PL235163B1 - Nucleotide sequence of synthetic Cas9 gene, a cassette directing sgRNA for editing of plant genome and the efficient system for side-directed mutagenesis of a selected plant genome - Google Patents

Abstract

The subject matter of the application is a synthetic Cas9 endonuclease gene having the nucleotide sequence shown as SEQ ID NO:1. The application also includes an empty sgRNA targeting cassette and a filled sgRNA targeting cassette. In addition, the subject of the application is also a kit for the production of the Cas9/sgRNA system and a method for editing the genome of a monocotyledonous plant, in particular barley.

Description

Description of the invention

The invention relates to the nucleotide sequence of the synthetic Cas9 gene, the sgRNA targeting cassette for editing the plant genome, and the efficient Cas9 / sgRNA system for the site-directed mutagenesis of a selected region of the plant genome.

The CRISPR / Cas9 system is a unique tool that allows introducing changes to the nucleotide sequence in a specific region of the genome (Jinek et al., 2012; Cong et al., 2013; Bortesi et al., 2015; Ma et al., 2015). Such changes are also referred to as genome editing.

The introduced change / mutation of the nucleotide sequence may consequently cause: i) a change in the regulation of the gene, ii) a complete switch-off of expression, iii) a change in the amino acid sequence of the encoded protein, iv) a change in the nucleotide sequence of the regulatory RNA. The obtained changes are permanent, i.e. they are passed on to progeny plants reproduced generatively or vegetatively and are indistinguishable from a naturally occurring mutation or a mutation resulting from mutagenesis (Pan et al. 2016; Mao et al. 2017). The bacterial CRISPR / Cas9 system is used to edit the genome, therefore the efficiency and precision of this process depend on the structure of the modified genome. In the case of the Arabidopsis thaliana model plant with a small genome, the method was optimized by e.g. on the use of various promoters to increase Cas9 gene expression (reviewed by Mao et al. 2017). Another way was to use the Cas9 nucleotide sequence containing optimized codon sequences adapted to the genome of the plant being changed (Bortesi and Fischer, 2015). It has also been shown that temperature stress may be a factor influencing system performance (LeBlanc et al. 2018).

Gene editing technology is increasingly used both in research and to obtain improved crop plants (Liu et al., 2017; Sun et al., 2016). The usefulness of this technology is especially important in the case of very large and complex cereal genomes, for which it is practically impossible to obtain and identify mutations at a specific locus using conventional mutagenesis methods. In addition, the described technology allows you to generate specific mutations directly in advanced breeding material or in varieties, eliminating the need for time-consuming and labor-intensive introduction of mutations to these materials or varieties in the process of crossing plants.

The object of the invention is to provide an efficient, synthetic CRISPR / Cas9 system, hereinafter referred to as the Cas9 / sgRNA system, suitable for obtaining a mutation at a defined site of a plant genome, with particular reference to monocotyledonous plants including cereals.

Surprisingly, it turned out that the use of the newly designed Cas9 gene sequence and the sequence of the sgRNA cassette according to the invention together with the sequences of the modified genes in the Cas9 / sgRNA system make it possible to obtain mutations in these genes with high efficiency. Depending on the proposed construction, mutations can be obtained in one or more native genes simultaneously.

The essence of the invention

The invention relates to a synthetic Cas9 endonuclease gene having the nucleotide sequence shown as SEQ. No. 1.

Another subject matter of the invention is an empty sgRNA targeting cassette consisting of:

a) a U6 promoter having the nucleotide sequence shown as SEQ. no.2 and

b) a gRNA coding sequence having the nucleotide sequence shown as SEQ. No. 3 connected operationally with the one in between

c) a sequence encoding a site for cloning a sequence complementary to the edited sequence, preferably containing a restriction enzyme recognition sequence.

Preferably, the sequence encoding a site for cloning a sequence complementary to the edited sequence comprises two Bsal restriction sites for directionally cloning a sequence complementary to the edited sequence. Preferably, the empty sgRNA targeting cassette has the sequence shown as SEQ. no 4.

Another subject matter of the invention is a filled sgRNA targeting cassette consisting of:

a) a U6 promoter having the nucleotide sequence shown as SEQ. no.2 and

b) a gRNA coding sequence having the nucleotide sequence shown as SEQ. No. 3 connected operationally with the one in between

PL235 163 Β1

c) a sequence complementary to the edited sequence, preferably cloned into two sites for the restriction enzyme Bsal.

Preferably, the filled sgRNA targeting cassette according to the invention is characterized in that it comprises as a sequence complementary to the edited sequence a sequence selected from the group consisting of:

a) a sequence comprising a 21 nucleotide sequence complementary to the edited target gene sequence (spacer), preferably in the form of a double-stranded DNA oligonucleotide having the following structure:

5'-CTTGNNNNNNNNNNNNNNNNNNN-3 '

3'-NNNNNNNNNNNNNNNNNNNNCAAA-5 'where N is any selected nucleotide,

b) a PTG sequence (polycistronic tRNA / g RN A) with the structure shown in Fig. 4 containing several individual sequences containing a 20 or 21 nucleotide sequence complementary to the edited target gene sequence (spacers) separated by short tRNA sequences.

Preferably, the filled sgRNA targeting cassette according to the invention is characterized in that it contains a 21 nucleotide sequence complementary to the edited target gene sequence (spacer) selected from: SEQ. no.5-7.

Preferably, the filled sgRNA targeting cassette according to the invention is characterized in that it comprises a PTG sequence having the nucleotide sequence shown as SEQ. no.8.

Another object of the invention is a kit for the production of a Cas9 / sgRNA system for editing the genome of a monocotyledonous plant, in particular of barley, characterized in that it comprises a synthetic Cas9 endonuclease gene according to the invention as defined above and an empty sgRNA targeting cassette according to the invention as defined above.

A further object of the invention is a Cas9 / sgRNA system for editing the genome of a monocotyledonous plant, in particular of barley, characterized in that it comprises the synthetic Cas9 endonuclease gene according to the invention as defined above and the filled sgRNA targeting cassette according to the invention as defined above.

Preferably, the kit according to the invention or the system according to the invention is in the form of a plasmid vector.

Another object of the invention is a method for editing the genome of a monocotyledonous plant, in particular of barley, characterized in that the Cas9 / sgRNA system according to the invention as defined above is introduced into the plant cell, the transformed cells are then isolated and transgenic plants are regenerated therefrom.

