, were selected for further analyses (Fig. 10).

For the RDR vector (Fig. 3a-1 and Fig. 7a), we observed 3 HDR genotypes (Fig. 10). Line 288-6 was a heterozygous with one allele of precise gene replacement, one wild- type (Line 288-6) (Fig. 3a-2, 3a-3 and Fig. 8). Line 289-4 had one allele with the expected substitutions around both target one and at W548L locus, whereas the other allele was wild-type (Fig. 3a-2, 3a-3 and Fig. 8). Line 291-3 only had one allele with the expected substitutions around target two (Fig. 3a-2, 3a-3 and Fig. 8). The efficiency of precise HDR was 1.7% (1/58) (Fig. 10). In this vector, DRT with ribozyme units at each end could be released at RNA level through self-cleavage of the ribozymes. The achievement of precise HDR event in this experiment clearly demonstrated that TT- HDR was feasible and could be employed for targeted gene replacement in plant cells.

For the TDT vector (Fig. 3b-1 and Fig. 7b), among the 87 plants recovered, PCR-RE and sequencing analyses identified 4 independent heterozygous lines with one allele containing the expected precise gene replacement (Line 183-2, 185-5, 198-1 , and 278- 4), whereas the other allele was either wild-type or had partial HDR at S627I locus (Line 198-1) (Fig. 3b-2, 3b-3; Fig. 10 and Fig. 8). We also observed that another line had the expected substitutions at both W548L and S627I loci, but with a 28 bp deletion around target two (Line 193), which started from 17 bp upstream of stop codon of ALS (Fig. 3b-2, 3b-3 and Fig. 8). The efficiency of precise HDR was 4.6% (4/87) (Fig. 10), indicating that this is an efficient strategy to achieve gene replacement by CRISPR/LbCpfl The higher frequency of HDR events in this treatment may be due to the fact that the DRT with target sites at each end in this vector could be released by LbCpfl/crRNA at both DNA and RNA levels.

Following the same transformation, culture and selection procedure, we bombarded the control vector (Fig. 7c). Among 32 plants recovered, we didn’t detect any plants with either partial or precise HDR events. Compared to the TDT vector (Fig. 3b-1), DRT in this vector could only be released by LbCpfl/crRNA at DNA level. These results, together with the observation in RNP assays in rice calli, further support that the TT- HDR indeed occurs in plant cells.

We also performed Agrobacterium-medi atedi transformation to investigate whether the constructs (RDR and TDT) can achieve TT-H DR-mediated targeted gene replacement in rice. Each vector was transformed into about 300 calli. The RDR vector (Fig. 7a) resulted in 17 plants, but no HDR events were observed (Fig. 11). For the TDT vector (Fig. 7b), we identified two partial HDR events among the 35 T₀ plants (Fig. 11). The HDR efficiency can likely be improved if stronger promoters are used for producing more abundant RNA transcripts.

To analyze the stability and heritability of the HDR events, we analyzed the edited plants at Ti generation. The edited loci in all of the analyzed lines, except for lines 183- 2 and 198-1 , which died after transferring into soil, displayed Mendelian segregation (Fig. 12). Furthermore, transgene-free lines with precisely edited ALS were recovered following segregation in Ti generation (data not shown). Moreover, we did not observe any off-target effects in these tested lines (Fig. 13).

Taken together, we here demonstrated that RNA transcripts can serve as repair template for HDR in rice, which provides an alternative mechanism underlying more effective delivery DRT into plant cells. We showed that TT-HDR technology enables precise gene replacement in rice, greatly expanding our ability to improve agriculturally important traits. The TT-HDR technology makes DNA-free HDR feasible, providing a potential path for bypassing some regulatory obstacles in commercializing crops with improved traits through CRISPR-mediated HDR technology.

Materials and Methods

Synthesis of the designed donor repair template

We designed a donor repair template (DRT) fragment that contained the following features (Fig. 1 b). Firstly, the fragment contained the desired mutations (W548L and S627I substitutions) in the ALS gene, which render rice plants resistance to ALS- inhibiting herbicides. Secondly, the donor fragment had several synonymous substitutions at the target 1 and target 2 loci, respectively, which prevent the introduced replacement from further cleavage by LbCpfl/crRNAs once HDR is successfully achieved. Thirdly, the 381 bp core sequences in the DRT was flanked with a 97 bp left homologous arm and a 121 bp right homologous arm, respectively, which are identical to the stretches of wild type ALS sequences. Moreover, an EcoRV restriction site between the two target sites in the donor fragment was abolished to facilitate detection of gene replacement events. Finally, the designed DRT fragment was synthesized by BGI (Beijing Genomics Institute, China).

Preparation of ssDNA and ssRNA donor repair template

The single-stranded DNA (ssDNA) fragment was amplified using primer set donorF/donorR (Figure 15) from the synthesized DRT by asymmetric PCR and the products were purified by Columns (TIANGEN, China) followed by ethanol precipitation. T7-DRT DNA fragment was amplified using primer set T7-donorF/T7- donorR from a synthesized DRT and was used as the templates for in vitro transcription of ssRNA (Figure 15). The in vitro transcription was performed using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, USA). The in vitro transcribed ssRNAs were subjected to either DNase I, or RNase H, or RNase A treatments as described in the manufacture’s protocol and further purified using NucAway™ Spin Columns (Life Technologies Inc., USA).

Preparation of RCR-RCR2-RDR transcripts

The ribozyme-crRNA-ribozyme (RCR) units were assembled through overlapping PCR reactions. To generate RCR1 , we conducted the first round of PCR (PCR1) using RCR1 F2/RCR-common-R primer set and the plasmid pRS316-RGR-GFP (Zhang et al., 2017; Gao and Zhao, 2014) as a template. The second PCR reaction used the primer set RCRF1/RCR-common-R and the product from PCR1 as the template (Figure 15). The same procedure was used to obtain the RCR2 unit with the primer set RCR2- F2/RCR-common-R, and RCR-F1/RCR-common-R, respectively (Figure 15. RCR1- RCR2 unit was obtained through three rounds of overlapping PCR reactions. The first PCR was performed with primer set RCR-Common-F/RCR1-10 random-R using RCR1 unit as the template (Figure 15). The second PCR was performed with primer set RCR2-10 random-F/Sacl-RCR2-R using the RCR2 unit as the template (Figure 15). Products of PCR 1 & 2 were used as templates for the third PCR reaction with the primer set RCR-Common-F/Sacl-RCR2-R to generate the RCR1-RCR2 unit. The RCR1-RCR2 unit was cloned into p£AS Y-Blunt vector (TransGen Biotech, China) for sequencing.

RDR-Nos fragment was obtained through five rounds of overlapping PCR reactions. The Hammerhead ribozyme (HH) fragment was obtained by PCR through annealed primer set HHF/HHR (Figure 15). The second PCR was performed with primer set donor-HH-F/donor-HH-R using the synthesized DRT as template (Figure 15). The third PCR was performed using primer set HDVF/HDVR with the plasmid pRS316-RGR- GFP (Gao and Zhao, 2014; Zhang et al., 2017) as the template (Supplementary Table 1). The fourth PCR was performed using primer set Nos-HDVF/Not-NosR with the plasmid pCXUN-Cas9 (Sun et al., 2016) as template (Figure 15). Products of PCR 1 & 2& 3 & 4 were used as templates for the fifth PCR reaction with the primer set Not- HHF/Not-NosR to generate the RDR-Nos fragment (Figure 15). The fragment was cloned into the Not\ site of pEASY- RCR1-RCR vector using the Assembly Kit (TransGen Biotech, China). The final plasmid was named as pEASY-RCR1-RCR-RDR- Nos. PCR primers for vector construction were listed in Figure 15. PCR products named RCR1-RCR2-RDR were amplified from the vector pEASY- RCRI-RCR-RDR-Nos by appropriate primer set, and used as the templates for in vitro transcription (Figure 15). In vitro transcription was performed using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs, USA). The in vitro transcribed products were subjected to DNase I, RNase H, or RNase A treatments as described in the manufacture’s protocol and further purified using NucAway™ Spin Columns (Life Technologies Inc., USA).

A ribonucleoprotein (RNP) complex comprising of LbCpfl-crRNA and RNA transcripts was generated as the following: 10 pg LbCpfl protein and 10 pg RNA transcripts including crRNAs and DRT RNA transcripts in a 1 :1 molar ratio were pre-mixed in 1 *NEB Buffer 3 supplemented with 1 pi of RNase inhibitor (New England Biolabs, USA) to the final volume of 20 pi and incubated at room temperature for 15 min.

CRISPR/Cpf1-RNP-mediated TT-HDR by using RNA transcripts as donor repair template in rice calli

The pre-assembled RNPs were precipitated onto 0.6 mm gold particles (Bio-Rad, USA) using a water soluble cationic lipid TranslT-2020 (Mirus, USA) as follows: 50 pi of gold particles (water suspension of 20 mg ml ¹) and 2 pi of TranslT-2020 water solution were added to 20 pi preassembled RNPs, mixed gently, and incubated on ice for 10 min. RNP/RNA-coated gold particles were then pelleted in a microfuge at 8,000g for 30 s and the supernatant was discarded. The pellet was re-suspended in 50 pi of sterile water by brief sonication. Immediately after sonication, coated gold particles were loaded onto a macro-carrier (10 pi each) and allowed to air dry. Calli of a japonica rice (cv Zhonghua 11) developed from mature embryos were bombarded using a PDS- 1000/He Gun (Bio-Rad, USA) with a rupture pressure of 600 psi following the protocol described previously (Li et al., 1993).

For each treatment, we bombarded 10 calli with three biological replicates. Thirty-six hours after bombardment, DNA from the calli was extracted using a DNA Quick Plant System (Tiangen, China). PCR amplification was performed using EASY Taq polymerase (TransGen Biotech, China) by using 200 ng of genomic DNA as template. Each callus was tested individually by PCR and sequencing. The PCR products were generated using the allele-specific primer set ALSTestF/T2MR (Figure 15) with up stream primer located in the genome sequence of ALS gene outside of left homologous arm, whereas the down-stream primer was an allele-specific primer (Fig. 1a and 15). The obtained amplicons were cloned into the cloning vector pEasy-Blunt (TransGen Biotech, Beijing, China). At least 10 positive colonies for each sample were sequenced.

Droplet Digital PCR (ddPCR)

Primers and probes were designed following the criteria specified by the instrument manufacturer. Candidate primers were designed using Primer 5 with manually adjusted settings to have annealing temperature of 56°C, whilst fluorescently labeled probes for amplicon detection were selected to have annealing temperatures of ³ 59°C. The edited OsALS gene probes were 5’ FAM™ (6-fluorescein) labeled and the wild type OsALS gene probes were 5’ HEX™ (hexachloro-fluorescein) labeled (Figure 15 and Figure 2). Both types of probes were quenched with Iowa Black Hole Quencher® 1 (The Beijing Genomics Institute, China).

For each sample tested, a ddPCR cocktail was generated that contained 11 mI 2x ddPCR Supermix for Probes (no dUTP) (Bio-Rad Laboratories, CA), 900 nM of each primer pair and 250 nM of each probe. 25 ng genome DNA was added to the mixture and the final volume was adjusted to 22 mI with sterile ultrapure water. Droplets were produced from 20 mI of the complete reaction mixture drawn together with 70 mI Bio- Rad Droplet Generation Oil in the microcapillary droplet generator cartridge following the manufacturer’s instructions. Droplets (40 mI) were transferred slowly and carefully from the droplet generation cassette to a ddPCR™ 96-Well plates, sealed with pierceable foil and placed into the thermocycler. The amplification program incorporated an initial 95°C denaturation for 10 minutes, followed by 40 cycles of 94°C (30 seconds) and 56°C for 1 minute. The 40 cycles were followed by a step at 98°C for 10 minutes and then at 4°C forever. A temperature ramp rate of 2°C /second was utilized between all changes in temperature to follow the instrument manufacturer guidelines. After amplification, the samples were transferred to a Bio-Rad QX200 droplet reader.

Detection of successful HDR events and evaluation of the HDR efficiencies at target 1 and target 2 loci by ddPCR, respectively

For every treatment, 1 pg genomic DNA from each callus (total 10 calli for each treatment) were pooled for evaluating the frequency of HDR events. Because of the limit of amplification length using ddPCR, mutated target 1 and mutated target 2 were detected separately. At target 1 locus, the T1 F/T1 R primer set was used to amplify the products and the probes T 1 -Edit and T1-WT were used to detect PCR products of edited and wild type OsALS gene, respectively (Figures 5 and 15). We designed T1 F which is located on the genome outside of the left homologous arm of DRT, whereas T1 R is inside the DRT (Figure 5). At target 2 locus, the same experiment was performed with primer pair T2F/T2R and probes T2-Edit and T2-WT (Figures 5 and 15). Also, we designed T2F which is located inside the DRT whereas T2R is located on the genome outside of the right homologous arm of DRT (Figure 5). Droplets were counted and the frequencies of HDR events were generated by using the Bio-Rad QuantaSoft™ software (v1.6.6.0320) (Figure 6).

For each sample, the frequency of HDR events was calculated. A box-and-whisker plot was made based on the ratio of HDR events using the software OriginPro 9 (OriginLab, USA). To compare the efficiency of different treatments, a Student’s f-test was employed to evaluate the significance of the difference existing between two experiments using the OriginPro 9. Significance (p-value) was evaluated at the 1% level for all comparisons. For each experiment, the standard deviation (SD) of the mean was calculated based on nine biological replicates (Figure 9).

Construction of the CRISPR/LbCpfl related vectors

The pCXUN-LbCpfl vector used in this study was constructed based on the vector pCXUN-Cas9 (Sun et al. , 2016) by replacing the ubiquitin-Cas9 with the ubiquitin- LbCpfl from the LbCpf1-OsU6 (Wang et al., 2017). The backbone of pCXUN-Ubi- LbCpf1-Nos contains a hygromycin resistant gene (hpf). The Sad and Kpn\ sites in pCXUN-Ubi-LbCpf1-Nos were used for introducing the OsU3-RCR1-RCR2 expression cassette and the DNA donor repair template (DRT), respectively (Figure 7).

OsU3 promoter was amplified using primer set OsU3F/OsU3R (Figure 15) from the plasmid pCXUN-Cas9-OsU3 (Sun et al., 2016). Because OsU3 promoter was used in this experiment, we also placed an adenine nucleotide before the first nucleotide of the RCR sequences. The full length OsU3-RCR1-RCR2 cassette was obtained through two rounds of overlapping PCR reactions. The first PCR was performed with primer set OsU3F/OsU3-RCR1 R using the OsU3 promoter sequence as the template (Figure 15). The second PCR was performed with primer set RCR-Common-F/Sacl-RCR2-R using vector pEASY- RCR1-RCR as the template (Figure 15). Products of PCR 1 & 2 were used as templates for the third PCR reaction with the primer set Sacl-OsU3-F/Sacl- RCR2-R to generate the OsU3-RCR1-RCR2 cassette. At the 5’-end of the primer pair of Sacl-OsU3-F/Sacl-RCR2-R, the sequences are homologous to the sequences outsides of Sad site in pCXUN-Ubi-LbCpf1-Nos. OsU3-RCR1-RCR2 fragment was subsequently cloned into the Sacl-linearized pCXUN-Ubi-LbCpf1-Nos, by using pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing, China). The vector harboring both Ubi-LbCpf1-Nos and OsU3-RCR1-RCR2 was named as pCXUN-OsU3-RCR1-RCR2-Ubi-LbCpf1-Nos.

The vector pCXUN-OsU3-RCR1-RCR2-RDR-Nos-Ubi-LbCpf1-Nos, was obtained by overlapping PCR reactions. Kpn-RDR-Nos fragment was amplified with the primer set Kpn-HHF/Kpn-NosR from vector p£ASY-RCR1-RCR-RDR-Nos as the template (Figure 15). The fragment was cloned into the Kpn\ site of pCXUN-OsU3-RCR1-RCR2-Ubi- LbCpf1-Nos using the Assembly Kit (TransGen Biotech, Beijing, China). The final plasmid was named as pCXUN-OsU3-RCR1-RCR2-RDR-Nos-Ubi-LbCpf1-Nos (Figure 7a). PCR primers for vector construction were listed in Figure 15.

The vector pCXUN-Osll3-RCR1-RCR2-armed donor (with targets)-Nos-Ubi-LbCpf1- Nos, was obtained by overlapping PCR reactions. The fragment of donor (with targets)- Nos was assembled through overlapping PCR reactions. PCR1 products was obtained by PCR using primer set Kpn-donorF/donor-R with synthesized donor fragment as the template, and PCR2 was performed with the primer set Nos-donorF/Kpn-NosR using the plasmid pCXUN-Ubi-LbCpf1-Nos as template (Figure 15). Products of PCR 1 & 2 were used as templates for the third rounds of PCR reaction with the primer set Kpn- donorF/Kpn-NosR to generate the donor-Nos fragment. The fragment was cloned into the Kpn\ site of pCXUN-OsU3-RCR1-RCR2-Ubi-LbCpf1-Nos using the Assembly Kit (TransGen Biotech, Beijing, China). The final plasmid was named pCXUN-OsU3- RCR1-RCR2-armed donor (with targets)-Nos-Ubi-LbCpf1-Nos (Figure 7b). PCR primers for vector construction were listed in Figure 15.

To generate a control plasmid, donor fragment was amplified by PCR using primer set Pme-donorF/Pme-donorR with the synthesized donor fragment as template, and cloned into the Pme I site of pCXUN-OsU3-RCR1-RCR2, which located at the other side of Ubi-LbCpf1-Nos cassette, by using the Assembly Kit (TransGen Biotech, Beijing, China). The final plasmid was named pCXUN-OsU3-RCR1-RCR2-Ubi-LbCpf1- Nos-armed donor (with targets) (Figure 7c). PCR primers for vector construction were listed in Figure 15.