Preferably, the Cas9 / sgRNA system is introduced into the cells of immature barley embryos using Agrobacterium tumefaciens.

All claimed products are non-naturally occurring, artificial, isolated DNA molecules or compositions thereof.

In a preferred embodiment of the invention, the Cas9 endonuclease gene sequence is intended for use in plants, especially in monocots, in particular in cereals, and allows efficient translation and production of an active endonuclease protein in plant cells. Detailed description of the invention

The subject of the application is an efficient, synthetic system for generating mutations at a defined site of a plant genome. The system includes: i) a newly designed nucleotide sequence of the synthetic Cas9 gene and ii) a sequence of an sgRNA targeting cassette.

The Cas9 nucleotide sequence has been designed with special regard to its efficient expression and translation in monocotyledons (cereals) and is described in Example 1.1. The codon sequence and nuclear localization signals NLSs were modified and an intron sequence was introduced.

The sgRNA targeting cassette contains DNA with a nucleotide sequence complementary to the sgRNA (single guided RNA) and is described in Example 1.2.

The final vector with the Cas9 / sgRNA system containing both components of the system: i.e. the synthetic Cas9 gene and the sgRNA targeting cassette was built as follows: the sequence of the synthetic Cas9 gene was inserted into the pBract211 vector between the Ubił promoter

Using the nos terminator, and then, in the vector enriched in this way, a Gateway cloning cassette was inserted in the SphI restriction site, thus obtaining the so-called vector. Gateway Destination.

At the same time, the second vector, the so-called Gateway Entry: DNA complementary to the sgRNA was inserted into the linearized pCR8 / GW / TOPO vector with a fragment containing two Bsal restriction sites allowing for directional incorporation of the so-called spacer, resulting in a complete sgRNA cassette.

Both vectors, ie Gateway Destination and Gateway Entry with the complete sgRNA targeting cassette, were subjected to the Gateway recombination process resulting in a vector with the Cas9 / sgRNA system.

The system performance was verified in the following two series of experiments.

Series I: Editing cassette performance verification performed on two native barley genes: HvCKX1 and Nud (example 2).

To check the effectiveness of the Cas9 / sgRNA system, a vector was constructed in which the complete sgRNA targeting cassette contained the sgRNA with the sequence of the so-called a spacer complementary to the selected 21-nucleotide fragment of the barley HvCKX1 or Nud gene. This vector was used for the genetic transformation of immature barley embryos with Agrobacterium tumefaciens.

Series II: checking the effectiveness of simultaneous editing of two genes. For this purpose, a vector was constructed with an sgRNA targeting cassette, which contained an sgRNA with two simultaneously two spacer sequences, such that the sequence of the first spacer was complementary to the selected 21-nucleotide fragment of the barley HvCKX1 gene and the sequence of the second spacer was complementary to the selected 21-nucleotide fragment of the barley gene HvCKX1. The sgRNA sequences along with the spacers were separated by short tRNA-Gly sequences derived from barley. This vector was used for the genetic transformation of immature barley embryos with Agrobacterium tumefaciens.

Results

In series I independent experiments, approximately 100 transgenic plants were obtained from independent transformation events (Example 2.1). It was documented that the mutational events in the case of the applied solution occurred with a very high frequency; for the HvCKX1 gene, they occurred in 66% of the transgenic plants and for the Nud gene in 64% of the transgenic plants (Example 2.2). Moreover, it has been documented that almost all such induced sequence changes / mutations are stably passed on to the next generative generations (Example 2.3). In the second series of experiments, the effectiveness of the sgRNA targeting cassette in the simultaneous induction of mutational events in two different genes: HvCKX1 and HvCKX3 was documented. More than 70 transgenic plants were obtained from independent transformation events, 9 of which were mutated in both the HvCKX1 and HvCKX3 genes.

Example 1. Cas9 / sgRNA system for editing the genome of a monocotyledonous plant and its components

In the described embodiment, the designed Cas9 / sgRNA system for editing the genome of a monocotyledonous plant consists of a synthetic sequence of the Cas9 endonuclease gene, a sgRNA (single guided RNA) targeting sequence, and additionally a pBract211 vector sequence, including but not limited to the sequence of the right and left border of T-DNA determining the integration of T-DNA in the plant genome and the sequence of promoters and terminators conditioning the expression of the cassette in monocotyledonous plants.

Plasmid vectors were obtained, constituting exemplary implementations of the Cas9 / sgRNA system intended for editing selected barley genes (ie the HvCKX1 gene, the Nud gene and simultaneously the HvCKX1 and HvCKX3 genes).

A plasmid vector exemplary of the Cas9 / sgRNA system for editing the HvCKX1 gene is schematically shown in Fig. 1, and its sequence is shown as SEQ. No. 9.

An analogous plasmid vector exemplary embodiment of the Cas9 / sgRNA system for editing the Nud gene is schematically shown in Fig. 1, and its sequence is shown as SEQ. No. 10.

A plasmid vector embodying the Cas9 / sgRNA system for the simultaneous editing of the HvCKX1 and HvCKX3 genes is schematically shown in Fig. 2, and its sequence is shown as SEQ. No. 11.

PL235 163 Β1

1.1. Obtaining the nucleotide sequence of the synthetic Cas9 endonuclease gene

The synthetic Cas9 gene was derived from native Cas9 endonuclease proteins, including Cas9 from Streptococcus pyogenes. The codon selection was optimized for efficient translation in monocot cells. The 5 'end region was enriched with the intron sequence derived from Arabidopsis thaliana and the nuclear localization signal. Sequence No. 1 shows the nucleotide sequence of the Cas9 gene of the invention.

1.2. Obtaining the nucleotide sequence of the sgRNA cassette

The sgRNA targeting cassette is an element of the CRISPR system responsible for targeting the Cas9 endonuclease to a specific DNA sequence in the genome of the edited plant.

This cassette consists of: i) the promoter of the U6RNA gene derived from wheat (Triticum aesń'vu / 77) [1], ii) a fragment with Bsal restriction sites enabling directional incorporation of a 20-21 nucleotide DNA sequence complementary to the edited gene (the so-called spacer ) in a single or multiple (polycistronic) system; and iii) gRNA (guided RNA) sequences.