Rice transformation

For induction of the rice calli from matured seeds, the mature seeds were firstly sterilized by 75% alcohol and 20% NaCIO followed by washes with sterilized deionized water. Then, the sterilized rice seeds were placed onto the induction medium for about one month at 28°C in the dark. Finally, the induced rice calli after subculture were used for transformation.

For rice transformation by bombardment, the vectors pCXUN-OsU3-RCR1- RCR2- RDR-Nos-Ubi-LbCpf1-Nos, pCXUN-Osll3-RCR1-RCR2-armed donor (with targets)- Nos-Ubi-LbCpf1-Nos and pCXUN-OsU3-RCR1-RCR2-Ubi-LbCpf1-Nos- armed donor (with targets) (Figure 7) were linearized by Sac II, then transformed into calli of a japonica rice (cv Zhonghua 11) by particle bombardment followed the protocol described previously (Li et al., 1993). Particle bombardments were performed using a PDS1000/He particle bombardment system (Bio-Rad, USA).

For rice Agrobacterium transformation, the vectors RDR (pCXUN-OsU3-RCR1- RCR2- RDR-Nos-Ubi-LbCpf1-Nos) (Figure 7a) and TDT (pCXUN-OsU3- RCR1-RCR2-armed donor (with targets)-Nos-Ubi-LbCpf1-Nos) (Figure 7b) were transformed into calli of a japonica rice (cv Zhonghua 11) by Agrobacterium-med atedi transformation as described previously (Hiei et al., 1994).

After transformation, the calli were selected on 1^st selection medium containing 50 mg/L hygromycin for two weeks at 28°C in the dark to allow the growth of calli with the construct, either transiently expressed or stably integrated. Then the well-grown calli were transferred to the 2^nd selection media containing 0.4 mM BS at 28°C in dark for two weeks. After two rounds of selection, the vigorously resistant calli were transferred to regeneration media with 0.4 pM BS for about 3~4 weeks to regenerate green seedlings at 28°C in the light (16 L: 8D). After regeneration, the green seedlings were transferred to the rooting medium to generate green plants at 28°C in the light (16 L: 8D).

Molecular characterization of the edited plants

Rice genomic DNA from leaf tissues was extracted using a DNA Quick Plant System (Tiangen, Beijing, China). PCR amplification was performed using EASY Tag polymerase (TransGen Biotech, Beijing, China) and 200 ng of genomic DNA as template. All plants were tested individually with PCR-RE and sequencing. The PCR products amplified by the primer pair ALSTestF/ALSTestR (Figure 15) were digested with EcoRV and then directly sequenced to screen for the plants with modified ALS gene. The sequence chromatograms were analyzed by a web-based tool (http://dsdecode.scgene.com/) to confirm the genotype and zygosity of the tested plants (Liu et al., 2015). Some PCR products were also cloned into the cloning vector pEasy-Blunt Zero (TransGen Biotech, China), and at least 10 positive colonies for each sample were sequenced. Primers for detection of the presence of LbCpfl, RCR and hptll were listed in Figure 15.

To investigate off-target effects, we selected 3 and 2 potential off-target sites based on the predictions of the CRISPR-GE (http://skl.scau.edu.cn/), for the target 1 and target 2, respectively (Figure 13). Site-specific genomic PCR and Sanger sequencing was used to determine the off-target effects. The primer sets were as listed in Figure 15.

Segregation and statistical analysis

T₀ plants with ~30 seeds or more were harvested for segregation analysis. Genomic DNAs were extracted from Ti seedlings using a DNA Quick Plant System (Tiangen, China) from leaf tissues. PCR amplification was performed using EASY Taq polymerase (TransGen Biotech, China) and 200 ng of genomic DNA as template. The PCR products amplified by the primer pair ALSTestF/ALSTestR (Figure 1a and 15) were directly sequenced to perform segregation analysis of HDR events in Ti seedlings. The PCR products amplified by the primer pair LbCpfl F/LbCpf1 R (Figure 15) were used to detect LbCpfl in Ti seedlings. c2 -test was performed to test whether the segregations of edited events were somatic and in accordance with Mendelian genetics.

EXAMPLE II: Modification of ALS gene mediated by RNA transcript as a repair template using CRISPR/Cpf1 system.

1. Construction of expression vectors

1. Plasmid pCXUN - Construction of LbCpfl -Nos

(1) The plasmid pCXUN-Cas9 was digested with restriction endonucleases Bam HI and HirrM to obtain a vector backbone 1 of about 9282 bp. (2) The LbCpf1-OsU6 vector was digested with restriction endonucleases Bam HI and Hind\\\ to obtain a Ubi- LbCpfl expression cassette of about 5846 bp .

(3) The vector backbone 1 and the Ubi- LbCpfl expression cassette were ligated with T4 ligase to obtain plasmid pCXUN-LbCpfl - Nos.

2. OsU3-RCR1-RCR2 - Construction of expression cassette

(1) Using the plasmid pRS316-RCR-GFP as a template, primers consisting of primer RCR1 F2 (SEQ ID NO: 54) and primer RCR-common-R (SEQ ID NO: 55) were used for the first round of PCR amplification to obtain the first round of PCR amplification products.

(2) using the first round of PCR amplification products obtained in step (1) as a template, using a primer pair consisting of primer RCRF1 (SEQ ID NO: 56) and primer RCR-common-R (SEQ ID NO: 57) for a second round of PCR amplification to obtain a second round of PCR amplification products. (RCR1).

(3) Using the plasmid pRS316-RCR-GFP as a template, the primer pair RCR2-F2 (SEQ ID NO: 58) and the primer RCR-common-R (SEQ ID NO: 59) were used for the first round of PCR amplification to obtain the first round of PCR amplification products.

(4) Step (3) obtained in the first round PCR products as template, using primers RCR-F1 primer (SEQ ID NO: 56) and RCR-common-R primer (SEQ ID NO: 57) pair consisting of a second round of PCR amplification, to obtain a second round of PCR amplification Add product (RCR 2).

(5) Using pCXUN-Cas9-OsU3 as a template, the primer pair consisting of primer OsU3F (SEQ ID NO: 60) and primer OsU3-RCR1 R (SEQ ID NO: 62) was used for PCR amplification to obtain the first round of PCR amplification product (OsU3 promoter sequence).

(6) using the second round of PCR amplification product (RCR1) obtained in step (2) as a template, using a primer pair consisting of primer RCR-Common-F (SEQ ID NO: 63) and primer RCR1-10 random-R (SEQ ID NO: 64) for the second round of PCR amplification. A second round of PCR amplification products was obtained.

(5) obtained in (7) The first round of PCR amplification products of step and step (6) (OsU3 promoter sequence) obtained in accordance with a second round of PCR amplification products molar ratio of 1 : 1 mixture as a template, using primers The primer pair consisting of OsU3F (SRQ ID NO: 60) and primer RCR1-10 random-R (SEQ ID NO: 64) was subjected to a third round of PCR amplification to obtain a third round of PCR product (OsU3-RCR1 expression cassette ).

(8) using the second round of PCR amplification product (RCR 2) obtained in step (4) as a template, using a primer pair consisting of primer RCR2-10 random-F (SEQ ID NO: 65) and primer Sacl-RCR2-R (SEQ ID NO: 68) for the fourth round of PCR amplification , a fourth round of PCR amplification products was obtained.

(9) mixing the third round of PCR product obtained in step (7) (OsU3-RCR1 expression cassette) and the fourth round of PCR amplification product obtained in step (8) according to a molar ratio of 1 :1 as a template, using primer Sad -OsU3-F (SEC ID NO: 67) and the primer Sacl-RCR2-R (SEC ID NO: 68) were subjected to a fifth round of PCR amplification to obtain a fifth round of PCR amplification products (OsU3-RCR1-RCR2 expression cassette).

3 - The synthesis of RPR fragments

(1) The primer HHF (SEC ID NO: 77) and the primer HHR (SEC ID NO: 78) are annealed to form an HH fragment (first round product) .

(2) The ALS gene fragment (SEC ID NO: 4) modified by chemical synthesis was used as a template, and the primer pair consisting of primer donor-HH-F (SEC ID NO: 79) and primer donor-HH-F (SEQ ID NO: 80) was used for PCR amplification to obtain the second round of product.

(3) Using the plasmid pRS316-RGR-GFP as a template, primers consisting of primer HDVF (SEQ ID NO: 81) and primer HDVR (SEQ ID NO: 82) were used for PCR amplification to obtain a third round of product.

(4) Using plasmid pCXUN-Cas9 as a template, primers consisting of primer Nos- HDVF (SEQ ID NO: 83) and primer KPN-NosR (SEQ ID NO: 84) were used for PCR amplification to obtain the fourth round of product.

(5) After mixing the first round product, the second round product, the third round product and the fourth round product according to a molar ratio of 1 : 1 : 1 : 1 , the primer consisting of the primer Kpn-HHF (SEQ ID NO: 85) and the primer Kpn-NosR (SEQ ID NO: 86) is used. PCR amplification was performed to obtain an RDR fragment.

4 - Armed donor (with targets) - synthesis of Nos fragments

(1) The ALS gene fragment (SEQ ID NO: 4 ) modified by chemical synthesis was used as a template, and the primer pair consisting of the primer Kpn-donorF (SEQ ID NO: 69) and the primer donor-R (SEQ ID NO: 88) was used for PCR amplification to obtain the first round product.

(2) In pCXUN- Ubi-LbCpf1-Nos plasmid as a template, using primers consisting of primers Nos-donorF (SEQ ID NO: 71) and Kpn-NosR (SEQ ID NO: 72) for PCR amplification, to obtain a second round products.

( 3 ) The first round of product and the second round of product were mixed at a molar ratio of 1 :1 and used as a template to carry out PCR amplification using a primer pair consisting of a primer Kpn-donorF (SEQ ID NO: 69) and a primer Kpn-NosR (SEQ ID NO: 72) to obtain an armed donor (with targets)- Nos fragment . 5 - Synthesis of vector pCXUN-OsU3-RCR1-RCR2- RDR-Nos- Ubi-LbCpf1-Nos - the“RPR” vector

The OsU3-RCR1-RCR2 expression cassette prepared in the step 2 and the plasmid pCXUN-LbCpfl -Nos prepared in the step 1 were ligated by the homologous recombinase (Full-Gold, Beijing, China) to obtain the recombinant vector pCXUN- OsU3-RCR1-RCR2-Ubi-LbCpf1-Nos, step 3 was inserted into a recombinant vector fragment RDR pCXUN-OsU3-RCR1-RCR2 -Kpn I site of the Ubi-LbCpf1-Nos obtain vector pCXUN-Osll3-RCR1-RCR2-RDR-Nos-llbi-LbCpf1-Nos .

The vector pCXUN-Osll3-RCR1-RCR2-RDR-Nos-llbi-Lb Cpf1-Nos was sequenced. Its sequence is shown in SEQ ID NO: 1. SEQ ID NO: 1 in the sequence listing starts from the 5' end, and positions 13 to 713 are the nucleotide sequences of the OsU3-RCR1-RCR2 expression cassette, wherein positions 13 to 393 are the nucleotide sequence of the OsU3 promoter, 394 to 436 and are Hammerhead (HH) type nuclease nucleotide sequence 601 to 559 of the nucleotide sequence 481 to 548 and 646 to 713 are hepatitis delta virus (HDV) nucleases, positions 437 to 480 are the nucleotide sequence of crRNAI , and positions 602 to 645 are the nucleotide sequence of crRNA2 . Positions 724 to 1433 are RDR fragments, wherein the nucleotide sequences of the Hammerhead (HH) type nuclease are located at positions 724 to 766, the nucleotide sequence of the hepatitis D virus (HDV) nuclease at positions 1366 to 1433 , and the DRT sequence at positions 767 to 1365 . The Nos terminator is at positions 1434 to 1686 and positions 1789 to 2041 is the reverse complement of the nucleotide sequence encoding the Nos terminator; positions 2061-5909 are the reverse complement of a nucleotide sequence encoding LbCpfl and positions 5912 to 7897 are the reverse complement of the Ubi promoter.

In the RDR fragment, positions 767 to 863 are upstream homology arms, positions 864 to 1244 are mutation segments, and positions 1245 to 1365 are downstream homology arms.

6 - Synthesis of the vector pCXUN-OsU3-RCR1-RCR2- armed donor (with targets) -Nos- Ubi-LbCpf1-Nos - the“TDT” vector

The OsU3-RCR1-RCR2 expression cassette prepared in step 2 and the plasmid pCXUN-LbCpf1-Nos prepared in step 1 were ligated by the homologous recombinase (Full-Gold, Beijing, China) to obtain the recombinant vector pCXUN-OsU3-RCR1- RCR2 - Ubi-LbCpf1-Nos, the armed donor (with targets) obtained in step 4 -Nos fragment was inserted into a recombinant vector pCXUN -OsU3-RCR1-RCR2 - Kpn I site of the Ubi-LbCpf1-Nos obtain vector pCXUN-OsU3-RCR1 -RCR2-armed donor (with targets)-Nos- Ubi-LbCpf1-Nos .

The vector pCXUN-Osll3-RCR1-RCR2-armed donor (with targets)-Nos-Ubi-LbCpf1- Nos (SEC ID NO: 2) differs from the vector pCXUN-OsU3-RCR1-RCR2-RDR-Nos- Ubi-LbCpf1-Nos (SEC ID NO: 1) in that SEQ ID NO: 1 comprises ribozyme sequences. In SEQ ID NO: 1 from the 5 'end, the first 1-701 is the OsU3-RCR1-RCR2 sequence, wherein positions 1-381 of the nucleotide sequence is the OSU3 promoter, positions 382 to 424 and 547 to 589 are the nucleotide sequences of Hammerhead (HH) ribozyme sequence, and nucleotide sequences 469 to 536 and 634 to 701 are the hepatitis D virus (HDV) ribozyme sequences. Positions 425 to 468 of the nucleotide sequence are crRNAI and positions 590 to 453 are crRNA2. Positions 709 to 1361 are the armed donor (with targets) fragment and positions 736 to 1334 are DRT sequences. Positions 1362 to 1614 is the nucleotide sequence of the Nos terminator.

7 - Synthesis of the carrier pCXUN-OsU3-RCR1-RCR2- Ubi -LbCpf1-Nos -armed donor (with targets) - the“control” vector.

A chemically synthesized ALS gene fragment (SEQ ID NO: 4) was used as a template, and a primer pair consisting of a primer Pme-donorF and a primer Pme-donorR was used for PCR amplification to obtain a PCR- amplified product ( arm-DRT ) .

The OsU3-RCR1-RCR2 expression cassette prepared in the step 2 and the plasmid pCXUN - LbCpfl prepared in the step 1 were ligated with a homologous recombinase (Full-Gold, Beijing, China) to obtain a recombinant vector pCXUN- LbCpf1-OsU3-RCR1-RCR2. The armed-DR T was inserted into a recombinant vector pCXUN-LbCpf1-OsU3-RCR1- RCR2 at a Pme I site, resulting in the vector pCXUN- OsU3-RCR1-RCR2- Ubi -LbCpfl -Nos -armed donor (with targets).

The vector pCXUN-OsU3-RCR1-RCR2- Ubi-LbCpf1-Nos- armed donor (with targets) is shown in SEC ID NO: 3.

8 - Detection of RNA template-mediated repair in rice callus Japonica cv Zhonghua 11 rice seeds are selected, seed coats are peeled, washed and sterilized, uniform sterilization NB entering a solid medium containing 2,4-D 2 mg / liter, 28 deg°C dark for 40 -50 days to induce callus production. The calli obtained in step 1 are added to MS medium containing 0.3M mannitol and 0.3M sorbitol osmotic treatment for 4-6 hours.

Using pCXUN-OsU3-RCR1-RCR2- RDR-Nos- Ubi-LbCpf1-Nos vector as template and using the primers T7- F and primer T7-Nos-R consisting of primers for PCR amplification, to obtain in vitro transcription templates. The RCR1-RCR2-RDR fragment was prepared according to the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB) instructions, incubated at 37 °C for 6 h, and in vitro transcribed to obtain transcripts (crRNAs and RNA repair templates) .

Table 1 : Reaction system:

4. The transcription product obtained in Step 3 was added to 2mI_ DNase I, and 30 mI_ RNase-Free ddH₂ O processes, the DNA is removed and the Lb Cpf1 protein is assembled. After purification and incubation at room temperature for 15min, RNP is formed. The system is assembled as follows:

Table 2: Assembly system:

5. The RNP obtained in step is used to transform rice callus by gene gun, using 0.6pm powder and a bombardment pressure of 900 psi.

6. After step 5 and culture of the rice callus at 28°C in dark culture, genomic DNA was extracted after 36h. Genomic CDNA was used as a template and PCR amplification was performed using the following primers: ALSTestF and primer T2MR. The amplification products were sequenced to detect the occurrence of ALS gene homologous recombination. 7. The result is shown in Figure 1d. Wherein, WT ALS is the wild type ALS gene; the Donor is a repair template sequence (SEQ ID NO: 6); underlined sequences are the target 1 and target 2 sequences; italics base is designated the mutated PAM site and the Eco RV cleavage site, and the italicized base are the bases replaced by the target.

The results show that in the obtained callus, intact homologous recombination was detected in the callus RDR35 , and some homologous recombination was found in the callus of RDR41. The results show that RNA as a repair template can be successfully used to mediate homologous recombination repair of genomic DNA.

9 - Acquisition of genetically modified rice.

1. Japonica cv Zhonghua 11 rice seeds are selected, seed coats are peeled, washed and sterilized, uniform sterilization NB entering a solid medium containing 2,4-D 2 mg / liter, 28 deg°C dark for 40 -50 days to induce callus production.

2. The calli obtained in step 1 are treated with 0.3M mannitol and 0.3M sorbitol hypertonic medium M S 4-6 hours. Calli are bombarded with the pCXUN-OsU3- RCR1-RCR2-RDR-Nos-Ubi-LbCpf1-Nos by gene gun bombardment, using 0.6pm powder and a pressure of 900 psi bombardment. After bombardment calli are transferred to MS medium containing 0.3M mannitol and 0.3M sorbitol in a dark cultured for 16 hours after 28 °C NB screening medium ( NB solid medium containing 2 mg / L of 2,4-D and 50 mg / L hygromycin ) was continuously dark cultured at 28 °C for 2 weeks .

3. After the completion of step 2, select a good positive growth (bright yellow callus). NB moved with sterile forceps to a pre-differentiation medium (containing 1 mg/liter NAA, 5 mg/I of the ABA, 2 mg / 1 kinetin and on a NB solid medium of 50 mg / L hygromycin , dark culture was continued for 2 weeks at 28 °C).

4. After completing step 3, the vigorously growing callus are added to a MS differentiation medium ( MS solid medium containing 0.02 mg / L NAA , 2 mg / L kinetin and 0.4 pM bispyribac sodium ) and grown at 28 °C in continuous light culture.

5. After completing step 4, seedlings with a length of 2-5mm are transferred to a MS solid medium 28 °C lighted culture 2-3 weeks, then transferred to soil in a greenhouse for growth (temperature 28-30 deg.] C, 16 h light / 8 h dark), to give T o transgenic plants (transfected pCXUN-OsU3-RCR1-RCR2- RDR-Nos- Ubi- LbCpf1-Nos).

10 - Genotypic identification of transgenic rice.

Test plants: wild-type: Japonica cv Zhonghua 11 rice (WT), T₀ transgenic plants (transfected pCXUN-OsU3-RCR1-RCR2- RDR-Nos- Ubi-LbCpf1-Nos), T₀ transgenic plants (transfected pCXUN-OsU3 -RCR1-RCR2-armed donor (with targets) -Nos- Ubi- LbCpf1-Nos) and T ₀ transgenic plants (transfected pCXUN-OsU3-RCR1-RCR2- Ubi - LbCpf1-Nos - armed donor (with targets)).

The genomic DNA of the plant to be tested was extracted, and the primer pair consisting of primer ALStestF and primer ALStestR was used for PCR amplification using genomic DNA as a template. The PCR amplification product was digested with Eco RV, and the wild control was cut by Eco RV and produced. Two types of 481 bp and 322 bp fragments, which could not be completely digested by Eco RV, were identified as homologous recombination successful plants. The PCR product will be completely or partially excised for cloning and sequencing. The statistical results are shown in Figures 10 and Figure 3.

EXAMPLE III: Genome modification of animal cells using the CRISPR/Cpfl system.

A. Plate cells

1) Approximately 18-24 hours before transfection, plate cells in 2.5 ml complete growth medium per well in a 6-well plate. Ideally cells should be ^³ 80% confluent prior to transfection.

For adherent cells: Plate cells at a density of 0.8-3.0x105 cells/ml.

For suspension cells: Plate cells at a density of 2.5-5.0x105 cells/ml.

2) Incubate cell cultures overnight.

B. Prepare TranslT-2020 Reagent (alternatively, you may also use lipofectin to enhance transfection): RBC (ribonucleoprotein complex) complex (Immediately before transfection)

1) Warm TranslT-2020 reagent (Mirus, USA) to room temperature and vortex gently before using.

2) RBC (RNP complex) formation. A RBC comprising of LbCpfl-crRNA and RNA transcripts was generated as the following: 2 pg LbCpfl protein and 2 pg RNA transcripts including crRNAs and DRT RNA transcripts in a 1 :1 molar ratio were pre- mixed in 1 *NEB Buffer 3 supplemented with 0.2 pi of RNase inhibitor (New England Biolabs, USA) to the final volume of 4 mI and incubated at room temperature for 15 min.

3) Place 250 mI of Opti-MEM I Reduced-Serum Medium in a sterile tube.

4) Add the RBC to step 3 and pipet gently to mix completely.

5) Add 7.5-10 mI TranslT-2020 reagent to the diluted RBC mixture.

6) Pipet gently to mix completely.

7) Incubate at room temperature for 15-30 minutes to allow sufficient time for complexes to form.

C. Distribute the complexes to cells in complete growth medium

1) Add the TranslT-2020 reagent: RBC complexes (prepared in Step B) drop-wise to different areas of the wells.

2) Gently rock the culture vessel back-and-forth and from side-to-side to evenly distribute the TranslT-2020 reagent: RBC complexes.

3) Incubate for 24-72 hours. It is not necessary to replace the complete growth medium with fresh medium.

4) Harvest cells and assay as required.

SEQUENCE LISTING

SEQ ID NO: 1 : pCXUN-OsU3-RCR1-RCR2-RDR-Nos- Ubi-LbCpf1-Nos (the “RDR” vector).

Bold = HH ribozyme

Bold and underlined = 1-6 nucleotides of HH ribozyme

Double underlined = HDV ribozyme

Wavy underlined = cRNA1 target sequences

Dashed jjnderJined = cRNA2 target sequences

First italic section = OsU3-RCR1-RCR2

Second italic section = RDR

Third italic section = LbCpfl

CAPITALS = Ubiquitin promoter

gaattcgagctc

aaggaatctttaaacatacgaacagatcacttaaagttcttctgaagcaacttaaagttatcaggcatgcatggatcttgga ggaatcagatgtgcagtcagggaccatagcacaagacaggcgtcttctactggtgctaccagcaaatgctggaagccg ggaacactgggtacgttggaaaccacgtgatgtgaagaagtaagataaactgtaggagaaaagcatttcgtagtgggc catgaagcctttcaggacatgtattgcagtatgggccggcccattacgcaattggacgacaacaaagactagtattagta ccacctcggctatccacatagatcaaagctgatttaaaagagttgtgcagatgatccgtggca

aaattactgatgagtccgtgaggacgaaacgagtaagctcgtctaatttctactaagtgtagat

ggtatggtggtgcaatgggagga

aaccaacataatcccaacctcctcactaacaccaactaaacaacatacttcaacataacaaataaaacaaatacaacc aaattactgatgagtccgtgaggacgaaacgagtaagctcgtctaatttctactaagtgtagat

aaccaacataatcccaacctcctcactaacaccaactaaacaacatacttcaacataacaaataaaac

cggtaccaca

catcaactgatgagtccgtgaggacgaaacgagtaagctcgtcttgatggggatggtagcttcctcatgaacattca ggagctggcattgatccgcattgagaacctccctgtgaaggtgatggtgttgaacaaccaacacctaggcatggtcgtcc agttggaggataggttttacaaggcgaatagggcgcatacatacttgggcaacccggaatgtgagagcgagatatatcc agattttgtgactattgctaaggggttcaatattcctgcagtccgtgtaacaaagaagagtgaagtccgtgccgccatcaag aagatgctcgagactccagggccatacttgttggacatcatcgtcccgcaccaggagcatgtgctgcctatgatcccaatt gggggcgcattcaaggacatgatcctggatggtgatggcaggactgtgtattaatctataatctgtatgttggcaaagcac cagcccggcctatgtctgacgtgaatgactcataaagagtggtatgcctatgatgtttgtatgtgctctatcaataactaaggt atcaactataaaccatatactcttctattttacttatttaatatacttaacataataatcctaattaacttcctgctaaaccaacat aatcccaacctcctcactaacaccaactaaacaacatacttcaacataacaaataaaac

gatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaatt acgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacattt aatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatc ggtacccctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgta aaacgacggccagtgaattcccgatctagtaacatagatgacaccgcgcgcgataatttatcctagtttgcgcgctatatttt gttttctatcgcgtattaaatgtataattgcgggactctaatcataaaaacccatctcataaataacgtcatgcattacatgtta attattacatgcttaacgtaattcaacagaaattatatgataatcatcgcaagaccggcaacaggattcaatcttaagaaac tttattgccaaatgtttgaacgatcggggaaattcggatcc

ttactttttcttttttgcctggccggcctttttcgtggccgccggccttttgtgcttcacgctggtctgggcgtactccagccactcct tgttagagatggcgatcttcaccttatccagcttctcgtcctcggccttcttgaa

ctggccgatggcccacagcacctttctggcgatgttataggcgccattggcgtcggcgttctttggcaggatggcattctcct gggcctcatagttccggctatcgtagaagatgccgtcggagttcttcacagggctgatcagaaaatccacgtcggtgcgg cctgtgatgctgttccgcatctgcagcatcaggctcatcagggccataaagctagagtagaaggccttgtcggactgctcg cacagcagggctctgatatcgccctgctgataattgatgccgtacttgttgaacagctccttataggcgctggtcaggcaca cctcctcccagtcgaacacgttgttcttcttaggattccggaagattctgatccggttgccgtaggagtacagcttccacttctt gatgtaatcggcgtctgtgcgagagaagttcttatagtccagggcaaactcgaacagatcctcctcgggcacgtacatga tcctgtcaaaggagctgatgaacttcttggaatcggcgatgctggtatacttggttttcagcaggttcacaaagccggtagat ggatcgatcttggatgtcagccaggcagggatgtaaaagatgaagccgttctgggtagacatggacttaaagctctcga acttattggtgatctgatagcccttcagggcgccgcctgttgcacaaggattagacttcttgtccaccatgtagttcagcttatc gatcagcatcttctcgaacttctgatacacctgcttctccaccttcacgcggctattcttaaagccagagttcaggtcctccag ggcgatcacggcatcgtacttctccaccagctcgcagatcttgtgcaccacctgagagatatagccggccttcagctcctt gatattctcgatggaggtccagttctggcgggcctcgaacctctccttctccttcttgtccagcagagagtggtaatctgtcttg atcctgatgccgttgaagttgttgatgatctcgttcagggaatactgctccacgatgttgcccttgccgtccaccaccacgat atacagcagattgcgctcgcccctatcgatgccgatcacataggggttatcgtcgtgcttcagcagcacgcgcacctctgt attgatcttgaagatgttcttggggcacttattgatggcgattgggatgtgcagctcgtactggtcctcagaaaacctcttatcc ttatacacgtcgtaggacagggttgtggttttcttgggattatctggattcttgttggcgataggggagttggctgggtgcacca ccagctcctccttcttcagggaggcgcgcctcatgaacagctctgctcctccgctcagcctgatctgtccgtgattgttctcgt caaacagcagcttgaagtacatggtgtgcagattgggtgtgccgtgagacttatcggaaaagtccttgttatagatctgga acatatacagcttgccctcctccaccagcttatccacctccttcttgctggcagactcgaagctcaccttatagccctgctcct ccacctctctgtaaaagccggcgatgtccttatacttctctgtctcagaaaagttgaaatcgtaggcattggaccactttggat accgggagatgctatccttaaagaagtcgatcagcttgtgacagtcattcaggttaaacatatcgcccttcttgaatgtgcc attcttgtagatcttctggatgtcctcgctggggttatagtaggccatccacttcttagaaaagaacacctttggcagcatctta ttagggccgggcagcagcttatagttgatcttctcgtaattgccgttcacatcgtccttgtcgatcttctgcaggcacttggcgt acttcttatccatgatggccagatagtacttggagccgtatctcaggatggtggcccgatagtctgtctccttatccttgtccca gccgcccatgaactgagggttctgaaaatacagcttgaacttatccttagagtagggcttctgggtcacataattgcggatg gcatcgtagatgtggtccaccttcagcaggatgtcgtaggccagcacaaaatcgccatagaaggactcgtccctgtttgtc tccttgccctcgccaaagaaggccttgatgtaattctcgaagctcttcacagaatccagcaggtccttcatgatggccacca cggcgtcgttcttcttcaggctcttctccagcacaaaatcggcgtcgaacagcttctcagaggagccatacaccttgtagat ctcatccaccttctggatgatgatctccttcagcttctccaccacagacagatcggcgtcggcgtactcctgcagctgctcca gagaaaaggagccgatcttcttgaaggactttctccgatcgtcctcgtacttctcggtcaccacggccttcttcttcaggtgg atatcgtcatactcggcattccacttgtcccggatcacgttccactcgccgaagatatccttggagattgtgctgatggcggg gccgttcttcacaaagatgccggcgctagagtactcgtcaaaattcttgaacagcttctccagcttcttgatggagctgaag atctcgctgttcttgttcagggtgtttctaaacacctccagcacctcctcatcggatgtatagccctcgccgtagaagctcaga gactcccgatcgctcagcacctgcttatacagtggcttaaacttaggcagcttctgcttggttttctgattatacaggttgatgta ctcgttcaggcccttgatcttctcgccgctctcggtcacgaagccgccgatgatggcgttatacacgtcgatgccctcctgtg tcagcacaaagttaaagaactcgccctcaaagaaatcctccacatcatagtcgctgttcaggatcttctccttgatctcctgc acctcgtgcttatcaaagatggcgtccaccttctcgaagatgtccatattagagatgtagcgggtcagattctcgttgataca cctgaaggcgatggatgtgctcttggcctcctcggaaaacatattctctctgttatcaaagaagccggtgaaggctgtggta aagccattgaagctgttcaccagggcgatctcgtccttatcgtccaggaactctggcaggattgtctcgatgatatccttctta aacagggacttgtagccctcgttgcccttgaaggccttggcgatctccttccgcagattgatctccaggttctccagctcctta ttctccttctcggttctggttttcttccggaacaggctgatgtaattgttcagattcttcagcttgatgctgtgcagcacgtcgttgat aaaagacagatagtagcgatccagcagcttcttcacgcccttataatcctcggctctcttctcgtcctccaccagcagccgc ttattgtcgatgttctcctgggtcttgcccacagggatggccttgaacctcagggtcttagacagggagtagcagtttgtaaa cttctccagcttgct

ggctgctgggactccgtggataccgaccttccgcttcttctttggggccatcttatcgtcatcgtctttgtaatcaatatcatgat ccttgtagtctccgtcgtggtccttatagtccat

GGCTGCAGAAGTAACACCAAACAACAGGGTGAGCATCGACAAAAGAAACAGTACC AAGCAAAT AAAT AGCGT AT GAAGGCAGGGCT AAAAAAATCCACAT AT AGCTGCT G CAT ATGCCAT CATCCAAGT AT AT CAAG AT CAAAAT AATT AT AAAACAT ACTT GTTT AT T AT AAT AGAT AGGT ACTCAAGGTT AGAGCAT AT GAAT AGATGCT GCAT AT GCCATC AT GT AT ATGCAT CAGT AAAACCCACAT CAACAT GTATACCTATCCT AG ATCG AT ATT TCCATCCAT CTT AAACTCGT AACT AT G AAG AT GTAT G ACACACACAT ACAGTTCCA AAATT AAT AAAT ACACCAGGT AGTTT G AAACAGT ATT CT ACTCCG AT CT AGAACG AA TGAACGACCGCCCAACCACACCACATCATCACAACCAAGCGAACAAAAAGCATCT CT GT AT ATGCATCAGT AAAACCCGCATCAACAT GT AT ACCT ATCCT AGATCGAT ATT TCCATCCAT CAT CTT CAATTCGT AACT AT GAAT ATGT ATGGCACACACAT ACAGAT C CAAAATT AAT AAATCCACCAGGT AGTTT G AAA CAG AATT CT ACTCCG ATCT AG AAC GACCGCCCAACCAGACCACATCATCACAACCAAGACAAAAAAAAGCATGAAAAGA

TGACCCGACAAACAAGTGCACGGCATATATTGAAATAAAGGAAAAGGGCAAACCA

AACCCT ATGCAACG AAACAAAAAAAAT CAT G AAATCGATCCCGTCTGCGG AACGG

CTAGAGCCATCCCAGGATTCCCCAAAGAGAAACACTGGCAAGTTAGCAATCAGAA

CGTGTCTGACGTACAGGTCGCATCCGTGTACGAACGCTAGCAGCACGGATCTAAC

ACAAACACGGATCT AACACAAACAT GAACAGAAGT AGAACT ACCGGGCCCT AACC

ATGGACCGGAACGCCGATCTAGAGAAGGTAGAGAGGGGGGGGGGGGGAGGACG

AGCGGCGTACCTTGAAGCGGAGGTGCCGACGGGTGGATTTGGGGGAGATCTGG

TT GT GT GT GT GTGCGCTCCGAACAACACGAGGTTGGGGAAAGAGGGT GT GGAGG

GGGTGTCTATTTATTACGGCGGGCGAGGAAGGGAAAGCGAAGGAGCGGTGGGAA

AGGAATCCCCCGTAGCTGCCGTGCCGTGAGAGGAGGAGGAGGCCGCCTGCCGT

GCCGGCTCACGTCTGCCGCTCCGCCACGCAATTTCTGGATGCCGACAGCGGAGC

AAGTCCAACGGTGGAGCGGAACTCTCGAGAGGGGTCCAGAGGCAGCGACAGAG

ATGCCGTGCCGTCTGCTTCGCTTGGCCCGACGCGACGCTGCTGGTTCGCTGGTT

GGTGTCCGTTAGACTCGTCGACGGCGTTTAACAGGCTGGCATTATCTACTCGAAA

CAAG AAAAAT GTTTCCTT AGTTTTTTT AATTT CTT AAAGGGT ATTT GTTT AATTTTT A

GTCACTTT ATTTT ATTCT ATTTT AT AT CT AAATT ATT AAAT AAAAAAACT AAAAT AGAG

TTTT AGTTTT CTT AATTT AGAGGCT AAAAT AGAAT AAAATAGAT GT ACT AAAAAAATT

AGTCTAT AAAAACCATT AACCCT AAACCCTAAAT GG ATGT ACT AAT AAAATGG AT G A

AGT ATT AT AT AGGT GAAGCT ATTTGCAAAAAAAAAGGAGAACACATGCACACT AAA

AAG AT AAAACT GT AG AGTCCT GTT GT C AAAAT ACT CAATT GTCCTTT AG ACCAT GTC

T AACT GTT C ATTT AT AT GATT CT CT AAAACACT GAT ATT ATT GT AGT ACT AT AG ATT A

T ATT ATTCGT AG AGT AAAGTTT AAAT ATATGT AT AAAG ATAGAT AAACT GCACTT CA

AACAAGT GT GACAAAAAAAAT AT GTGGT AATTTTTT AT AACTT AGACATGCAATGCT

CATT AT CTCT AGAGAGGGGCACGACCGGGTCACGCTGCA

aagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcaca tccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggc gaatgctagagcagcttgagcttggatcagattgtcgtttcccgccttcagtttaaactatcagtgtttgacaggatatattggc gggtaaacctaagagaaaagagcgtttattagaataacggatatttaaaagggcgtgaaaaggtttatccgttcgtccattt gtatgtgcatgccaaccacagggttcccctcgggatcaaagtactttgatccaacccctccgctgctatagtgcagtcggct tctgacgttcagtgcagccgtcttctgaaaacgacatgtcgcacaagtcctaagttacgcgacaggctgccgccctgccct tttcctggcgttttcttgtcgcgtgttttagtcgcataaagtagaatacttgcgactagaaccggagacattacgccatgaaca agagcgccgccgctggcctgctgggctatgcccgcgtcagcaccgacgaccaggacttgaccaaccaacgggccga actgcacgcggccggctgcaccaagctgttttccgagaagatcaccggcaccaggcgcgaccgcccggagctggcc aggatgcttgaccacctagccctggcgacgttgtgacagtgaccaggctagaccgcctggcccgcagcacccgcgac ctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggcagagccgtgggccgacacca ccacgccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaatcatcgaccgcacc cggagcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcaccccggcacagatcg cgcacgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcg accctgtaccgcgcacttgagcgcagcgaggaagtgacgcccaccgaggccaggcggcgcggtgccttccgtgagg acgcattgaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcacc aggacggccaggacgaaccgtttttcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagc cgcccgcgcacgtctcaaccgtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggc cagcttggccgctgaagaaaccgagcgccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcatgcg gtcgctgcgtatatgatgcgatgagtaaataaacaaatacgcaaggggaacgc