The sequence of the sgRNA targeting cassette (SEQ ID NO: 4) is shown in Figure 3; the sequence of the U6 promoter is underlined (sequence No. 2), the fragment with two Bsal restriction sites for directional insertion of the spacer sequence with the restriction enzyme Bsal (the Bsal restriction sites in bold) is underlined, the gRNA sequence is marked with two underlining (sequence No. 3) .

After placing the sgRNA cassette in the Gateway Entry vector and cleaving with the Bsal enzyme, it is possible to insert:

i) any single target DNA sequence of the edited gene (spacer) in the form of a double-stranded DNA oligonucleotide having the following structure:

5 '-CTTGNNNNNNNNNNNNNNNNNNN-3'

3'-NNNNNNNNNNNNNNNNNNNCAAA-5 'where N is any chosen nucleotide, ii) several single target sequences of the edited genes (spacers), separated by short tRNA sequences (PTG - polycistronic tRNA / gRNA) with the structure shown in Fig. 4.

Two single sgRNA cassettes were prepared for two barley genes: HvCKX1 and Nud, and one PTG fragment cassette for simultaneous editing of the HvCKX1 and HvCKX3 genes. The following target sequences were used for these genes:

HvCKXl 5'-GATCACCGCGGCGTCTCCTA-3 '(Seq. No.5)

HvCKX3 5'-GCAAGTTCATCCAGAGCCCCA-3 '(Seq. No. 6)

Nud 5'-GTTGGGCACCTTTGAGACGG-3 '(Seq. No. 7)

Fig. 5 shows the sequence of the PTG fragment (SEQ. No. 8) with the spacing (no underlines), the tRNA fragments (single underline) and the gRNA (double underline) listed:

1.3. Obtaining a binary vector with the Cas9 / sgRNA system for genetic transformation of monocots.

The synthetic Cas9 gene was placed in the pBract211 binary vector, designed for genetic transformation of monocots. Then this vector, by inserting the appropriate sequence (so-called Gateway cassettes), was adapted for cloning in the Gateway system. The finished sgRNA targeting cassette is placed immediately after the nose terminator of the synthetic Cas9 gene in the Gateway cloning site.

The diagram of the pBract211 vector with the Cas9 / sgRNA system is shown in Fig. 6, which shows the diagram of the pBract211 vector containing the synthetic Cas9 gene and the Gateway cloning cassette (GW cassette) (A), and the diagram of the complete Cas9 / sgRNA cassette consisting of and ) the Ubi promoter, the synthetic Cas9 gene and the nos terminator and ii) the sgRNA targeting cassette (B).

Example 2. Verification of the effectiveness of the gene editing cassette in plants 2.1.

2.1. Obtaining transgenic plants

The evaluation of the effectiveness of the Cas9 / sgRNA system in inducing targeted mutations was performed on the basis of the barley plants. For this purpose, the vector pBract211 containing the appropriate

PL235 163 Β1 Cas9 / sgRNA system was used for genetic transformation of immature barley embryos with Agrobacterium tumefaciens. Three independent genetic transformation experiments were performed using three different sgRNA targeting cassettes, one for the HvCKX1 gene (ckx1-sgRNA), one for the Nud gene (nud-sgRNA) and a third for the simultaneous editing of the HvCKX1 and HvCKX3 genes (PTG-ckx1-3). the transformation carried out resulted in 71 transgenic plants containing the ckx1-sgRNA cassette, 28 plants containing the nud-sgRNA cassette and 72 plants containing the PTG-ckx1-3 cassette. All the transgenic plants in the three experiments resulted from independent transformation events.

2.2. Detection of mutations induced by the Cas9 / sgRNA system

Mutations induced in barley genes by the Cas9 / sgRNA system were identified by PCR combined with restriction analysis. Additionally, the presence of the mutation was verified by sequencing cloned genomic DNA fragments, including the edited sequence. The presence of mutations in the HvCKX1 gene was observed in 47 out of 71 tested transgenic plants, while mutations in the Nud gene were observed in 18 out of 28 tested transgenic plants. Thus, in both experiments, the efficiency of the Cas9 / sgRNA system was above 60% (Table 1).

Table 1. Results of genetic transformation of barley using three different sgRNA targeting cassettes

SgRNA targeting cassette 9 Number of transformed embryos Number of transform events Number of transgenic plants Number of mutant plants (%) ckxl-sgRNA 1143 137 71 47 (66%) nud-sgRNA 1209 96 28 18 (64%) PTG-ckxl-3 1320 142 72 9 (12.5%) ¹

'Number of plants with mutations in both the HvCKX1 and HvCKX3 genes.

2.3. Analysis of the inheritance of mutations induced by the Cas9 / sgRNA system

The inheritance of mutations induced by the Cas9 / sgRNA system was studied in the first generative generation of transgenic plants. 20 T1 lines whose mother plants had a mutation in the HvCKX1 gene were selected for analysis. 10 plants from each line were tested for the presence of the mutation. The inheritance of mutations in the HvCKX1 gene was observed in 93 plants (47%) in 18 T1 lines.

Literature:

Bortesi, L. and Fischer, R. (2015) The CRISPR / Cas9 system for plant genome editing and beyond. Biotechnol. Adv. 33 (1), 41-52.

Cong, L., Ran, F.A., Cox, D. et al. (2013) Multiplex genome engineering using CRISPR / Cas systems. Science, 339 (6121), 819-823.

Jinek, M., Chyliński, K., Fonfara, I., Hauer, M., Doudna, J.A. and Charpentier, E. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337 (6096), 816-821. LeBlanc, Ch., Zhang F., Mendez J., Lozano Y., Chatpar K., Irish V.F., and Jacob Y. (2018) Increased efficiency of targeted mutagenesis by CRISPR / Cas9 in plants using heat stress. Plant J. 93: 377-386. Liu, H. "Ding, Y.D., Zhou, Y.Q., Jin, W.Q" Xie, K.B. and Chen, L.L. (2017) CRISPR-P 2.0: an improved CRISPR-Cas9 tool for genome editing in plants. Moth. Plant, 10, 530-532.

Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., et al. (2015). A Robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Moth. Plant 8, 12741284. doi: 10.1016 / IU. 2015.04.007.