atgaaggttatcgctgtacttaaccagaaaggcgggtcaggcaagacgaccatcgcaacccatctagcccgcgccctg caactcgccggggccgatgttctgttagtcgattccgatccccagggcagtgcccgcgattgggcggccgtgcgggaag atcaaccgctaaccgttgtcggcatcgaccgcccgacgattgaccgcgacgtgaaggccatcggccggcgcgacttcg tagtgatcgacggagcgccccaggcggcggacttggctgtgtccgcgatcaaggcagccgacttcgtgctgattccggt gcagccaagcccttacgacatatgggcaaccgccgacctggtggagctggttaagcagcgcattgaggtcacggatgg aaggctacaagcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgcatcggcggtgaggttgccgaggcgctggc cgggtacgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcacaac cgttcttgaatcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcatt tga

gttaatgaggtaaagagaaaatgagcaaaagcacaaacacgctaagtgccggccgtccgagcgcacgcagcagca aggctgcaacgttggccagcctggcagacacgccagccatgaagcgggtcaactttcagttgccggcggaggatcac accaagctgaagatgtacgcggtacgccaaggcaagaccattaccgagctgctatctgaatacatcgcgcagctacca gagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaaggaggcggcatggaaaatcaagaacaac caggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttggccaggcgtaagcggctgggttgtctgcc ggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgac

ggtcgcaaaccatccggcccggtacaaatcggcgcggcgctgggtgatgacctggtggagaagttgaaggccgcgca ggccgcccagcggcaacgcatcgaggcagaagcacgccccggtgaatcgtggcaagcggccgctgatcgaatccg caaagaatcccggcaaccgccggcagccggtgcgccgtcgattaggaagccgcccaagggcgacgagcaaccag attttttcgttccgatgctctatgacgtgggcacccgcgatagtcgcagcatcatggacgtggccgttttccgtctgtcgaagc gtgaccgacgagctggcgaggtgatccgctacgagcttccagacgggcacgtagaggtttccgcagggccggccggc atggccagtgtgtgggattacgacctggtactgatggcggtttcccatctaaccgaatccatgaaccgataccgggaagg gaagggagacaagcccggccgcgtgttccgtccacacgttgcggacgtactcaagttctgccggcgagccgatggcg gaaagcagaaagacgacctggtagaaacctgcattcggttaaacaccacgcacgttgccatgcagcgtacgaagaa ggccaagaacggccgcctggtgacggtatccgagggtgaagccttgattagccgctacaagatcgtaaagagcgaaa ccgggcggccggagtacatcgagatcgagctagctgattggatgtaccgcgagatcacagaaggcaagaacccgga cgtgctgacggttcaccccgattactttttgatcgatcccggcatcggccgttttctctaccgcctggcacgccgcgccgcag gcaaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggagagttcaagaagttctgttt caccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggcggggcaggctggcccg atcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaa

tgtacggagcagatgctagggcaaattgccctagcaggggaaaaaggtcgaaaaggtctctttcc

tgtggatagcacgtacattgggaacccaaagccgtacattgggaaccggaacccgtacattgggaacccaaagccgta cattgggaaccggtcacacatgtaagtgactgatataaaagagaaaaaaggcgatttttccgcctaaaactctttaaaact tattaaaactcttaaaacccgcctggcctgtgcata

actgtctggccagcgcacagccgaagagctgcaaaaagcgcctacccttcggtcgctgcgctccctacgccccgccgc ttcgcgtcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccaggcaatctaccagggcgcggaca agccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgcgtttcggtgatgacggtgaaa acctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcag ggcgcgtcagcgggtgttggcgggtgtcgggg

cgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactga gagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagg

cgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcgg taatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagga accgtaaaaaggccgcgttgctggcgtt

tttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggact ataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtcc gcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagc tgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaa gacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagt tcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcgg aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg cgcagaaaaaaaggatctcaa

gaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcattctagg ta

ctaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcga catactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctc ccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagtt cctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatcc acatcggccagatcgttattcagtaagtaatccaattcggctaagcggctgtctaagctattcgtatagggacaatccgata tgtcgatggagtgaaagagcctgatgcactccgcatacagctcgataatcttttcagggctttgttcatcttcatactcttccga gcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacctttggaaca ggcagctttccttccagccatagcatcatgtccttttcccgttcaacatcataggtggtccctttataccggctgtccgtcattttt aaatataggttttcattttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtatttttcga tcagttttttcaattccggtgatattctcattttagccat

ttattatttccttcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcactgttcctt gcattctaaaaccttaaataccagaaaacagctttttcaaagttgttttcaaagttggcgtataacatagtatcgacggagcc gattttgaaaccgcggtgatcacaggcagcaacgctctgtcatcgttacaatcaacatgctaccctccgcgagatcatccg tgtttcaaacccggcagcttagttgccgttcttccgaatagcatcggtaacatgagcaaagtctgccgccttacaacggctc tcccgctgacgccgtcccggactgatgggctgcctgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttg gctggctggtggcaggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgt actgaattaacgccgaattaattcgggg

gatctggattttagtactggattttggttttaggaattagaaattttattgatagaagtattttacaaatacaaatacatactaagg gtttcttatatgctcaacacatgagcgaaaccctataggaaccctaattcccttatctgggaactactcacacattattatgga gaaa

ctcgagcttgtcgatcgacagatccggtcggcatctact

ctatttctttgccctcggacgagtgctggggcgtcggtttccactatcggcgagtacttctacacagccatcggtccagacgg ccgcgcttctgcgggcgatttgtgtacgcccgacagtcccggctccggatcggacgattgcgtcgcatcgaccctgcgcc caagctgcatcatcgaaattgccgtcaaccaagctctgatagagttggtcaagaccaatgcggagcatatacgcccgga gtcgtggcgatcctgcaagctccggatgcctccgctcgaagtagcgcgtctgctgctccatacaagccaaccacggcctc cagaagaagatgttggcgacctcgtattgggaatccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggcc attgtccgtcaggacattgttggagccgaaatccgcgtgcacgaggtgccggacttcggggcagtcctcggcccaaagc atcagctcatcgagagcctgcgcgacggacgcactgacggtgtcgtccatcacagtttgccagtgatacacatggggat cagcaatcgcgcatatgaaatcacgccatgtagtgtattgaccgattccttgcggtccgaatgggccgaacccgctcgtct ggctaagatcggccgcagcgatcgcatccatagcctccgcgaccggttgtagaacagcgggcagttcggtttcaggca ggtcttgcaacgtgacaccctgtgcacggcgggagatgcaataggtcaggctctcgctaaactccccaatgtcaagcac ttccggaatcgggagcgcggccgatgcaaagtgccgataaacataacgatctttgtagaaaccatcggcgcagctattt acccgcaggacatatccacgccctcctacatcgaagctgaaagcacgagattcttcgccctccgagagctgcatcaggt cggagacgctgtcgaacttttcgatcagaaacttctcgacagacgtcgcggtgagttcaggctttttcat

atctcattgccccccggatctgcgaaagctcgagagagatagatttgtagagagagactggtgatt

tcagcgtgtcctctccaaatgaaatgaacttccttatatagaggaaggtcttgcgaaggatagtgggattgtgcgtcatccct tacgtcagtggagatatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggt gggggtccatctttgggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgtaggt gccaccttccttttctactgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgatattaccctt tgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccat gttatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatcttt gggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgtaggtgccaccttccttttct actgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgatattaccctttgttgaaaagtctc a

atagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccatgttggcaagctgct ctagccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactgga aagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccgg ctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattac

SEQ ID NO: 2 pCXUN-OsU3-RCR1-RCR2-armed donor (with targets)-Nos- Ubi- LbCpf1-Nos (the“TDT” vector).

Bold = HH ribozyme

Bold and underlined = 1-6 nucleotides of HH ribozyme

Double underlined = HDV ribozyme

Wavy underlined = cRNA1 target sequences

Dashed jjnderJined = cRNA2 target sequences

First italic section = Armed donor (with targets) at each end Second italic section = LbCpfl

CAPITALS = Ubiquitin promoter

gaattcgagctc

aaggaatctttaaacatacgaacagatcacttaaagttcttctgaagcaacttaaagttatcaggcatgcatggatcttgga ggaatcagatgtgcagtcagggaccatagcacaagacaggcgtcttctactggtgctaccagcaaatgctggaagccg ggaacactgggtacgttggaaaccacgtgatgtgaagaagtaagataaactgtaggagaaaagcatttcgtagtgggc catgaagcctttcaggacatgtattgcagtatgggccggcccattacgcaattggacgacaacaaagactagtattagta ccacctcggctatccacatagatcaaagctgatttaaaagagttgtgcagatgatccgtggca

aaattactgatgagtccgtgaggacgaaacgagtaagctcgtctaatttctactaagtgtagat

ggtatggtggtgcaatgggagga

ggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgcttcggcatggcgaatgggacgaatacgacc aaattactq^^^^^g^^gacgaaacgagtaagctcgtctaatttctactaagtgtagat

aaccaacataatcccaacctcctcactaacaccaactaaacaacatacttcaacataacaaataaaac

cggtacctttgggtatggtggtgcaatgggaggattgatggggatggtagcttcctcatgaacattcaggagctggcattga tccgcattgagaacctccctgtgaaggtgatggtgttgaacaaccaacacctaggcatggtcgtccagttggaggatagg ttttacaaggcgaatagggcgcatacatacttgggcaacccggaatgtgagagcgagatatatccagattttgtgactatt gctaaggggttcaatattcctgcagtccgtgtaacaaagaagagtgaagtccgtgccgccatcaagaagatgctcgaga ctccagggccatacttgttggacatcatcgtcccgcaccaggagcatgtgctgcctatgatcccaattgggggcgcattca aggacatgatcctggatggtgatggcaggactgtgtattaatctataatctgtatgttggcaaagcaccagcccggcctatg tctgacgtgaatgactcataaagagtggtatgcctatgatgtttgtatgtgctctatcaataactaaggtgtcaactatgaacc atatgctcttctgttttacttgtttgatgtgcttggcatggtaatcctaattagcttcctgctgtttgacctgaatgacccataaaga gtg

gatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaatt acgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacattt aatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatc ggtacccctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgt aaaacgacggccagtgaattcccgatctagtaacatagatgacaccgcgcgcgataatttatcctagtttgcgcgctatatt ttgttttctatcgcgtattaaatgtataattgcgggactctaatcataaaaacccatctcataaataacgtcatgcattacatgtt aattattacatgcttaacgtaattcaacagaaattatatgataatcatcgcaagaccggcaacaggattcaatcttaagaa actttattgccaaatgtttgaacgatcggggaaattcggatcc

ttactttttcttttttgcctggccggcctttttcgtggccgccggccttttgtgcttcacgctggtctgggcgtactccagccactcct tgttagagatggcgatcttcaccttatccagcttctcgtcctcggccttcttgaa

ctggccgatggcccacagcacctttctggcgatgttataggcgccattggcgtcggcgttctttggcaggatggcattctcct gggcctcatagttccggctatcgtagaagatgccgtcggagttcttcacagggctgatcagaaaatccacgtcggtgcgg cctgtgatgctgttccgcatctgcagcatcaggctcatcagggccataaagctagagtagaaggccttgtcggactgctcg cacagcagggctctgatatcgccctgctgataattgatgccgtacttgttgaacagctccttataggcgctggtcaggcaca cctcctcccagtcgaacacgttgttcttcttaggattccggaagattctgatccggttgccgtaggagtacagcttccacttctt gatgtaatcggcgtctgtgcgagagaagttcttatagtccagggcaaactcgaacagatcctcctcgggcacgtacatga tcctgtcaaaggagctgatgaacttcttggaatcggcgatgctggtatacttggttttcagcaggttcacaaagccggtagat ggatcgatcttggatgtcagccaggcagggatgtaaaagatgaagccgttctgggtagacatggacttaaagctctcga acttattggtgatctgatagcccttcagggcgccgcctgttgcacaaggattagacttcttgtccaccatgtagttcagcttatc gatcagcatcttctcgaacttctgatacacctgcttctccaccttcacgcggctattcttaaagccagagttcaggtcctccag ggcgatcacggcatcgtacttctccaccagctcgcagatcttgtgcaccacctgagagatatagccggccttcagctcctt gatattctcgatggaggtccagttctggcgggcctcgaacctctccttctccttcttgtccagcagagagtggtaatctgtcttg atcctgatgccgttgaagttgttgatgatctcgttcagggaatactgctccacgatgttgcccttgccgtccaccaccacgat atacagcagattgcgctcgcccctatcgatgccgatcacataggggttatcgtcgtgcttcagcagcacgcgcacctctgt attgatcttgaagatgttcttggggcacttattgatggcgattgggatgtgcagctcgtactggtcctcagaaaacctcttatcc ttatacacgtcgtaggacagggttgtggttttcttgggattatctggattcttgttggcgataggggagttggctgggtgcacca ccagctcctccttcttcagggaggcgcgcctcatgaacagctctgctcctccgctcagcctgatctgtccgtgattgttctcgt caaacagcagcttgaagtacatggtgtgcagattgggtgtgccgtgagacttatcggaaaagtccttgttatagatctgga acatatacagcttgccctcctccaccagcttatccacctccttcttgctggcagactcgaagctcaccttatagccctgctcct ccacctctctgtaaaagccggcgatgtccttatacttctctgtctcagaaaagttgaaatcgtaggcattggaccactttggat accgggagatgctatccttaaagaagtcgatcagcttgtgacagtcattcaggttaaacatatcgcccttcttgaatgtgcc attcttgtagatcttctggatgtcctcgctggggttatagtaggccatccacttcttagaaaagaacacctttggcagcatctta ttagggccgggcagcagcttatagttgatcttctcgtaattgccgttcacatcgtccttgtcgatcttctgcaggcacttggcgt acttcttatccatgatggccagatagtacttggagccgtatctcaggatggtggcccgatagtctgtctccttatccttgtccca gccgcccatgaactgagggttctgaaaatacagcttgaacttatccttagagtagggcttctgggtcacataattgcggatg gcatcgtagatgtggtccaccttcagcaggatgtcgtaggccagcacaaaatcgccatagaaggactcgtccctgtttgtc tccttgccctcgccaaagaaggccttgatgtaattctcgaagctcttcacagaatccagcaggtccttcatgatggccacca cggcgtcgttcttcttcaggctcttctccagcacaaaatcggcgtcgaacagcttctcagaggagccatacaccttgtagat ctcatccaccttctggatgatgatctccttcagcttctccaccacagacagatcggcgtcggcgtactcctgcagctgctcca gagaaaaggagccgatcttcttgaaggactttctccgatcgtcctcgtacttctcggtcaccacggccttcttcttcaggtgg atatcgtcatactcggcattccacttgtcccggatcacgttccactcgccgaagatatccttggagattgtgctgatggcggg gccgttcttcacaaagatgccggcgctagagtactcgtcaaaattcttgaacagcttctccagcttcttgatggagctgaag atctcgctgttcttgttcagggtgtttctaaacacctccagcacctcctcatcggatgtatagccctcgccgtagaagctcaga gactcccgatcgctcagcacctgcttatacagtggcttaaacttaggcagcttctgcttggttttctgattatacaggttgatgta ctcgttcaggcccttgatcttctcgccgctctcggtcacgaagccgccgatgatggcgttatacacgtcgatgccctcctgtg tcagcacaaagttaaagaactcgccctcaaagaaatcctccacatcatagtcgctgttcaggatcttctccttgatctcctgc acctcgtgcttatcaaagatggcgtccaccttctcgaagatgtccatattagagatgtagcgggtcagattctcgttgataca cctgaaggcgatggatgtgctcttggcctcctcggaaaacatattctctctgttatcaaagaagccggtgaaggctgtggta aagccattgaagctgttcaccagggcgatctcgtccttatcgtccaggaactctggcaggattgtctcgatgatatccttctta aacagggacttgtagccctcgttgcccttgaaggccttggcgatctccttccgcagattgatctccaggttctccagctcctta ttctccttctcggttctggttttcttccggaacaggctgatgtaattgttcagattcttcagcttgatgctgtgcagcacgtcgttgat aaaagacagatagtagcgatccagcagcttcttcacgcccttataatcctcggctctcttctcgtcctccaccagcagccgc ttattgtcgatgttctcctgggtcttgcccacagggatggccttgaacctcagggtcttagacagggagtagcagtttgtaaa cttctccagcttgct

ggctgctgggactccgtggataccgaccttccgcttcttctttggggccatcttatcgtcatcgtctttgtaatcaatatcatgat ccttgtagtctccgtcgtggtccttatagtccat

GGCTGCAGAAGTAACACCAAACAACAGGGTGAGCATCGACAAAAGAAACAGTACC AAGCAAAT AAAT AGCGT AT GAAGGCAGGGCT AAAAAAATCCACAT AT AGCTGCT G CAT ATGCCAT CATCCAAGT AT ATC AAGAT CAAAAT AATT AT AAAACAT ACTT GTTT AT T AT AAT AGAT AGGT ACTCAAGGTT AGAGCAT AT GAAT AGATGCT GCAT AT GCCATC ATGTAT ATGCAT CAGT AAAACCCAC AT CAACAT GTATACCTATCCT AG ATCG AT ATT TCCATCCAT CTT AAACTCGT AACT AT G AAGAT GTAT G ACACACACAT ACAGTTCCA AAATT AAT AAAT ACACCAGGT AGTTT G AAACAGT ATT CT ACTCCG AT CT AG AACGAA TGAACGACCGCCCAACCACACCACATCATCACAACCAAGCGAACAAAAAGCATCT CT GT AT ATGCATCAGT AAAACCCGCATCAACAT GT AT ACCT ATCCT AGATCGAT ATT TCCATCCAT CAT CTT CAATTCGT AACT AT GAAT ATGT ATGGCACACACAT ACAGAT C CAAAATT AAT AAATCCACCAGGT AGTTT G AAA CAG AATT CT ACTCCG ATCT AG AAC GACCGCCCAACCAGACCACATCATCACAACCAAGACAAAAAAAAGCATGAAAAGA TGACCCGACAAACAAGTGCACGGCATATATTGAAATAAAGGAAAAGGGCAAACCA AACCCTATGCAACGAAACAAAAAAAATCATGAAATCGATCCCGTCTGCGGAACGG CTAGAGCCATCCCAGGATTCCCCAAAGAGAAACACTGGCAAGTTAGCAATCAGAA CGTGTCTGACGTACAGGTCGCATCCGTGTACGAACGCTAGCAGCACGGATCTAAC ACAAACACGGATCT AACACAAACAT GAACAGAAGT AGAACT ACCGGGCCCT AACC ATGGACCGGAACGCCGATCTAGAGAAGGTAGAGAGGGGGGGGGGGGGAGGACG AGCGGCGTACCTTGAAGCGGAGGTGCCGACGGGTGGATTTGGGGGAGATCTGG TT GT GT GT GT GTGCGCTCCGAACAACACGAGGTTGGGGAAAGAGGGT GT GGAGG GGGTGTCTATTTATTACGGCGGGCGAGGAAGGGAAAGCGAAGGAGCGGTGGGAA AGGAATCCCCCGTAGCTGCCGTGCCGTGAGAGGAGGAGGAGGCCGCCTGCCGT GCCGGCTCACGTCTGCCGCTCCGCCACGCAATTTCTGGATGCCGACAGCGGAGC AAGTCCAACGGTGGAGCGGAACTCTCGAGAGGGGTCCAGAGGCAGCGACAGAG ATGCCGTGCCGTCTGCTTCGCTTGGCCCGACGCGACGCTGCTGGTTCGCTGGTT GGTGTCCGTTAGACTCGTCGACGGCGTTTAACAGGCTGGCATTATCTACTCGAAA

CAAG AAAAAT GTTTCCTT AGTTTTTTT AATTT CTT AAAGGGT ATTT GTTT AATTTTT A

GTCACTTT ATTTT ATTCT ATTTT AT AT CT AAATT ATT AAAT AAAAAAACT AAAAT AGAG

TTTT AGTTTT CTT AATTT AGAGGCT AAAAT AGAAT AAAATAGAT GT ACT AAAAAAATT

AGTCTAT AAAAACCATT AACCCT AAACCCTAAAT GG ATGT ACT AAT AAAATGG AT G A

AGT ATT AT AT AGGT GAAGCT ATTTGCAAAAAAAAAGGAGAACACATGCACACT AAA

AAG AT AAAACT GT AG AGTCCT GTT GT C AAAAT ACT CAATT GTCCTTT AGACCAT GTC

T AACT GTT C ATTT AT AT GATT CT CT AAAACACT GAT ATT ATT GT AGT ACT AT AG ATT A

T ATT ATTCGT AG AGT AAAGTTT AAAT ATATGT AT AAAG ATAGAT AAACT GCACTT CA

AACAAGT GT GACAAAAAAAAT AT GTGGT AATTTTTT AT AACTT AGACATGCAATGCT

CATT AT CTCT AGAGAGGGGCACGACCGGGTCACGCTGCA

aagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcaca tccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggc gaatgctagagcagcttgagcttggatcagattgtcgtttcccgccttcagtttaaactatcagtgtttgacaggatatattggc gggtaaacctaagagaaaagagcgtttattagaataacggatatttaaaagggcgtgaaaaggtttatccgttcgtccattt gtatgtgcatgccaaccacagggttcccctcgggatcaaagtactttgatccaacccctccgctgctatagtgcagtcggct tctgacgttcagtgcagccgtcttctgaaaacgacatgtcgcacaagtcctaagttacgcgacaggctgccgccctgccct tttcctggcgttttcttgtcgcgtgttttagtcgcataaagtagaatacttgcgactagaaccggagacattacgccatgaaca agagcgccgccgctggcctgctgggctatgcccgcgtcagcaccgacgaccaggacttgaccaaccaacgggccga actgcacgcggccggctgcaccaagctgttttccgagaagatcaccggcaccaggcgcgaccgcccggagctggcc aggatgcttgaccacctagccctggcgacgttgtgacagtgaccaggctagaccgcctggcccgcagcacccgcgac ctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggcagagccgtgggccgacacca ccacgccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaatcatcgaccgcacc cggagcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcaccccggcacagatcg cgcacgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcg accctgtaccgcgcacttgagcgcagcgaggaagtgacgcccaccgaggccaggcggcgcggtgccttccgtgagg acgcattgaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcacc aggacggccaggacgaaccgtttttcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagc cgcccgcgcacgtctcaaccgtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggc cagcttggccgctgaagaaaccgagcgccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcatgcg gtcgctgcgtatatgatgcgatgagtaaataaacaaatacgcaaggggaacgc

atgaaggttatcgctgtacttaaccagaaaggcgggtcaggcaagacgaccatcgcaacccatctagcccgcgccctg caactcgccggggccgatgttctgttagtcgattccgatccccagggcagtgcccgcgattgggcggccgtgcgggaag atcaaccgctaaccgttgtcggcatcgaccgcccgacgattgaccgcgacgtgaaggccatcggccggcgcgacttcg tagtgatcgacggagcgccccaggcggcggacttggctgtgtccgcgatcaaggcagccgacttcgtgctgattccggt gcagccaagcccttacgacatatgggcaaccgccgacctggtggagctggttaagcagcgcattgaggtcacggatgg aaggctacaagcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgcatcggcggtgaggttgccgaggcgctggc cgggtacgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcacaac cgttcttgaatcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcatt tga

gttaatgaggtaaagagaaaatgagcaaaagcacaaacacgctaagtgccggccgtccgagcgcacgcagcagca aggctgcaacgttggccagcctggcagacacgccagccatgaagcgggtcaactttcagttgccggcggaggatcac accaagctgaagatgtacgcggtacgccaaggcaagaccattaccgagctgctatctgaatacatcgcgcagctacca gagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaaggaggcggcatggaaaatcaagaacaac caggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttggccaggcgtaagcggctgggttgtctgcc ggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgac

ggtcgcaaaccatccggcccggtacaaatcggcgcggcgctgggtgatgacctggtggagaagttgaaggccgcgca ggccgcccagcggcaacgcatcgaggcagaagcacgccccggtgaatcgtggcaagcggccgctgatcgaatccg caaagaatcccggcaaccgccggcagccggtgcgccgtcgattaggaagccgcccaagggcgacgagcaaccag attttttcgttccgatgctctatgacgtgggcacccgcgatagtcgcagcatcatggacgtggccgttttccgtctgtcgaagc gtgaccgacgagctggcgaggtgatccgctacgagcttccagacgggcacgtagaggtttccgcagggccggccggc atggccagtgtgtgggattacgacctggtactgatggcggtttcccatctaaccgaatccatgaaccgataccgggaagg gaagggagacaagcccggccgcgtgttccgtccacacgttgcggacgtactcaagttctgccggcgagccgatggcg gaaagcagaaagacgacctggtagaaacctgcattcggttaaacaccacgcacgttgccatgcagcgtacgaagaa ggccaagaacggccgcctggtgacggtatccgagggtgaagccttgattagccgctacaagatcgtaaagagcgaaa ccgggcggccggagtacatcgagatcgagctagctgattggatgtaccgcgagatcacagaaggcaagaacccgga cgtgctgacggttcaccccgattactttttgatcgatcccggcatcggccgttttctctaccgcctggcacgccgcgccgcag gcaaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggagagttcaagaagttctgttt caccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggcggggcaggctggcccg atcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaa

tgtacggagcagatgctagggcaaattgccctagcaggggaaaaaggtcgaaaaggtctctttcc

tgtggatagcacgtacattgggaacccaaagccgtacattgggaaccggaacccgtacattgggaacccaaagccgta cattgggaaccggtcacacatgtaagtgactgatataaaagagaaaaaaggcgatttttccgcctaaaactctttaaaact tattaaaactcttaaaacccgcctggcctgtgcata

actgtctggccagcgcacagccgaagagctgcaaaaagcgcctacccttcggtcgctgcgctccctacgccccgccgc ttcgcgtcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccaggcaatctaccagggcgcggaca agccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgcgtttcggtgatgacggtgaaa acctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcag ggcgcgtcagcgggtgttggcgggtgtcgggg

cgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactga gagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagg

cgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcgg taatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagga accgtaaaaaggccgcgttgctggcgtt

tttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggact ataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtcc gcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagc tgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaa gacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagt tcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcgg aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg cgcagaaaaaaaggatctcaa

gaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcattctagg ta

ctaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcga catactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctc ccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagtt cctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatcc acatcggccagatcgttattcagtaagtaatccaattcggctaagcggctgtctaagctattcgtatagggacaatccgata tgtcgatggagtgaaagagcctgatgcactccgcatacagctcgataatcttttcagggctttgttcatcttcatactcttccga gcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacctttggaaca ggcagctttccttccagccatagcatcatgtccttttcccgttcaacatcataggtggtccctttataccggctgtccgtcattttt aaatataggttttcattttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtatttttcga tcagttttttcaattccggtgatattctcattttagccat

ttattatttccttcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcactgttcctt gcattctaaaaccttaaataccagaaaacagctttttcaaagttgttttcaaagttggcgtataacatagtatcgacggagcc gattttgaaaccgcggtgatcacaggcagcaacgctctgtcatcgttacaatcaacatgctaccctccgcgagatcatccg tgtttcaaacccggcagcttagttgccgttcttccgaatagcatcggtaacatgagcaaagtctgccgccttacaacggctc tcccgctgacgccgtcccggactgatgggctgcctgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttg gctggctggtggcaggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgt actgaattaacgccgaattaattcgggg

gatctggattttagtactggattttggttttaggaattagaaattttattgatagaagtattttacaaatacaaatacatactaagg gtttcttatatgctcaacacatgagcgaaaccctataggaaccctaattcccttatctgggaactactcacacattattatgga gaaa

ctcgagcttgtcgatcgacagatccggtcggcatctact ctatttctttgccctcggacgagtgctggggcgtcggtttccactatcggcgagtacttctacacagccatcggtccagacgg ccgcgcttctgcgggcgatttgtgtacgcccgacagtcccggctccggatcggacgattgcgtcgcatcgaccctgcgcc caagctgcatcatcgaaattgccgtcaaccaagctctgatagagttggtcaagaccaatgcggagcatatacgcccgga gtcgtggcgatcctgcaagctccggatgcctccgctcgaagtagcgcgtctgctgctccatacaagccaaccacggcctc cagaagaagatgttggcgacctcgtattgggaatccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggcc attgtccgtcaggacattgttggagccgaaatccgcgtgcacgaggtgccggacttcggggcagtcctcggcccaaagc atcagctcatcgagagcctgcgcgacggacgcactgacggtgtcgtccatcacagtttgccagtgatacacatggggat cagcaatcgcgcatatgaaatcacgccatgtagtgtattgaccgattccttgcggtccgaatgggccgaacccgctcgtct ggctaagatcggccgcagcgatcgcatccatagcctccgcgaccggttgtagaacagcgggcagttcggtttcaggca ggtcttgcaacgtgacaccctgtgcacggcgggagatgcaataggtcaggctctcgctaaactccccaatgtcaagcac ttccggaatcgggagcgcggccgatgcaaagtgccgataaacataacgatctttgtagaaaccatcggcgcagctattt acccgcaggacatatccacgccctcctacatcgaagctgaaagcacgagattcttcgccctccgagagctgcatcaggt cggagacgctgtcgaacttttcgatcagaaacttctcgacagacgtcgcggtgagttcaggctttttcat

atctcattgccccccggatctgcgaaagctcgagagagatagatttgtagagagagactggtgatt

tcagcgtgtcctctccaaatgaaatgaacttccttatatagaggaaggtcttgcgaaggatagtgggattgtgcgtcatccct tacgtcagtggagatatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggt gggggtccatctttgggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgtaggt gccaccttccttttctactgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgatattaccctt tgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccat gttatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatcttt gggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgtaggtgccaccttccttttct actgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgatattaccctttgttgaaaagtctc aatagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccatgttggcaagctgc tctagccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactgg aaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccg gctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattac

SEQ ID NO: 3 pCXUN-OsU3-RCR1-RCR2- Ubi-LbCpf1-Nos- armed donor (with targets) (the“control” vector).

Bold = HH ribozyme

Bold and underlined = 1-6 nucleotides of HH ribozyme

Double underlined = HDV ribozyme

Wavy underlined = cRNA1 target sequences

Dashed jjnderJined = cRNA2 target sequences

First italic section = OsU3-RCR1-RCR2

Second italic section = LbCpfl

Third italic section = Armed donor (with targets at each end)

FIRST CAPITALS SECTION = OsU3 promoter

SECOND CAPITALS SECTION = Ubiquitin promoter

Gaattcgagctc

AAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAG

TTA TCAGGCA TGCA TGGA TCTTGGAGGAATCAGA TGTGCAGTCAGGGACCA TAGC

ACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATGCTGGAAGCCGGGAACA

CTGGGTACGTTGGAAACCACGTGATGTGAAGAAGTAAGATAAACTGTAGGAGAAA

AGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCG

GCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTCGGCTAT

CCACA TAG A TCAAAGCTGA TTTAAAAGAGTTG TGCAGA TGA TCCG TGGCA aaattactgatgagtccgtgaggacgaaacgagtaagctcgtctaatttctactaagtgtagat

ggtatggtggtgcaatgggagga

aaccaacataatcccaacctcctcactaacaccaactaaacaacatacttcaacataacaaataaaacaaatacaacc aaattactgatgagtccgtgaggacgaaacgagtaagctcgtctaatttctactaagtgtagat

aaccaacataatcccaacctcctcactaacaccaactaaacaacatacttcaacataacaaataaaac

cggtacccctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgt aaaacgacggccagtgaattccc

gatctagtaacatagatgacaccgcgcgcgataatttatcctagtttgcgcgctatattttgttttctatcgcgtattaaatgtata attgcgggactctaatcataaaaacccatctcataaataacgtcatgcattacatgttaattattacatgcttaacgtaattca acagaaattatatgataatcatcgcaagaccggcaacaggattcaatcttaagaaactttattgccaaatgtttgaacgatc ggggaaattcggatcc

ttactttttcttttttgcctggccggcctttttcgtggccgccggccttttgtgcttcacgctggtctgggcgtactccagccactcct tgttagagatggcgatcttcaccttatccagcttctcgtcctcggccttcttgaa