Mao, Y., Botella, J.R. and Zhu, J.K. (2017) Heritability of targeted gene modifications induced by plantoptimized CRISPR systems. Cell. Moth. Life Sei. 74 (6), 1075-1093.

Pan, C., Ye, L., Qin, L., Liu, X., He, Y., Wang, J., et al. (2016). CRISPR / Cas9mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci. Rep. 6: 24765. doi: 10.1038 / srep24765.

Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu JL etal. Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology 2013, 31 (8): 686-688. Sun, Y.W., Li, J.Y. and Xia, L.Q. (2016) Precise genome modification via sequence-specific nucleasesmediated gene targeting for crop improvement. Front. Plant Sci. 7, 1928.

PL235 163 Β1

SEQLTXT.txt

SEQUENCE LISTING <110> Institute of Plant Breeding and Acclimatization - State

research Institute

University of Warsaw <12Θ> Nucleotide sequence of the synthetic Cas9_ gene, sgRNA targeting cassette for editing the plant genome and efficient system for targeted mutagenesis of a selected region of the plant genome <130> PK / 5270 / RW <160> 11 <170> Patentln version 3.5 <210> 1 <211> 4464 <212> DNA <213> artificial <220>

<223> Synthetic Cas9 endonuclease gene <400> 1

cccgggatcc gccatggccc cgaagaagaa gaggaaggtg ggcatggaca agaagtactc 60 catcggcctg gacatcggga ccaactctgt aggctgggcg gtgatcacgg acgagtataa 12Θ ggtgccctcg aagaagttca aggtcctggg aaacaccgac cgtcacagca tcaagaagaa 180 cctgataggg gccctcctgt tcgactcagg taaatttctg tgttccttat tctctcaaaa 240 tcttcgattt tgttttcgtt cgatcccaat ttcgtatatg ttctttggtt tagattctgt 3Θ0 taatcttaga tcgaagacga ttttctgggt ttgatcgtta gatatcatct taattctcga 360 ttagggtttc atagatatca tccgatttgt tcaaataatt tgagttttgt cgaataatta 420 ctcttcgatt tgtgatttct atctagatct ggtgttagtt tctagtttgt gcgatcgaat 480 ttgtcgatta atctgagttt ttctgattaa caggtgagac tgctgaggcg accaggctga 540 agcgtactgc aagaagacgt tacacgaggc ggaagaacag gatctgctac ctccaagaga 6ΘΘ tcttcagcaa cgagatggcg aaggtcgacg actcgttctt ccaccgtctc gaggagagct 660 tcctggtcga ggaagacaag aagcacgaga ggcacccgat cttcgggaac atcgtggacg 720 aagtggcgta ccacgagaag taccccacaa tctaccacct ccggaagaag ctggttgact 780 ccactgacaa ggctgacctg cgtctcatct acctggccct Page cgcccacatg 1 atcaagttca 840

PL235 163 Β1 ggggtcactt cctcatagag ggggacctga tcatccagct ggtgcaaacc tacaaccagc gagttgacgc caaggctatc ctgtctgcta taatcgctca gcttccgggg gagaagaaga gtctgggcct gaccccgaac ttcaaaagca agctgtcaaa ggacacatac gacgacgacc agtacgccga cctctttctg gcagctaaga tcctccgagt caacactgag atcaccaagg acgacgagca ccaccaggac ctgactctcc agaagtacaa ggagatcttc ttcgaccagt gcggggcttc tcaagaggag ttctacaagt gcaccgagga gctgctcgtt aagttgaaca ttgataacgg gtcgatcccg caccagatcc gccaagagga cttctacccc ttcctcaagg cattccgaat cccgtactac gtcgggcctc tgacccgcaa gtcggaggag actatcacac gcgcatctgc ccagagtttc atcgagagga agaaggtgct cccgaagcac tctctgctct cgaaggtcaa gtacgtgacc gagggtatgc agaaggccat cgtcgatctc ctgttcaaga aggaggacta cttcaagaag atcgagtgct acaggttcaa cgcaagcctg gggacctacc acttcctcga caacgaggag aatgaggaca tcttcgagga ccgcgagatg atcgaggagc acaaggtgat gaagcaactg aagaggcgca agctgatcaa cggcatcagg gacaagcaga

SEQLTXT.txt accctgacaa tagcgacgtc gacaagctgt tcttcgagga gaaccccatc aacgctagtg ggctgtccaa aagccgacgc ttggagaact acggactctt cgggaacctg attgccctga acttcgacct cgccgaggac gcgaaactcc tggacaacct gttagcacag atcggggacc acctgtctga cgccatcctc ctgtccgaca ccccactgag cgcctctatg ataaaacgct tgaaggctct cgtacgtcag cagctgcctg cgaagaacgg ctacgccggg tacattgatg tcatcaagcc catcctcgag aagatggacg gagaggacct gctccgaaag cagaggacct atctcggtga gctccatgct atcctgcgac acaaccgcga gaagattgag aagatcctga ttgcaagggg caacagccgg ttcgcttgga cctggaactt tgaggaggtc gtggacaagg tgaccaactt cgataagaac ctcccgaacg acgagtactt caccgtgtac aacgagctga ggaagcctgc gttcctgagc ggggagcaaa ccaaccgcaa ggtgaccgtc aagcagctca tcgactccgt cgagatctcg ggcgttgagg atgacctgct caagattatc aaggacaagg ttctagagga catcgtgctc acgctgaccc gcctcaagac ctacgcccac ctcttcgacg ggtatactgg atgggggagg ctctcaagga gcggcaagac gatcctcgac ttcctgaagt

90Θ

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1740

18Θ0

1860

1920

1980

2040

2100

2160

2220

2280

2340

2400

Page 2

PL235 163 Β1 ctgacggctt cgctaaccgc aacttcatgc aggaagacat ccagaaggcg caggtaagcg ctaacctcgc cggctccccc gcaatcaaaa acgagctggt taaagtgatg ggcaggcaca gcgagaatca gacgactcag aagggccaga aggagggtat caaggaactc ggcagccaga agctccagaa tgagaagctg tacctctact accaagagct ggacatcaac aggctgtccg gtttcctcaa ggacgactcg atcgacaaca gcaagtctga caacgtgcca agcgaggagg agctcctgaa cgccaagctc atcacccagc gaggaggcct gtcggagctt gataaggcgg ggcagatcac gaagcacgtg gcgcagatcc agaacgacaa gctcatccgc gaggtgaagg acttccgcaa ggatttccag ttctacaagg acgacgccta cctgaacgcg gtagttggca agagcgagtt cgtgtacggc gactacaagg cggagcagga gatcggcaag gcgaccgcca tcttcaagac cgagatcacg ctggccaacg ctaatggtga gactggcgag atcgtgtggg aggttctgag tatgcctcag gtgaacatcg tcagcaagga gtcaatcctc ccgaagagga actgggaccc aaagaagtac ggcgggttcg tggtggcgaa ggtcgagaag ggtaagtcca gcatcaccat catggagcgg tccagcttcg agggctataa ggaggtcaag aaggacctca

SEQLTXT.txt agctcatcca cgacgactcg ctcacgttca gccagggtga ttctctgcat gagcatatcg agggaatcct ccagaccgtc aaggtcgtgg agcctgagaa catcgtgatc gagatggcgc agaactcgcg cgagcgcatg aagagaattg tcctcaagga gcacccagtt gagaacaccc acctgcagaa cggcagggac atgtacgtgg actacgacgt tgaccatatc gtgccccaaa aggtgctcac gaggtccgac aagaaccgcg tcgtgaagaa gatgaagaac tactggcggc gcaagttcga caacttgaca aaggcagaga gcttcattaa gaggcagctc gtcgagacta tggatagcag gatgaacacg aagtacgacg tcatcaccct caagtccaag ctggtgagcg tccgcgagat caacaactac caccacgccc cggccctcat caagaagtac cctaagctcg tgtacgacgt gcgcaagatg atcgcgaagt agtacttctt ctacagcaac atcatgaact gcgagatcag aaagaggccg ctcattgaga ataagggaag ggacftcgcg actgtgcgca tcaagaagac cgaggtccag accggcggct acagcgataa gctgattgcg aggaagaagg atagcccaac cgtggcttac agtgtgctgg agaagctcaa gtccgtgaag gagctgctcg agaagaaccc aatcgacttc ctcgaggcca tcatcaagct gccaaagtac tccctgttcg

Page 3

246Θ

2520

2580

2640

2700

2760

2820

2880

2940

3000

3060

3120

3180

3240

330Θ

3360

3420

3480

3540

3600

3660

3720

3780

3840

3900

3960

PL235 163 Β1

SEQLTXT.txt agcttgagaa cggccgcaag aggatgctcg cttctgcagg agagttgcag aaaggcaacg 4020 agcttgcgct cccgtccaag tacgtcaact tcctgtacct cgccagccac tacgagaagc 4080 tg ^ agggctc tccagaggac aatgagcaga agcagctatt cgtcgagcag cacaagcact 4140 acctggacga gatcatcgag cagatcagcg agttcagcaa gagggtgatc ctcgccgacg 420Θ cgaacctgga taaggtgctc tccgcctaca acaagcaccg cgacaagcca attcgcgaac 4260 aggccgaaaa cattatacac ctgtttacgc tcacaaacct gggtgcgcca gcagccttca 4320 agtacttcga taccaccatc gaccgcaagc ggtacacctc cactaaggag gtgctcgacg 4380 cgaccctgat ccaccagtct atcaccggtc tctacgaaac aaggatcgac ctgtctcagc 4440 tcggaggtga ttgagctcac <tagt 4464 <210> 2 <212> <220> artificial

<223> U6 promoter <400> 2

gaccaagccc gttattctga cagttctggt gctcaacaca tttatattta tcaaggagca 60 cattgttact cactgctagg agggaatcga actaggaata ttgatcagag gaactacgag 120 agagctgaag ataactgccc tctagctctc actgatctgg gtcgcatagt gagatgcagc 180 ccacgtgagt tcagcaacgg tctagcgctg ggcttttagg cccgcatgat cgggcttttg 240 tcgggtggtc gacgtgttca cgattgggga gagcaacgca gcagttcctc ttagtttagt 300 cccacctcgc ctgtccagca gagttctgac cggtttataa actcgcttgc tgcatcagac 360 ttg 363

<210> 3 <211> 83 <212> DNA <213> artificial <22Θ>

<223> gRNA coding sequence

Page 4

PL235 163 Β1

SEQLTXT.txt <400> 3 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttt 83 <210> 4 <211> 467 <212> DNA <213> artificial <220>

<223> Cassette sgRNA <400> 4 gaccaagccc gttattctga cagttctggt gctcaacaca tttatattta tcaaggagca 60 cattgttact cactgctagg agggaatcga actaggaata ttgatcagag gaactacgag 120 agagctgaag ataactgccc tctagctctc actgatctgg gtcgcatagt gagatgcagc 180 ccacgtgagt tcagcaacgg tctagcgctg ggcttttagg cccgcatgat cgggcttttg 240 tcgggtggtc gacgtgttca cgattgggga gagcaacgca gcagttcctc ttagtttagt 300 cccacctcgc ctgtccagca gagttctgac cggtttataa actcgcttgc tgcatcagac 360 ttgcgagacc aacacaaggt ctccgtttta gagctagaaa tagcaagtta aaataaggct 420 agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttt 467 <210> 5 <211> 20 <212> DNA <213> <213> artificial <213>

<223> HvCKXl <400> 5 gatcaccgcg gcgtctccta <210>

<211>

<212>

<213>

<220>

<223>

DNA artificial

HvCKX3

Page 5

PL235 163 Β1 <400> 6 gcaagttcat ccagagcccc a

SEQLTXT.txt <210> 7 <211> 20 <212> DNA <213> artificial <220>

<223> Nud <400> 7 gttgggcacc tttgagacgg <210> 8 <211> 356 <212> DNA <213> artificial <220>

<223> PTG <400> 8 caacaaagcg catctggtgt agtggtatca tagtaccctc ccacggtact gaccagggtt 60 cgattccctg gatgcgcaga tcaccgcggc gtctcctagt tttagagcta gaaatagcaa 12Θ gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgccaacaa 18Θ agcgcatctg gtgtagtggt atcatagtac cctcccacgg tactgaccag ggttcgattc 240 cctggatgcg cagcaagttc atccagagcc ccagttttag agctagaaat agcaagttaa 308 aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttt 356 <210> 9 <211> 11887 <212> DNA <213> artificial <220>