ctggccgatggcccacagcacctttctggcgatgttataggcgccattggcgtcggcgttctttggcaggatggcattctcct gggcctcatagttccggctatcgtagaagatgccgtcggagttcttcacagggctgatcagaaaatccacgtcggtgcgg cctgtgatgctgttccgcatctgcagcatcaggctcatcagggccataaagctagagtagaaggccttgtcggactgctcg cacagcagggctctgatatcgccctgctgataattgatgccgtacttgttgaacagctccttataggcgctggtcaggcaca cctcctcccagtcgaacacgttgttcttcttaggattccggaagattctgatccggttgccgtaggagtacagcttccacttctt gatgtaatcggcgtctgtgcgagagaagttcttatagtccagggcaaactcgaacagatcctcctcgggcacgtacatga tcctgtcaaaggagctgatgaacttcttggaatcggcgatgctggtatacttggttttcagcaggttcacaaagccggtagat ggatcgatcttggatgtcagccaggcagggatgtaaaagatgaagccgttctgggtagacatggacttaaagctctcga acttattggtgatctgatagcccttcagggcgccgcctgttgcacaaggattagacttcttgtccaccatgtagttcagcttatc gatcagcatcttctcgaacttctgatacacctgcttctccaccttcacgcggctattcttaaagccagagttcaggtcctccag ggcgatcacggcatcgtacttctccaccagctcgcagatcttgtgcaccacctgagagatatagccggccttcagctcctt gatattctcgatggaggtccagttctggcgggcctcgaacctctccttctccttcttgtccagcagagagtggtaatctgtcttg atcctgatgccgttgaagttgttgatgatctcgttcagggaatactgctccacgatgttgcccttgccgtccaccaccacgat atacagcagattgcgctcgcccctatcgatgccgatcacataggggttatcgtcgtgcttcagcagcacgcgcacctctgt attgatcttgaagatgttcttggggcacttattgatggcgattgggatgtgcagctcgtactggtcctcagaaaacctcttatcc ttatacacgtcgtaggacagggttgtggttttcttgggattatctggattcttgttggcgataggggagttggctgggtgcacca ccagctcctccttcttcagggaggcgcgcctcatgaacagctctgctcctccgctcagcctgatctgtccgtgattgttctcgt caaacagcagcttgaagtacatggtgtgcagattgggtgtgccgtgagacttatcggaaaagtccttgttatagatctgga acatatacagcttgccctcctccaccagcttatccacctccttcttgctggcagactcgaagctcaccttatagccctgctcct ccacctctctgtaaaagccggcgatgtccttatacttctctgtctcagaaaagttgaaatcgtaggcattggaccactttggat accgggagatgctatccttaaagaagtcgatcagcttgtgacagtcattcaggttaaacatatcgcccttcttgaatgtgcc attcttgtagatcttctggatgtcctcgctggggttatagtaggccatccacttcttagaaaagaacacctttggcagcatctta ttagggccgggcagcagcttatagttgatcttctcgtaattgccgttcacatcgtccttgtcgatcttctgcaggcacttggcgt acttcttatccatgatggccagatagtacttggagccgtatctcaggatggtggcccgatagtctgtctccttatccttgtccca gccgcccatgaactgagggttctgaaaatacagcttgaacttatccttagagtagggcttctgggtcacataattgcggatg gcatcgtagatgtggtccaccttcagcaggatgtcgtaggccagcacaaaatcgccatagaaggactcgtccctgtttgtc tccttgccctcgccaaagaaggccttgatgtaattctcgaagctcttcacagaatccagcaggtccttcatgatggccacca cggcgtcgttcttcttcaggctcttctccagcacaaaatcggcgtcgaacagcttctcagaggagccatacaccttgtagat ctcatccaccttctggatgatgatctccttcagcttctccaccacagacagatcggcgtcggcgtactcctgcagctgctcca gagaaaaggagccgatcttcttgaaggactttctccgatcgtcctcgtacttctcggtcaccacggccttcttcttcaggtgg atatcgtcatactcggcattccacttgtcccggatcacgttccactcgccgaagatatccttggagattgtgctgatggcggg gccgttcttcacaaagatgccggcgctagagtactcgtcaaaattcttgaacagcttctccagcttcttgatggagctgaag atctcgctgttcttgttcagggtgtttctaaacacctccagcacctcctcatcggatgtatagccctcgccgtagaagctcaga gactcccgatcgctcagcacctgcttatacagtggcttaaacttaggcagcttctgcttggttttctgattatacaggttgatgta ctcgttcaggcccttgatcttctcgccgctctcggtcacgaagccgccgatgatggcgttatacacgtcgatgccctcctgtg tcagcacaaagttaaagaactcgccctcaaagaaatcctccacatcatagtcgctgttcaggatcttctccttgatctcctgc acctcgtgcttatcaaagatggcgtccaccttctcgaagatgtccatattagagatgtagcgggtcagattctcgttgataca cctgaaggcgatggatgtgctcttggcctcctcggaaaacatattctctctgttatcaaagaagccggtgaaggctgtggta aagccattgaagctgttcaccagggcgatctcgtccttatcgtccaggaactctggcaggattgtctcgatgatatccttctta aacagggacttgtagccctcgttgcccttgaaggccttggcgatctccttccgcagattgatctccaggttctccagctcctta ttctccttctcggttctggttttcttccggaacaggctgatgtaattgttcagattcttcagcttgatgctgtgcagcacgtcgttgat aaaagacagatagtagcgatccagcagcttcttcacgcccttataatcctcggctctcttctcgtcctccaccagcagccgc ttattgtcgatgttctcctgggtcttgcccacagggatggccttgaacctcagggtcttagacagggagtagcagtttgtaaa cttctccagcttgct

ggctgctgggactccgtggataccgaccttccgcttcttctttggggccatcttatcgtcatcgtctttgtaatcaatatcatgat ccttgtagtctccgtcgtggtccttatagtccat

GGCTGCAGAAGTAACACCAAACAACAGGGTGAGCATCGACAAAAGAAACAGTACC AAGCAAAT AAAT AGCGT AT GAAGGCAGGGCT AAAAAAATCCACAT AT AGCTGCT G CAT ATGCCAT CATCCAAGT AT ATC AAGAT CAAAAT AATT AT AAAACAT ACTT GTTT AT T AT AAT AGAT AGGT ACTCAAGGTT AGAGCAT AT GAAT AGATGCT GCAT AT GCCATC AT GT AT ATGCAT CAGT AAAACCCACAT CAACAT GTATACCTATCCT AG ATCG AT ATT TCCATCCAT CTT AAACTCGT AACT AT G AAGAT GTAT G ACACACACAT ACAGTTCCA AAATT AAT AAAT ACACCAGGT AGTTT G AAACAGT ATT CT ACTCCG AT CT AGAACGAA TGAACGACCGCCCAACCACACCACATCATCACAACCAAGCGAACAAAAAGCATCT CT GT AT ATGCATCAGT AAAACCCGCATCAACAT GT AT ACCT ATCCT AGATCGAT ATT TCCATCCAT CAT CTT CAATTCGT AACT AT GAAT ATGT ATGGCACACACAT ACAGAT C CAAAATT AAT AAATCCACCAGGT AGTTT G AAA CAG AATT CT ACTCCG ATCT AG AAC GACCGCCCAACCAGACCACATCATCACAACCAAGACAAAAAAAAGCATGAAAAGA TGACCCGACAAACAAGTGCACGGCATATATTGAAATAAAGGAAAAGGGCAAACCA AACCCT ATGCAACG AAACAAAAAAAAT CAT G AAATCGATCCCGTCTGCGG AACGG CTAGAGCCATCCCAGGATTCCCCAAAGAGAAACACTGGCAAGTTAGCAATCAGAA CGTGTCTGACGTACAGGTCGCATCCGTGTACGAACGCTAGCAGCACGGATCTAAC ACAAACACGGATCT AACACAAACAT GAACAGAAGT AGAACT ACCGGGCCCT AACC ATGGACCGGAACGCCGATCTAGAGAAGGTAGAGAGGGGGGGGGGGGGAGGACG AGCGGCGTACCTTGAAGCGGAGGTGCCGACGGGTGGATTTGGGGGAGATCTGG TT GT GT GT GT GTGCGCTCCGAACAACACGAGGTTGGGGAAAGAGGGT GT GGAGG GGGTGTCTATTTATTACGGCGGGCGAGGAAGGGAAAGCGAAGGAGCGGTGGGAA AGGAATCCCCCGTAGCTGCCGTGCCGTGAGAGGAGGAGGAGGCCGCCTGCCGT GCCGGCTCACGTCTGCCGCTCCGCCACGCAATTTCTGGATGCCGACAGCGGAGC AAGTCCAACGGTGGAGCGGAACTCTCGAGAGGGGTCCAGAGGCAGCGACAGAG ATGCCGTGCCGTCTGCTTCGCTTGGCCCGACGCGACGCTGCTGGTTCGCTGGTT GGTGTCCGTTAGACTCGTCGACGGCGTTTAACAGGCTGGCATTATCTACTCGAAA CAAG AAAAAT GTTTCCTT AGTTTTTTT AATTT CTT AAAGGGT ATTT GTTT AATTTTT A GTCACTTT ATTTT ATTCT ATTTT AT AT CT AAATT ATT AAAT AAAAAAACT AAAAT AGAG TTTT AGTTTT CTT AATTT AGAGGCT AAAAT AGAAT AAAATAGAT GT ACT AAAAAAATT AGTCTAT AAAAACCATT AACCCT AAACCCTAAAT GG ATGT ACT AAT AAAATGG AT G A AGT ATT AT AT AGGT G AAGCT ATTTGCAAAAAAAAAGG AG AACACATGCACACT AAA AAGAT AAAACT GT AG AGTCCT GTT GT CAAAAT ACT CAATT GTCCTTT AG ACCAT GTC T AACT GTT C ATTT AT AT GATT CT CT AAAACACT GAT ATT ATT GT AGT ACT AT AG ATT A T ATT ATTCGT AG AGT AAAGTTT AAAT ATATGT AT AAAG ATAGAT AAACT GCACTT CA AACAAGT GT GACAAAAAAAAT AT GTGGT AATTTTTT AT AACTT AGACATGCAATGCT CATT AT CTCT AGAGAGGGGCACGACCGGGTCACGCTGCA

aagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcaca tccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggc gaatgctagagcagcttgagcttggatcagattgtcgtttcccgccttcagtttgtttaaacgtaaaacgacggccagtgaat tggagatcggtacttcgcgaatgcgtcgagatgacccaatgctctaga

aaccaacatttgggtatggtggtgcaatgggaggattgatggggatggtagcttcctcatgaacattcaggagctggcatt gatccgcattgagaacctccctgtgaaggtgatggtgttgaacaaccaacacctaggcatggtcgtccagttggaggata ggttttacaaggcgaatagggcgcatacatacttgggcaacccggaatgtgagagcgagatatatccagattttgtgact attgctaaggggttcaatattcctgcagtccgtgtaacaaagaagagtgaagtccgtgccgccatcaagaagatgctcga gactccagggccatacttgttggacatcatcgtcccgcaccaggagcatgtgctgcctatgatcccaattgggggcgcatt caaggacatgatcctggatggtgatggcaggactgtgtattaatctataatctgtatgttggcaaagcaccagcccggcct atgtctgacgtgaatgactcataaagagtggtatgcctatgatgtttgtatgtgctctatcaataactaaggtgtcaactatga accatatgctcttctgttttacttgtttgatgtgcttggcatggtaatcctaattagcttcctgctgtttgacctgaatgacccataa agagtggtatgccta

actagtccattgggtcatcggatgccgggaccgacgagtgcagaggcgtgcaagcgagcttggcgtaatcatggtcata gctgtttcctggtttaaacaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttattaga ataacggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtgcatgccaaccacagggttcccctcgg gatcaaagtactttgatccaacccctccgctgctatagtgcagtcggcttctg

acgttcagtgcagccgtcttctgaaaacgacatgtcgcacaagtcctaagttacgcgacaggctgccgccctgcccttttc ctggcgttttcttgtcgcgtgttttagtcgcataaagtagaatacttgcgactagaaccggagacattacgccatgaacaag agcgccgccgctggcctgctgggctatgcccgcgtcagcaccgacgaccaggacttgaccaaccaacgggccgaact gcacgcggccggctgcaccaagctgttttccgagaagatcaccggcaccaggcgcgaccgcccggagctggccagg atgcttgaccacctagccctggcgacgttgtgacagtgaccaggctagaccgcctggcccgcagcacccgcgacctact ggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggcagagccgtgggccgacaccaccac gccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaatcatcgaccgcacccgga gcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcaccccggcacagatcgcgca cgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcgaccc tgtaccgcgcacttgagcgcagcgaggaagtgacgcccaccgaggccaggcggcgcggtgccttccgtgaggacgc attgaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcaccagga cggccaggacgaaccgtttttcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagccgcc cgcgcacgtctcaaccgtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggccagct tggccgctgaagaaaccgagcgccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcatgcggtcgct gcgtatatgatgcgatgagtaaataaacaaatacgcaaggggaacgc

atgaaggttatcgctgtacttaaccagaaaggcgggtcaggcaagacgaccatcgcaacccatctagcccgcgccctg caactcgccggggccgatgttctgttagtcgattccgatccccagggcagtgcccgcgattgggcggccgtgcgggaag atcaaccgctaaccgttgtcggcatcgaccgcccgacgattgaccgcgacgtgaaggccatcggccggcgcgacttcg tagtgatcgacggagcgccccaggcggcggacttggctgtgtccgcgatcaaggcagccgacttcgtgctgattccggt gcagccaagcccttacgacatatgggcaaccgccgacctggtggagctggttaagcagcgcattgaggtcacggatgg aaggctacaagcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgcatcggcggtgaggttgccgaggcgctggc cgggtacgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcacaac cgttcttgaatcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcatt tga

gttaatgaggtaaagagaaaatgagcaaaagcacaaacacgctaagtgccggccgtccgagcgcacgcagcagca aggctgcaacgttggccagcctggcagacacgccagccatgaagcgggtcaactttcagttgccggcggaggatcac accaagctgaagatgtacgcggtacgccaaggcaagaccattaccgagctgctatctgaatacatcgcgcagctacca gagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaaggaggcggcatggaaaatcaagaacaac caggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttggccaggcgtaagcggctgggttgtctgcc ggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgac

ggtcgcaaaccatccggcccggtacaaatcggcgcggcgctgggtgatgacctggtggagaagttgaaggccgcgca ggccgcccagcggcaacgcatcgaggcagaagcacgccccggtgaatcgtggcaagcggccgctgatcgaatccg caaagaatcccggcaaccgccggcagccggtgcgccgtcgattaggaagccgcccaagggcgacgagcaaccag attttttcgttccgatgctctatgacgtgggcacccgcgatagtcgcagcatcatggacgtggccgttttccgtctgtcgaagc gtgaccgacgagctggcgaggtgatccgctacgagcttccagacgggcacgtagaggtttccgcagggccggccggc atggccagtgtgtgggattacgacctggtactgatggcggtttcccatctaaccgaatccatgaaccgataccgggaagg gaagggagacaagcccggccgcgtgttccgtccacacgttgcggacgtactcaagttctgccggcgagccgatggcg gaaagcagaaagacgacctggtagaaacctgcattcggttaaacaccacgcacgttgccatgcagcgtacgaagaa ggccaagaacggccgcctggtgacggtatccgagggtgaagccttgattagccgctacaagatcgtaaagagcgaaa ccgggcggccggagtacatcgagatcgagctagctgattggatgtaccgcgagatcacagaaggcaagaacccgga cgtgctgacggttcaccccgattactttttgatcgatcccggcatcggccgttttctctaccgcctggcacgccgcgccgcag gcaaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggagagttcaagaagttctgttt caccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggcggggcaggctggcccg atcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaa

tgtacggagcagatgctagggcaaattgccctagcaggggaaaaaggtcgaaaaggtctctttcc tgtggatagcacgtacattgggaacccaaagccgtacattgggaaccggaacccgtacattgggaacccaaagccgta cattgggaaccggtcacacatgtaagtgactgatataaaagagaaaaaaggcgatttttccgcctaaaactctttaaaact tattaaaactcttaaaacccgcctggcctgtgcata

actgtctggccagcgcacagccgaagagctgcaaaaagcgcctacccttcggtcgctgcgctccctacgccccgccgc ttcgcgtcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccaggcaatctaccagggcgcggaca agccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgcgtttcggtgatgacggtgaaa acctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcag ggcgcgtcagcgggtgttggcgggtgtcgggg

cgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactga gagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagg

cgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcgg taatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagga accgtaaaaaggccgcgttgctggcgtt

tttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggact ataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtcc gcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagc tgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaa gacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagt tcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcgg aaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacg cgcagaaaaaaaggatctcaa

gaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcattctagg ta

ctaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcga catactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctc ccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagtt cctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatcc acatcggccagatcgttattcagtaagtaatccaattcggctaagcggctgtctaagctattcgtatagggacaatccgata tgtcgatggagtgaaagagcctgatgcactccgcatacagctcgataatcttttcagggctttgttcatcttcatactcttccga gcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacctttggaaca ggcagctttccttccagccatagcatcatgtccttttcccgttcaacatcataggtggtccctttataccggctgtccgtcattttt aaatataggttttcattttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtatttttcga tcagttttttcaattccggtgatattctcattttagccat

ttattatttccttcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcactgttcctt gcattctaaaaccttaaataccagaaaacagctttttcaaagttgttttcaaagttggcgtataacatagtatcgacggagcc gattttgaaaccgcggtgatcacaggcagcaacgctctgtcatcgttacaatcaacatgctaccctccgcgagatcatccg tgtttcaaacccggcagcttagttgccgttcttccgaatagcatcggtaacatgagcaaagtctgccgccttacaacggctc tcccgctgacgccgtcccggactgatgggctgcctgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttg gctggctggtggcaggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgt actgaattaacgccgaattaattcgggg

gatctggattttagtactggattttggttttaggaattagaaattttattgatagaagtattttacaaatacaaatacatactaagg gtttcttatatgctcaacacatgagcgaaaccctataggaaccctaattcccttatctgggaactactcacacattattatgga gaaa

ctcgagcttgtcgatcgacagatccggtcggcatctact

ctatttctttgccctcggacgagtgctggggcgtcggtttccactatcggcgagtacttctacacagccatcggtccagacgg ccgcgcttctgcgggcgatttgtgtacgcccgacagtcccggctccggatcggacgattgcgtcgcatcgaccctgcgcc caagctgcatcatcgaaattgccgtcaaccaagctctgatagagttggtcaagaccaatgcggagcatatacgcccgga gtcgtggcgatcctgcaagctccggatgcctccgctcgaagtagcgcgtctgctgctccatacaagccaaccacggcctc cagaagaagatgttggcgacctcgtattgggaatccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggcc attgtccgtcaggacattgttggagccgaaatccgcgtgcacgaggtgccggacttcggggcagtcctcggcccaaagc atcagctcatcgagagcctgcgcgacggacgcactgacggtgtcgtccatcacagtttgccagtgatacacatggggat cagcaatcgcgcatatgaaatcacgccatgtagtgtattgaccgattccttgcggtccgaatgggccgaacccgctcgtct ggctaagatcggccgcagcgatcgcatccatagcctccgcgaccggttgtagaacagcgggcagttcggtttcaggca ggtcttgcaacgtgacaccctgtgcacggcgggagatgcaataggtcaggctctcgctaaactccccaatgtcaagcac ttccggaatcgggagcgcggccgatgcaaagtgccgataaacataacgatctttgtagaaaccatcggcgcagctattt acccgcaggacatatccacgccctcctacatcgaagctgaaagcacgagattcttcgccctccgagagctgcatcaggt cggagacgctgtcgaacttttcgatcagaaacttctcgacagacgtcgcggtgagttcaggctttttcat

atctcattgccccccggatctgcgaaagctcgagagagatagatttgtagagagagactggtgatt

tcagcgtgtcctctccaaatgaaatgaacttccttatatagaggaaggtcttgcgaaggatagtgggattgtgcgtcatccct tacgtcagtggagatatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggt gggggtccatctttgggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgtaggt gccaccttccttttctactgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgatattaccctt tgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccat gttatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatcttt gggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgtaggtgccaccttccttttct actgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgatattaccctttgttgaaaagtctc a

atagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccatgttggcaagctgct ctagccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactgga aagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccgg ctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattac SEQ ID NO: 4 - Armed donor (with targets)

aaccaacatttgggtatggtggtgcaatgggaggattgatggggatggtagcttcctcatgaacattcaggagctggcatt gatccgcattgagaacctccctgtgaaggtgatggtgttgaacaaccaacacctaggcatggtcgtccagttggaggata ggttttacaaggcgaatagggcgcatacatacttgggcaacccggaatgtgagagcgagatatatccagattttgtgact attgctaaggggttcaatattcctgcagtccgtgtaacaaagaagagtgaagtccgtgccgccatcaagaagatgctcga gactccagggccatacttgttggacatcatcgtcccgcaccaggagcatgtgctgcctatgatcccaattgggggcgcatt caaggacatgatcctggatggtgatggcaggactgtgtattaatctataatctgtatgttggcaaagcaccagcccggcct atgtctgacgtgaatgactcataaagagtggtatgcctatgatgtttgtatgtgctctatcaataactaaggtgtcaactatga accatatgctcttctgttttacttgtttgatgtgcttggcatggtaatcctaattagcttcctgctgtttgacctgaatgacccataa agagtggtatgccta