<223> System Cas9 / sgRNA editing HvCKXl gene <400> 9 tttttatccc cggaagcctg tggatagagg gtagttatcc acgtgaaacc gctaatgccc60 cgcaaagcct tgattcacgg ggctttccgg cccgctccaa aaactatcca cgtgaaatcg120 ctaatcaggg tacgtgaaat cgctaatcgg agtacgtgaa atcgctaata aggtcacgtg180

Page 6

PL235 163 Β1

SEQLTXT.txt aaatcgctaa tcaaaaaggc acgtgagaac gctaatagcc ctttcagatc aacagcttgc246 aaacacccct cgctccggca agtagttaca gcaagtagta tgttcaatta gcttttcaat30® tatgaatata tatatcaatt attggtcgcc cttggcttgt ggacaatgcg ctacgcgcac360 cggctccgcc cgtggacaac cgcaagcggt tgcccaccgt cgagcgccag cgcctttgcc42 © cacaacccgg cggccggccg caacagatcg ttttataaat tttttttttt gaaaaagaaa48Θ aagcccgaaa ggcggcaacc tctcgggctt ctggatttcc gatccccgga attagatctt540 ggcaggatat attgtggtgt aacgtatcac aagtttgtac aaaaaagcag gctccgcggc660 cgcccccttc acctagactc gacgcgtcct agagatccgt caacatggtg gagcacgaca660 ctctcgtcta ctccaagaat atcaaagata cagtctcaga agaccaaagg gctattgaga72Θ cttttcaaca aagggtaata tcgggaaacc tcctcggatt ccattgccca gctatctgtc780 acttcatcaa aaggacagta gaaaaggaag gtggcaccta caaatgccat cattgcgata840 aaggaaaggc tatcgttcaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac900 ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt96Θ gatgtgatat ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc1020 cttcctctat ataaggaagt tcatttcatt tggagaggac gaccccgata tgaaaaagcc1080 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga1140 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg12ΘΘ tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta1260 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag1320 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc1380 tgaaaccgaa ctgcccgctg ttctgcaggt aaatttctag tttttctcct tcattttctt1440 ggttaggacc cttttctctt tttatttttt tgagctttga tctttcttta aactgatcta1500 tWrttaatt gattggttat ggtgtaaata ttacatagct ttaactgata atctgattac1560 tttatttcgt gtgtctatga tgatgatgat aactgcagcc ggtcgcggag gccatggatg162 © cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgacga ccgcaagcga gcgggttcgg cccattcgacga ccgcaagcggtgcata

Page 7

PL235 163 Β1

SEQLTXT.txt actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc 18ΘΘ tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct 186Θ ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga 1920 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt 1980 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc 2Θ4Θ ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg 2100 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag 2160 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct 2220 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg 2280 aatagagtag atgccgaccg ggatccggag agctcgaatt tccccgatcg ttcaaacatt 2340 tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa 24Θ0 tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg 2460 agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 252Θ atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg 2580 gaattcatcg atgatatcag atcaagggtg ggcgcgccga accagctttc ttgtacaaag 2640 tggtgatccc cctcgaatcg aattccgtgc agcgtgaccc ggtcgtgccc ctctctagag 2700 ataatgagca ttgcatgtct aagttataaa aaattaccac atattttttt tgtcacactt 2760 gtttgaagtg cagtttatct atctttatac atatatttaa actttactct acgaataata 2820 taatctatag tactacaata atatcagtgt tttagagaat catataaatg aacagttaga 2880 catggtctaa aggacaattg agtattttga caacaggact ctacagtttt atctttttag 294Θ tgtgcatgtg ttctcctrtt tttttgcaaa tagcttcacc tatataatac ttcatccatt 3000 ttattagtac atccatttag ggtttagggt taatggtttt tatagactaa tttttttagt 3060 acatctattt tattctattt tagcctctaa attaagaaaa ctaaaactct attttagttt 3120 ttttatttaa taatttagat ataaaataga ataaaataaa gtgactaaaa attaaacaaa 3180 taccctttaa gaaattaaaa aaactaagga aacatttttc ttgtttcgag tagataatgc 3240 cagcctgtta aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg 330Θ

Page 8

PL235 163 Β1

SEQLTXT.txt cgtcgggcca agcgaagcag acggcacggc atctctgtcg ctgcctctgg acccctctcg336Θ agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt gcgtggcgga3420 gcggcagacg tgagccggca cggcaggcgg cctcctcctc ctctcacggc acggcagcta3480 cgggggattc ctttcccacc gctccttcgc tttcccttcc tcgcccgccg taataaatag3540 acaccccctc cacaccctct ttccccaacc tcgtgttgtt cggagcgcac acacacacaa3600 ccagatctcc cccaaatcca cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc3660 cccccccccc cctctctacc ttctctagat cggcgttccg gtccatggtt agggcccggt3720 agttctactt ctgttcatgt ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag3780 cgttcgtaca cggatgcgac ctgtacgtca gacacgttct gattgctaac ttgccagtgt3840 ttctctttgg ggaatcctgg gatggctcta gccgttccgc agacgggatc gatttcatga3900 ttttttttgt ttcgttgcat agggtttggt ttgccctttt cctttatttc aatatatgcc3960 gtgcacttgt ttgtcgggtc atcttttcat gctttttttt gtcttggttg tgatgatgtg4Θ20 gtctggttgg gcggtcgttc tagatcggag tagaattctg tttcaaacta cctggtggat4Θ8Θ ttattaattt tggatctgta tgtgtgtgcc atacatattc atagttacga attgaagatg4140 atggatgg aa atatcgatct aggataggta tacatgttga tgcgggtttt actgatgcat42Θ0 atacagagat gctttttgtt cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc4260 attcgttcta gatcggagta gaatactgtt tcaaactacc tggtgtattt attaattttg4320 gaactgtatg tgtgtgtcat acatcttcat agttacgagt ttaagatgga tggaaatatc4380 gatctaggat aggtatacat gttgatgtgg gttttactga tgcatataca tgatggcata444Θ tgcagcatct attcatatgc tctaaccttg agtacctatc tattataata aacaagtatg45Θ0 ttttataatt attttgatct tgatatactt ggatgatggc atatgcagca gctatatgtg4560 gattttttta gccctgcctt catacgctat ttatttgctt ggtactgttt cttttgtcga4620 tgctcaccct gttgtttggt gttacttcgc cccccgggat ccgccatggc cccgaagaag4680 aagaggaagg tgggcatgga caagaagtac tccatcggcc tggacatcgg gaccaactct4740 gtaggctggg cggtgatcac ggacgagtat aaggtgccct cgaagaagtt caaggtcctg48O0 ggaaacaccg accgtcacag catcaagaag aacctgatag gggccctcct gttcgactca4860