SEQ ID NO: 5 - Core sequence

Bold = Mutated target 1

Bold and underlined = W548L

Double underlined = Mutated EcoR V site

Italic = S627I

Dashed_underjine_d = Mutated target 2 cctaggcatggtcgtccagttggaggataggttttacaaggcgaatagggcgcatacatacttgggcaacccggaat gtgagagcgagatatatccagattttgtgactattgctaaggggttcaatattcctgcagtccgtgtaacaaagaagagtga agtccgtgccgccatcaagaagatgctcgagactccagggccatacttgttggacatcatcgtcccgcaccaggagcat gtgctgcctatgatcccaaffgggggcgcattcaaggacatgatcctggatggtgatggcaggactgtgtattaatctataat ctgtatgttggcaaagcaccagcccggcctatgtctgacgtg_aa_tgactcataaagagtg

SEQ ID NO: 6 -Wild type sequence of OsALS gene between target 1 and target 2 tttgggtatggtggtgcaatgggaggataggttttacaaggcgaatagggcgcatacatacttgggcaacccggaatgtg agagcgagatatatccagattttgtgactattgctaaggggttcaatattcctgcagtccgtgtaacaaagaagagtgaagt ccgtgccgccatcaagaagatgctcgagactccagggccatacttgttggatatcatcgtcccgcaccaggagcatgtgc tgcctatgatcccaagtgggggcgcattcaaggacatgatcctggatggtgatggcaggactgtgtattaatctataatctgt atgttggcaaagcaccagcccggcctatgtttgacctgaatgacccataaagagtg

SEQ ID NO: 7 - Lachnospiraceae bacterium ND2006 Cpf1 (LbCpfl) amino acid sequence with two NLS at the 5’ and 3’ end.

1 MAPKKKRKVG IHGVPAASKL EKFTNCYSLS KTLRFKAIPV GKTQENIDNK

51 RLLVEDEKRA EDYKGVKKLL DRYYLSFIND VLHSIKLKNL NNYISLFRKK

101 TRTEKENKEL ENLEINLRKE IAKAFKGNEG YKSLFKKDII ETILPEFLDD

151 KDEIALVNSF NGFTTAFTGF FDNRENMFSE EAKSTSIAFR CINENLTRYI 201 SNMDIFEKVD AIFDKHEVQE IKEKILNSDY DVEDFFEGEF FNFVLTQEGI 251 DVYNAIIGGF VTESGEKIKG LNEYINLYNQ KTKQKLPKFK PLYKQVLSDR 301 ESLSFYGEGY TSDEEVLEVF RNTLNKNSEI FSSIKKLEKL FKNFDEYSSA 351 GIFVKNGPAI STISKDIFGE WNVIRDKWNA EYDDIHLKKK AWTEKYEDD 401 RRKSFKKIGS FSLEQLQEYA DADLSVVEKL KEIIIQKVDE IYKVYGSSEK

451 LFDADFVLEK SLKKNDAVVA IMKDLLDSVK SFENYIKAFF GEGKETNRDE 501 SFYGDFVLAY DILLKVDHIY DAIRNYVTQK PYSKDKFKLY FQNPQFMGGW 551 DKDKETDYRA TILRYGSKYY LAIMDKKYAK CLQKIDKDDV NGNYEKINYK 601 LLPGPNKMLP KVFFSKKWMA YYNPSEDIQK IYKNGTFKKG DMFNLNDCHK 651 LIDFFKDSIS RYPKWSNAYD FNFSETEKYK DIAGFYREVE EQGYKVSFES 701 ASKKEVDKLV EEGKLYMFQI YNKDFSDKSH GTPNLHTMYF KLLFDENNHG 751 QIRLSGGAEL FMRRASLKKE ELVVHPANSP IANKNPDNPK KTTTLSYDVY 801 KDKRFSEDQY ELHIPIAINK CPKNIFKINT EVRVLLKHDD NPYVIGIDRG

851 ERNLLYIVW DGKGNIVEQY SLNEIINNFN GIRIKTDYHS LLDKKEKERF

901 EARQNWTSIE NIKELKAGYI SQVVHKICEL VEKYDAVIAL EDLNSGFKNS 951 RVKVEKQVYQ KFEKMLIDKL NYMVDKKSNP CATGGALKGY QITNKFESFK 1001 SMSTQNGFIF YIPAWLTSKI DPSTGFVNLL KTKYTSIADS KKFISSFDRI 1051 MYVPEEDLFE FALDYKNFSR TDADYIKKWK LYSYGNRIRI FRNPKKNNVF 1101 DWEEVCLTSA YKELFNKYGI NYQQGDIRAL LCEQSDKAFY SSFMALMSLM 1151 LQMRNSITGR TDVDFLISPV KNSDGIFYDS RNYEAQENAI LPKNADANGA 1201 YNIARKVLWA IGQFKKAEDE KLDKVKIAIS NKEWLEYAQT SVKH KRPAAT 1251 KKAGQAKKKK *

SEQ ID NO: 8 - Lachnospiraceae bacterium ND2006 Cpf1 (LbCpfl) nucleic acid sequence Bold = 3xFlag tag

Underlined = NLS

Italic = Cpf1 atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatgQ ccccaaagaagaagcggaaggtcggtatccacggagtcccagcagcc aacaaactaaaaaaatttacaaactgcta ctccctgtctaagaccctgaggttcaaggccatccctgtgggcaagacccaggagaacatcgacaataagcggctgct ggtggaggacgagaagagagccgaggattataagggcgtgaagaagctgctggatcgctactatctgtcttttatcaac gacgtgctgcacagcatcaagctgaagaatctgaacaattacatcagcctgttccggaagaaaaccagaaccgagaa ggagaataaggagctggagaacctggagatcaatctgcggaaggagatcgccaaggccttcaagggcaacgaggg ctacaagtccctgtttaagaaggatatcatcgagacaatcctgccagagttcctggacgataaggacgagatcgccctgg tgaacagcttcaatggctttaccacagccttcaccggcttctttgataacagagagaatatgttttccgaggaggccaaga gcacatccatcgccttcaggtgtatcaacgagaatctgacccgctacatctctaatatggacatcttcgagaaggtggacg ccatctttgataagcacgaggtgcaggagatcaaggagaagatcctgaacagcgactatgatgtggaggatttctttgag ggcgagttctttaactttgtgctgacacaggagggcatcgacgtgtataacgccatcatcggcggcttcgtgaccgagagc ggcgagaagatcaagggcctgaacgagtacatcaacctgtataatcagaaaaccaagcagaagctgcctaagtttaa gccactgtataagcaggtgctgagcgatcgggagtctctgagcttctacggcgagggctatacatccgatgaggaggtg ctggaggtgtttagaaacaccctgaacaagaacagcgagatcttcagctccatcaagaagctggagaagctgttcaag aattttgacgagtactctagcgccggcatctttgtgaagaacggccccgccatcagcacaatctccaaggatatcttcggc gagtggaacgtgatccgggacaagtggaatgccgagtatgacgatatccacctgaagaagaaggccgtggtgaccga gaagtacgaggacgatcggagaaagtccttcaagaagatcggctccttttctctggagcagctgcaggagtacgccga cgccgatctgtctgtggtggagaagctgaaggagatcatcatccagaaggtggatgagatctacaaggtgtatggctcct ctgagaagctgttcgacgccgattttgtgctggagaagagcctgaagaagaacgacgccgtggtggccatcatgaagg acctgctggattctgtgaagagcttcgagaattacatcaaggccttctttggcgagggcaaggagacaaacagggacga gtccttctatggcgattttgtgctggcctacgacatcctgctgaaggtggaccacatctacgatgccatccgcaattatgtga cccagaagccctactctaaggataagttcaagctgtattttcagaaccctcagttcatgggcggctgggacaaggataag gagacagactatcgggccaccatcctgagatacggctccaagtactatctggccatcatggataagaagtacgccaagt gcctgcagaagatcgacaaggacgatgtgaacggcaattacgagaagatcaactataagctgctgcccggccctaat aagatgctgccaaaggtgttcttttctaagaagtggatggcctactataaccccagcgaggacatccagaagatctacaa gaatggcacattcaagaagggcgatatgtttaacctgaatgactgtcacaagctgatcgacttctttaaggatagcatctcc cggtatccaaagtggtccaatgcctacgatttcaacttttctgagacagagaagtataaggacatcgccggcttttacaga gaggtggaggagcagggctataaggtgagcttcgagtctgccagcaagaaggaggtggataagctggtggaggagg gcaagctgtatatgttccagatctataacaaggacttttccgataagtctcacggcacacccaatctgcacaccatgtactt caagctgctgtttgacgagaacaatcacggacagatcaggctgagcggaggagcagagctgttcatgaggcgcgcct ccctgaagaaggaggagctggtggtgcacccagccaactcccctatcgccaacaagaatccagataatcccaagaa aaccacaaccctgtcctacgacgtgtataaggataagaggttttctgaggaccagtacgagctgcacatcccaatcgcc atcaataagtgccccaagaacatcttcaagatcaatacagaggtgcgcgtgctgctgaagcacgacgataacccctatg tgatcggcatcgataggggcgagcgcaatctgctgtatatcgtggtggtggacggcaagggcaacatcgtggagcagta ttccctgaacgagatcatcaacaacttcaacggcatcaggatcaagacagattaccactctctgctggacaagaaggag aaggagaggttcgaggcccgccagaactggacctccatcgagaatatcaaggagctgaaggccggctatatctctcag gtggtgcacaagatctgcgagctggtggagaagtacgatgccgtgatcgccctggaggacctgaactctggctttaaga atagccgcgtgaaggtggagaagcaggtgtatcagaagttcgagaagatgctgatcgataagctgaactacatggtgg acaagaagtctaatccttgtgcaacaggcggcgccctgaagggctatcagatcaccaataagttcgagagctttaagtcc atgtctacccagaacggcttcatcttttacatccctgcctggctgacatccaagatcgatccatctaccggctttgtgaacctg ctgaaaaccaagtataccagcatcgccgattccaagaagttcatcagctcctttgacaggatcatgtacgtgcccgagga ggatctgttcgagtttgccctggactataagaacttctctcgcacagacgccgattacatcaagaagtggaagctgtactcc tacggcaaccggatcagaatcttccggaatcctaagaagaacaacgtgttcgactgggaggaggtgtgcctgaccagc gcctataaggagctgttcaacaagtacggcatcaattatcagcagggcgatatcagagccctgctgtgcgagcagtccg acaaggccttctactctagctttatggccctgatgagcctgatgctgcagatgcggaacagcatcacaggccgcaccgac gtggattttctgatcagccctgtgaagaactccgacggcatcttctacgatagccggaactatgaggcccaggagaatgc catcctgccaaagaacgccgacgccaatggcgcctataacatcgccagaaaggtgctgtgggccatcggccagttcaa gaaggccgaggacgagaagctggataaggtgaagatcgccatctctaacaaggagtggctggagtacgcccagacc agcafaaagcacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagtaa

SEQ ID NO: 9 - Nucleotide sequence of T7 promoter

taatacgactcactata

SEQ ID NO: 10 - Nucleotide sequence of OsU3 promoter

Aaggaatctttaaacatacgaacagatcacttaaagttcttctgaagcaacttaaagttatcaggcatgcatggatcttgga ggaatcagatgtgcagtcagggaccatagcacaagacaggcgtcttctactggtgctaccagcaaatgctggaagccg ggaacactgggtacgttggaaaccacgtgatgtgaagaagtaagataaactgtaggagaaaagcatttcgtagtgggc catgaagcctttcaggacatgtattgcagtatgggccggcccattacgcaattggacgacaacaaagactagtattagta ccacctcggctatccacatagatcaaagctgatttaaaagagttgtgcagatgatccgtggc

SEQ ID NO: 11- Nucleotide sequence of FnCpfl

atgtccatctaccaggagttcgtgaacaagtactccctctccaagaccctccgcttcgagctcattccacagggcaagac cctcgagaatatcaaagcccgcggcctcattctcgacgatgaaaagcgcgccaaggactacaagaaggccaagcag atcatcgacaagtaccaccagttcttcatcgaggagatcctctcctccgtgtgcatttccgaggacctcctccagaactact ccgacgtgtacttcaagctcaagaagtccgacgacgacaacctccagaaggacttcaagtccgccaaggacaccatc aagaagcagatctccgagtacatcaaggactccgagaagttcaagaacctcttcaaccagaacctcatcgacgccaa gaagggccaagagtccgaccttatcctctggctcaagcagtccaaggacaacggcatcgagctcttcaaggccaactc cgacatcaccgacattgacgaggccctcgagatcatcaagtccttcaagggctggaccacctacttcaagggcttccac gagaaccgcaagaacgtgtactcctccaacgacatcccgacctccatcatctaccgcatcgtggacgacaacctcccg aagttcctcgagaacaaggccaagtacgagtccctcaaggataaagccccggaggccattaactacgagcagatcaa gaaggacctcgccgaggagctcacattcgacatcgactacaagacctccgaggtgaaccagagggtgttctccctcga cgaggtgttcgagatcgccaacttcaacaactacctcaaccagtccggcatcaccaagttcaacaccatcatcggcggc aagttcgtgaatggcgagaacaccaagcgcaagggcatcaacgagtacatcaacctctactcccagcagatcaacga caagaccctcaagaagtacaagatgtccgtgctcttcaagcagatcctctccgacaccgagtccaagtccttcgtgatcg acaagctcgaggacgattccgatgtggtgaccaccatgcagtccttctacgagcagatcgccgccttcaaaaccgtgga ggagaagtccatcaaggagaccctctccctcctcttcgatgacctcaaggcccagaaactcgacctctccaagatctact tcaagaacgacaagtccctcaccgatctctcccagcaggtgttcgacgactactccgtgattggcacagccgtgctcgag tatatcacccagcagatcgccccgaagaatctcgacaacccgtccaagaaggagcaagagctcatcgccaagaaga ccgagaaggccaagtacctctccctcgagaccatcaaactcgccctcgaggagttcaataagcaccgcgacatcgac aaacagtgccgcttcgaggaaatcctcgccaactttgccgccatcccgatgatcttcgacgagatcgcccagaacaagg acaacctcgcccagatctccatcaagtaccagaaccagggcaagaaggaccttctccaagcctccgctgaagacgat gtgaaggccatcaaggacctcctcgaccagaccaacaacctcctccacaagctcaagatcttccacatctcccagtccg aggacaaggccaacatcctcgacaaggacgagcacttctacctcgtgttcgaggagtgctacttcgagctcgccaacat cgtgccgctctacaacaagatccgcaactacatcacccagaagccgtactccgacgagaagttcaagctcaacttcga gaactccaccctcgctaatggctgggacaagaacaaggagccggacaacaccgccatcctcttcatcaaggacgaca agtactacctcggcgtgatgaacaagaagaacaacaagatcttcgacgacaaggccatcaaggagaacaagggcg agggctacaagaagatcgtgtacaagcttctcccgggcgccaataaaatgctcccgaaggtgttcttctccgccaagtcc atcaagttctacaacccgtccgaggacattctccgcatcaggaaccattccacccacaccaaaaacggctccccgcaa aagggctacgagaagttcgagttcaacatcgaggactgccgcaagttcatcgacttctacaagcagtccatctccaagc acccggagtggaaggatttcggcttccgcttttccgatacccagcgctacaactccatcgacgagttctaccgcgaggtgg agaaccaaggctacaagctcaccttcgagaacatctccgagtcctacatcgactccgtggtgaaccagggcaagctcta cctcttccagatctacaacaaggacttctccgcctattctaagggccgcccaaatctccataccctctactggaaggccctc ttcgacgagaggaacctccaagacgtggtgtacaagctcaatggcgaggccgagctcttttatcgcaagcagtccatcc cgaagaagattacccacccggccaaagaagctatcgccaacaagaacaaggacaacccgaagaaggagtccgtgt tcgagtacgacctcatcaaggacaagcgcttcaccgaggacaagttcttcttccactgcccgatcaccatcaacttcaagt cctccggcgccaacaagttcaacgacgagatcaacctcctcctcaaggagaaggccaacgacgtgcacatcctctcta ttgaccgcggcgaaaggcatctcgcctactatacactcgtggacggcaagggcaacatcatcaagcaggacaccttca acatcatcggcaacgaccgcatgaagaccaactaccacgacaagctcgccgccatcgagaaagatcgcgattccgc caggaaggactggaagaagatcaacaacatcaaggagatgaaggagggctacctctcccaagtggtgcacgagatt gccaagctcgtgatcgagtacaacgccatcgtggtgttcgaggacctcaacttcggcttcaaacgcggccgcttcaaggt tgagaagcaggtgtaccagaagctcgagaagatgctcatcgagaagctcaactacctcgtgttcaaggacaacgagtt cgacaagacaggcggagtgctcagggcctatcaactcacagccccgttcgagaccttcaagaagatgggcaagcag accggcatcatctattatgtgccggccggcttcacctctaaaatttgcccggtgaccggctttgtgaatcagctctacccgaa gtacgagtccgtgtccaagtcccaggagttcttctccaagttcgacaagatctgctacaacctcgacaagggctacttcga gttctccttcgactacaagaacttcggcgacaaggccgccaaaggcaagtggaccatcgcctcctttggatcccgcctca tcaatttccgcaactccgacaagaaccacaactgggacaccagggaggtgtatccgaccaaggagctcgagaagctc ctcaaggactactccatcgagtatggccacggcgagtgtattaaggccgccatttgcggcgaatccgacaagaagttctt cgccaagctcacctccgtgctcaacaccatcctccagatgcgcaactccaaaaccggaaccgagctcgactacctcatt tccccagtggccgatgtgaacggcaacttcttcgactctcgccaggccccaaaaaacatgccacaagacgccgatgcc aatggcgcctatcacatcggactcaagggactcatgcttctcggccgcatcaagaataaccaggagggcaagaagctc aacctcgtgatcaagaacgaggagtacttcgagttcgtgcagaaccgcaacaacgcagcagcgggtgggcgcgccg ggaagcggcggtcgcgggcgtcgaagcgctga

SEQ ID NO: 12 - Amino acid sequence of FnCpfl

MSIYQEFWK YSLSKTLRFE LIPQGKTLEN IKARGLILDD EKRAKDYKKA KQIIDKYHQF FIEEILSSVC ISEDLLQNYS DVYFKLKKSD DDNLQKDFKS AKDTIKKQIS EYIKDSEKFK NLFNQNLIDA KKGQESDLIL WLKQSKDNGI ELFKANSDIT DIDEALEIIK SFKGWTTYFK GFHENRKNVY SSNDIPTSII YRIVDDNLPK FLENKAKYES LKDKAPEAIN YEQIKKDLAE ELTFDIDYKT SEWQRVFSL DEVFEIANFN NYLNQSGITK FNTIIGGKFV NGENTKRKGI NEYINLYSQQ INDKTLKKYK MSVLFKQILS DTESKSFVID KLEDDSDWT TMQSFYEQIA AFKTVEEKSI KETLSLLFDD LKAQKLDLSK IYFKNDKSLT DLSQQVFDDY SVIGTAVLEY ITQQIAPKNL DNPSKKEQEL IAKKTEKAKY LSLETIKLAL EEFNKHRDID KQCRFEEILA NFAAIPMIFD EIAQNKDNLA QISIKYQNQG KKDLLQASAE DDVKAIKDLL DQTNNLLHKL KIFHISQSED KANILDKDEH FYLVFEECYF ELANIVPLYN KIRNYITQKP YSDEKFKLNF ENSTLANGWD KNKEPDNTAI LFIKDDKYYL GVMNKKNNKI FDDKAIKENK GEGYKKIVYK LLPGANKMLP KVFFSAKSIK FYNPSEDILR IRNHSTHTKN GSPQKGYEKF EFNIEDCRKF IDFYKQSISK HPEWKDFGFR FSDTQRYNSI DEFYREVENQ GYKLTFENIS ESYIDSVWQ GKLYLFQIYN KDFSAYSKGR PNLHTLYWKA LFDERNLQDV VYKLNGEAEL FYRKQSIPKK ITHPAKEAIA NKNKDNPKKE SVFEYDLIKD KRFTEDKFFF HCPITINFKS SGANKFNDEI NLLLKEKAND VHILSIDRGE RHLAYYTLVD GKGNIIKQDT FNIIGNDRMK TNYHDKLAAI EKDRDSARKD WKKINNIKEM KEGYLSQWH EIAKLVIEYN AIWFEDLNF GFKRGRFKVE KQVYQKLEKM LIEKLNYLVF KDNEFDKTGG VLRAYQLTAP FETFKKMGKQ TGIIYYVPAG FTSKICPVTG FWQLYPKYE SVSKSQEFFS KFDKICYNLD KGYFEFSFDY KNFGDKAAKG KWTIASFGSR LINFRNSDKN HNWDTREVYP TKELEKLLKD YSIEYGHGEC IKAAICGESD KKFFAKLTSV LNTILQMRNS KTGTELDYLI SPVADWGNF FDSRQAPKNM PQDADANGAY HIGLKGLMLL GRIKNNQEGK KLNLVIKNEE YFEFVQNRNN AAAGGRAGKR RSRASKR*

SEQ ID NO: 13 - Nucleotide sequence of AsCpfl atgacacagtttgaaggcttcaccaatctctaccaggtcagcaagacgctacgttttgagcttatcccgcagggaaaaac cctgaaacacattcaggaacaggggttcatagaggaagataaggcgcgtaacgaccattataaagaactgaagcctat aatcgaccgtatttataaaacgtacgcggatcagtgcctgcagctggttcagctggattgggagaatctgtccgcggctatt gatagctatcgcaaagagaagaccgaggaaacccgtaacgcactgattgaagagcaggcgacctatcggaatgcga tccatgattacttcatcggccgcaccgacaacctgaccgatgcaattaacaaacgtcacgcagagatttacaaaggtctg tttaaagcagagttattcaatggcaaggttctgaaacagctgggtacggtcaccaccaccgaacacgaaaacgcactgc tgaggagctttgataaatttaccacatatttcagcggtttctatgaaaatcgtaagaatgtatttagcgccgaagatatttcca ccgcaattcctcatcgtattgtgcaggataattttccgaagtttaaagaaaattgtcatatttttacccgtctgatcaccgcggt accgagcctgcgagagcattttgaaaacgttaagaaagccattggaatttttgtcagtaccagcattgaagaagtgttttcg ttcccgttctataaccaactgctgacccagacccagattgatctgtacaatcagctgctggggggcataagccgcgaggc aggtaccgaaaagataaagggactcaatgaggtgctgaatctggcaattcagaagaatgatgaaacggctcatatcatt gctagcctgccgcatcgtttcattcccctgtttaagcaaatcctgagcgatcgcaatacactgagctttatcctcgaagagttt aaatcggacgaagaagttatccagagcttttgcaaatacaaaaccctgctgcggaacgaaaatgtgctggagaccgct gaagcactgtttaatgaactgaactcgatcgacctcacccatatttttatatcccacaaaaaactggaaaccataagcagc gctctgtgtgaccattgggataccctgcgcaacgccctgtatgaacggcgtatcagcgagctgaccgggaaaatcacca aatccgcaaaggaaaaagttcagcgtagtctgaaacacgaggacatcaacctgcaagaaattattagcgcagcaggt aaagagctgagcgaagcattcaaacagaaaaccagcgaaatcctgagccatgcccatgctgcactggatcagccgct gccgaccaccctgaaaaaacaggaggaaaaggagattctgaaaagccaactggacagcctgctgggcctgtatcac ctgctggactggtttgcagtcgatgagagcaacgaggttgatcctgagttctccgctcgtctgaccggaatcaagctggag atggaaccgagtctgtcgttttacaataaagcgcgtaattacgcgaccaagaaaccgtatagcgtggaaaaattcaaact gaactttcagatgccgacccttgcaagcggatgggacgttaacaaagaaaaaaacaatggggcaattctgtttgtgaaa aatggcctctattatctgggtatcatgccgaaacagaaagggcgctacaaagccctgtcatttgagccgaccgagaaaa cctcagagggtttcgacaagatgtactacgattatttcccggatgcggcaaaaatgatacccaaatgtagcacccaactg aaggcagttacagcccactttcagacccataccaccccgatcctgctgtcgaacaattttatagagccgctggaaattacc aaagagatttatgatctgaataatccggaaaaggagcccaagaaatttcagacggcgtatgcaaaaaagaccgggga tcagaaaggttatcgtgaagcgctgtgcaaatggattgactttacccgtgactttctgtcaaaatataccaaaacgacgag cattgatctgagcagcctacgtccgagcagccaatataaggatctgggcgaatattacgccgaactgaatccgctgctct accatatttccttccaacgaatcgctgaaaaagaaataatggacgccgttgaaaccggcaaactgtatctgtttcaaatct acaacaaagatttcgccaaaggccatcacggtaagccgaacctgcataccctgtattggaccggtctgtttagcccgga gaatctggccaaaaccagcatcaagctgaacggacaggcagaactgttttaccgccccaaaagccgtatgaaaagga tggcacaccgcctgggcgaaaaaatgctgaataagaaactcaaagatcagaaaacgccgataccggataccctttatc aggagctgtatgattatgttaaccaccggctgagccatgacctgagcgacgaagcgcgtgcactgctgccgaacgtgatt accaaggaagtctcgcatgaaattattaaagatcggcgcttcaccagtgataaatttttcttccatgtaccgatcaccctgaa ttatcaagccgcaaatagcccttccaaatttaatcaacgcgtgaatgcgtacctgaaagagcatccggagaccccaatta ttggcatagaccgaggagaacgcaatctcatttatatcaccgtcattgatagcaccggtaagatcctggaacagcgtagc ctgaataccattcagcagtttgactaccagaaaaagctggacaacagagaaaaggaacgtgtagccgcccggcagg cttggagtgtggtgggtactatcaaggatctgaagcaggggtatctctcccaagttatccatgaaattgtcgatctaatgattc actatcaagcagtagtggtactggaaaatctgaatttcggtttcaaaagcaaacgtacagggatcgctgaaaaagccgtt tatcagcagttcgagaaaatgctgatagacaagctgaattgcctggttctgaaagattatccggcagagaaggtgggcg gtgtgctgaacccgtaccagctgactgatcaatttacgagctttgcaaaaatgggaacgcagagcggtttcctgttctatgtt ccggcgccatataccagcaagatagacccgctgacaggtttcgtagatccgtttgtctggaaaaccattaaaaatcatga aagtcgcaaacattttctggagggctttgattttctgcactatgacgtgaaaaccggcgacttcattctgcattttaaaatgaa ccgtaatctgtcctttcagcgcggcctgcctggctttatgccggcgtgggacattgtttttgaaaagaatgagacacagtttg atgccaaaggtaccccctttattgcggggaaacgcattgtgcccgttatagaaaatcaccgcttcaccggacggtatagg gacttgtacccggcaaatgaattgatagcgctgctggaggagaaaggtattgtctttcgggatggatcaaacatcctgccg aagctgctggagaacgatgacagccacgcaatagacaccatggtagcgctgatccgaagcgtgctgcagatgcgtaa cagtaatgcggctacgggggaagactacattaatagcccggtccgtgatctgaacggcgtttgtttcgatagcagatttca aaatccggagtggccgatggatgccgatgccaatggagcttaccatatcgctctcaaaggtcagctcctactgaaccattt gaaagaatcaaaagatctgaaactgcagaacggcatctcgaatcaggactggctggcctacattcaagaactgagaa actga

SEQ ID NO: 14 - Amino acid sequence of AsCpfl

MTQFEGFTNL YQVSKTLRFE LIPQGKTLKH IQEQGFIEED KARNDHYKEL KPIIDRIYKT YADQCLQLVQ LDWENLSAAI DSYRKEKTEE TRNALIEEQA TYRNAIHDYF IGRTDNLTDA INKRHAEIYK GLFKAELFNG KVLKQLGTVT TTEHENALLR SFDKFTTYFS GFYENRKNVF SAEDISTAIP HRIVQDNFPK FKENCHIFTR LITAVPSLRE HFENVKKAIG IFVSTSIEEV FSFPFYNQLL TQTQIDLYNQ LLGGISREAG TEKIKGLNEV LNLAIQKNDE TAHIIASLPH RFIPLFKQIL SDRNTLSFIL EEFKSDEEVI QSFCKYKTLL RNENVLETAE ALFNELNSID LTHIFISHKK LETISSALCD HWDTLRNALY ERRISELTGK ITKSAKEKVQ RSLKHEDINL QEIISAAGKE LSEAFKQKTS EILSHAHAAL DQPLPTTLKK QEEKEILKSQ LDSLLGLYHL LDWFAVDESN EVDPEFSARL TGIKLEMEPS LSFYNKARNY ATKKPYSVEK FKLNFQMPTL ASGWDWKEK NNGAILFVKN GLYYLGIMPK QKGRYKALSF EPTEKTSEGF DKMYYDYFPD AAKMIPKCST QLKAVTAHFQ THTTPILLSN NFIEPLEITK EIYDLNNPEK EPKKFQTAYA KKTGDQKGYR EALCKWIDFT RDFLSKYTKT TSIDLSSLRP SSQYKDLGEY YAELNPLLYH ISFQRIAEKE IMDAVETGKL YLFQIYNKDF AKGHHGKPNL HTLYWTGLFS PENLAKTSIK LNGQAELFYR PKSRMKRMAH RLGEKMLNKK LKDQKTPIPD TLYQELYDYV NHRLSHDLSD EARALLPNVI TKEVSHEIIK DRRFTSDKFF FHVPITLNYQ AANSPSKFNQ RWAYLKEHP ETPIIGIDRG ERNLIYITVI DSTGKILEQR SLNTIQQFDY QKKLDNREKE RVAARQAWSV VGTIKDLKQG YLSQVIHEIV DLMIHYQAW VLENLNFGFK SKRTGIAEKA VYQQFEKMLI DKLNCLVLKD YPAEKVGGVL NPYQLTDQFT SFAKMGTQSG FLFYVPAPYT SKIDPLTGFV DPFVWKTIKN HESRKHFLEG FDFLHYDVKT GDFILHFKMN RNLSFQRGLP GFMPAWDIVF EKNETQFDAK GTPFIAGKRI VPVIENHRFT GRYRDLYPAN ELIALLEEKG IVFRDGSNIL PKLLENDDSH AIDTMVALIR SVLQMRNSNA ATGEDYINSP VRDLNGVCFD SRFQNPEWPM DADANGAYHI ALKGQLLLNH LKESKDLKLQ NGISNQDWLA YIQELRN*

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Claims

CLAIMS:

1. A ribonucleoprotein complex (RBC) for use in genome editing, the complex comprising a nuclease, at least one target-DNA binding molecule and at least one RNA donor repair template.

2. The RBC of claim 1 , wherein the nuclease is a CRISPR enzyme.

3. The RBC of claim 2, wherein the CRIPSR enzyme is Cpf1.

4. The RBC of any preceding claim wherein the target-DNA binding molecule is a crRNA molecule.

5. The RBC of any preceding claim, wherein the RNA repair template comprises at least one mutation compared to a target sequence.

6. The RBC of any preceding claim, wherein the complex is DNA-free.

7. The RBC of any preceding claim, wherein the RNA donor repair template is single stranded.

8. A nucleic acid construct comprising at least one nucleic acid sequence encoding a target-DNA binding molecule and at least one nucleic acid sequence encoding a donor repair template (DRT) and preferably at least one nucleic acid sequence that allows self-cleavage of the target-DNA binding molecule and/or the DRT

9. The nucleic acid construct of claim 8, wherein the construct further comprises a nucleic acid sequence encoding a CRISPR enzyme, wherein preferably the CRISPR enzyme is operably linked to a second regulatory sequence.

10. The nucleic acid construct of claim 9, wherein the construct comprises a single regulatory element operably linked to all of the nucleic acid sequences encoding the target-DNA binding molecule, the donor repair template and the ribozyme.

11. The nucleic acid construct of claim 10, wherein the regulatory sequence is a polymerase promoter.

12. The nucleic acid construct of any of claims 8 to 11 , wherein the construct comprises at least one target-DNA binding molecule that is flanked by a 5’ and 3’ nucleic acid sequence that encodes a ribozyme enzyme and a donor repair template that is flanked by a 5’ and 3’ nucleic acid sequence that encodes a ribozyme enzyme.

13. The nucleic acid construct of any of claims 8 to 12, wherein the target-DNA binding molecule is a crRNA sequence.

14. A vector comprising the nucleic acid construct of any of claims 8 to 13.

15. A host cell comprising the nucleic acid construct of any of claims 8 to 13 or the vector of claim 14.

16. The host cell of claim 15, wherein the cell is a eukaryotic cell.

17. The host cell of claim 16, wherein the eukaryotic cell is a plant cell.

18. A transgenic organism expressing the nucleic acid construct of any of claims 8 to 13 or the vector of claim 14.

19. The transgenic organism of claim 18, wherein the organism is a plant.

20. A method of producing a transgenic organism as defined in claim 18 or 19, the method comprising:

(a) selecting a part of an organism;

(b) transfecting at least one cell of the part of the organism of part (a) with the nucleic acid construct of any of claims 8 to 13 or the vector of claim 14; and

(c) regenerating at least one organism derived from the transfected cell or cells.

21. The method of claim 20, wherein the organism is a plant.

22. An organism obtained or obtainable by the method of claim 20.

23. A method of performing genome editing in an organism, the method comprising introducing an RBC as defined in any of claims 1 to 7 or introducing and expressing a nucleic acid construct as defined in any of claims 8 to 13 or the vector of claim 14 into said organism.

24. A method of performing homology directed repair (HDR) in an organism, the method comprising introducing an RBC as defined in any of claims 1 to 7 or introducing and expressing a nucleic acid construct as defined in any of claims 8 to 13 or the vector of claim 14 into said organism.

25. The method of claim 23 or 24, wherein the organism is a eukaryote.

26. The method of claim 25, wherein the eukaryote is a plant.

27. Use of the RBC of any of claims 1 to 7 or the nucleic acid construct of any of claims 8 to 13 for use in homology-directed repair.

28. Use of the RBC of any of claims 1 to 8 or the nucleic acid construct of any of claims 8 to 13 for use in genome editing.