Page 9

PL235 163 Β1

SEQLTXT.txt ggtaaatttc tgtgttcctt attctctcaa aatcttcgat tttgttttcg ttcgatccca 4920 atttcgtata tgttctttgg tttagattct gttaatctta gatcgaagac gattttctgg 4980 gtttgatcgt tagatatcat cttaattctc gattagggtt tcatagatat catccgattt 5040 gttcaaataa tttgagtttt gtcgaataat tactcttcga tttgtgattt ctatctagat 5100 ctggtgttag tttctagttt gtgcgatcga atttgtcgat taatctgagt ttttctgatt 5160 aacaggtgag actgctgagg cgaccaggct gaagcgtact gcaagaagac gttacacgag 5220 gcggaagaac aggatctgct acctccaaga gatcttcagc aacgagatgg cgaaggtcga 5280 cgactcgttc ttccaccgtc tcgaggagag cttcctggtc gaggaagaca agaagcacga 5340 gaggcacccg atcttcggga acatcgtgga cgaagtggcg taccacgaga agtaccccac 5400 aatctaccac ctccggaaga agctggttga ctccactgac aaggctgacc tgcgtctcat 5460 ctacctggcc ctcgcccaca tgatcaagtt caggggtcac ttcctcatag agggggacct 5520 gaaccctgac aatagcgacg tcgacaagct gttcatccag ctggtgcaaa cctacaacca 558Θ gctcttcgag gagaacccca tcaacgctag tggagttgac gccaaggcta tcctgtctgc 5640 taggctgtcc aaaagccgac gcttggagaa cttaatcgct cagcttccgg gggagaagaa 5700 gaacggactc ttcgggaacc tgattgccct gagtctgggc ctgaccccga acttcaaaag 5760 caacttcgac ctcgccgagg acgcgaaact ccagctgtca aaggacacat acgacgacga 5820 cctggacaac ctgttagcac agatcgggga ccagtacgcc gacctctttc tggcagctaa 5880 gaacctgtct gacgccatcc tcctgtccga catcctccga gtcaacactg agatcaccaa 594Θ ggccccactg agcgcctcta tgataaaacg ctacgacgag caccaccagg acctgactct 60ΘΘ cctgaaggct ctcgtacgtc agcagctgcc tgagaagtac aaggagatct tcttcgacca 6060 gtcgaagaac ggctacgccg ggtacattga tggcggggct tctcaagagg agttctacaa 6120 gttcatcaag cccatcctcg agaagatgga cggcaccgag gagctgctcg ttaagttgaa 6180 cagagaggac ctgctccgaa agcagaggac ctttgataac gggtcgatcc cgcaccagat 6240 ccatctcggt gagctccatg ctatcctgcg acgccaagag gacttctacc ccttcctcaa 63Θ0 ggacaaccgc gagaagattg agaagatcct gacattccga atcccgtact acgtcgggcc 6360 tcttgcaagg ggcaacagcc ggttcgcttg gatgacccgc aagtcggagg agactatcac 6420

Page 10

PL235 163 Β1

SEQLTXT.txt accctggaac tttgaggagg tcgtggacaa gggcgcatct gcccagagtt tcatcgagag 6480 gatgaccaac ttcgataaga acctcccgaa cgagaaggtg ctcccgaagc actctctgct 6540 ctacgagtac ttcaccgtgt acaacgagct gacgaaggtc aagtacgtga ccgagggtat 6600 gcggaagcct gcgttcctga gcggggagca aaagaaggcc atcgtcgatc tcctgttcaa 6660 gaccaaccgc aaggtgaccg tcaagcagct caaggaggac tacttcaaga agatcgagtg 6720 cttcgactcc gtcgagatct cgggcgttga ggacaggttc aacgcaagcc tggggaccta 6780 ccatgacctg ctcaagatta tcaaggacaa ggacttcctc gacaacgagg agaatgagga 6840 cattctagag gacatcgtgc tcacgctgac cctcttcgag gaccgcgaga tgatcgagga 6900 gcgcctcaag acctacgccc acctcttcga cgacaaggtg atgaagcaac tgaagaggcg 6960 caggtatact ggatggggga ggctctcaag gaagctgatc aacggcatca gggacaagca 7020 gagcggcaag acgatcctcg acttcctgaa gtctgacggc ttcgctaacc gcaacttcat 7Θ80 gcagctcatc cacgacgact cgctcacgtt caaggaagac atccagaagg cgcaggtaag 714Θ cggccagggt gattctctgc atgagcatat cgctaacctc gccggctccc ccgcaatcaa 720Θ aaagggaatc ctccagaccg tcaaggtcgt ggacgagctg gttaaagtga tgggcaggca 7260 caagcctgag aacatcgtga tcgagatggc gcgcgagaat cagacgactc agaagggcca 7320 gaagaactcg cgcgagcgca tgaagagaat tgaggagggt atcaaggaac tcggcagcca 7380 gatcctcaag gagcacccag ttgagaacac ccagctccag aatgagaagc tgtacctcta 7440 ctacctgcag aacggcaggg acatgtacgt ggaccaagag ctggacatca acaggctgtc 7500 cgactacgac gttgaccata tcgtgcccca aagtttcctc aaggacgact cgatcgacaa 7560 caaggtgctc acgaggtccg acaagaaccg cggcaagtct gacaacgtgc caagcgagga 762Θ ggtcgtgaag aagatgaaga actactggcg gcagctcctg aacgccaagc tcatcaccca 768Θ gcgcaagttc gacaacttga caaaggcaga gagaggaggc ctgtcggagc ttgataaggc 774 © gggcttcatt aagaggcagc tcgtcgagac taggcagatc acgaagcacg tggcgcagat 7800 cctggatagc aggatgaaca cgaagtacga cgagaacgac aagctcatcc gcgaggtgaa 7860 ggtcatcacc ctcaagtcca agctggtgag cgacttccgc aaggatttcc agttctacaa 7920 ggtccgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg cggtagttgg 7980

Page 11

PL235 163 Β1

SEQLTXT.txt cacggccctc atcaagaagt accctaagct ggtgtacgac gtgcgcaaga tgatcgcgaa caagtacttc ttctacagca acatcatgaa cggcgagatc agaaagaggc cgctcattga ggataaggga agggacttcg cgactgtgcg cgtcaagaag accgaggtcc agaccggcgg gaacagcgat aagctgattg cgaggaagaa cgatagccca accgtggctt acagtgtgct caagaagctc aagtccgtga aggagctgct cgagaagaac ccaatcgact tcctcgaggc catcatcaag ctgccaaagt actccctgtt cgcttctgca ggagagttgc agaaaggcaa cttcctgtac ctcgccagcc actacgagaa gaagcagcta ttcgtcgagc agcacaagca cgagttcagc aagagggtga tcctcgccga caacaagcac cgcgacaagc caattcgcga gctcacaaac ctgggtgcgc cagcagcctt gcggtacacc tccactaagg aggtgctcga tctctacgaa acaaggatcg acctgtctca gaattcgatg agtcaagcag atcgttcaaa tcctgttgcc ggtcttgcga tgattatcat aataattaac atgtaatgca tgacgttatt caattataca tttaatacgc gatagaaaac tcgcgcgcgg tgtcatctat gttactagat acagttctgg tgctcaacac atttatattt gagggaatcg aactaggaat attgatcaga cgagagcgag ttcgtgtacg gcgactacaa gtcggagcag gagatcggca aggcgaccgc cttcttcaag accgagatca cgctggccaa gactaatggt gagactggcg agatcgtgt g caaggttctg agtatgcctc aggtgaacat cttcagcaag gagtcaatcc tcccgaagag ggactgggac ccaaagaagt acggcgggtt ggtggtggcg aaggtcgaga agggtaagtc cggcatcacc atcatggagc ggtccagctt caagggctat aaggaggtca agaaggacct cgagcttgag aacggccgca agaggatgct cgagcttgcg ctcccgtcca agtacgtcaa gctgaagggc tctccagagg acaatgagca ctacctggac gagatcatcg agcagatcag cgcgaacctg gataaggtgc tctccgccta acaggccgaa aacattatac acctgtttac caagtacttc gataccacca tcgaccgcaa cgcgaccctg atccaccagt ctatcaccgg gctcggaggt gattgagctc actagttcta catttggcaa taaagtttct taagattgaa ataatttctg ttgaattacg ttaagcatgt tatgagatgg gttttttgat tagagtcccg aaaatatagc gcgcaaacta ggataaatta cgaccggcat ggaccaagcc cgttattctg atcaaggagc acattgttac tcactgctag ggaactacga gataactgctgacc

8040

8100

8160

8220

8280

8340

8400

8460

8520

8580

8640

8700

8760

8820

8880

8940

9000

9060

9120

9180

9240

9300

9360

9420

9480

9540

Page 12

PL235 163 Β1

SEQLTXT.txt ctctagctct cactgatctg ggtcgcatag tgagatgcag cccacgtgag ttcagcaacg 9600 gtctagcgct gggcttttag gcccgcatga tcgggctttt gtcgggtggt cgacgtgttc 966Θ acgattgggg agagcaacgc agcagttcct cttagtttag tcccacctcg cctgtccagc 972Θ agagttctga ccggtttata aactcgcttg ctgcatcaga cttgatcacc gcggcgtctc 9780 ctagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 9840 agtggcaccg agtcggtgct ttttttcaag ctgatccttg acaggatata ttggcgggta 9900 aactaagtcg ctgtatgtgt ttgtttgaga tctcatgtga gcaaaaggcc agcaaaaggc 996Θ caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 10020 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 1ΘΘ80 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 10140 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 10200 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 10260 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 1Θ32Θ acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgag gtatgt 10380 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 10440 atttggtatc tgcgctctgc tgaagccagt taccttcgga agaagagttg gtagctcttg 1Θ5Θ0 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 10560 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 1062Θ gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 1068Θ ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgtgtaaca 10740 ttggtctagt gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc 108ΘΘ aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc 1Θ260 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac 1Θ920 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc 1098Θ atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg 11040 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt 11100

Page 13

PL235 163 Β1

SEQLTXT.txt cattcgtgat tgcgcctgag aacaggaatc gaatgcaacc tgaatcagga tattcttcta taaccatgca tcatcaggag cgtcagccag tttagtctga atgtttcaga aacaactctg tgattgcccg acattatcgc atttaatcgc ggccttgagc attactgttt atgtaagcag aatgtaacat cagagatttt tgagttgaag gatcagatca aaagttcaaa atcaccaact cactttctgg ctggatgatg cgagacgaaa tacgcgatcg ggcgcaggaa cactgccagc atacctggaa tgctgttttc tacggataaa atgcttgatg ccatctcatc tgtaacaaca gcgcatcggg cttcccatac gagcccattt atacccatat aagacgtttc ccgttgaata acagttttat tgttcatgat gagacacaac gtggctttgt cgcatcttcc cgacaacgca ggtccaccta caacaaagct gggcgattca ggcgatcccc ctgttaaaag gacaattaca gcatcaacaa tattttcacc cctgggatcg cagtggtgag gtcggaagag gcataaattc ttggcaacgc tacctttgcc aatcggtaga ttgtcgcacc aaatcagcat ccatgttgga tggctcataa caccccttgt gatatatttt tatcttgtgc tgaataaatc gaacttttgc gaccgttccg tggcaaagca ctcatcaacc gtggctccct atccaacagc ccgccgtcga gcgggct

11160

11220

11280

11340

11400

11460

11520

11580

11640

11700

11760

11820

11880

11887 <21Θ> 10 <211> 11887 <212> DNA <213> artificial <220>

<223> System Cas9 / sgRNA editing gene Nud <400> 10 tttttatccc cggaagcctg tggatagagg gtagttatcc acgtgaaacc gctaatgccc cgcaaagcct tgattcacgg ggctttccgg cccgctccaa aaactatcca cgtgaaatcg ctaatcaggg tacgtgaaat cgctaatcgg agtacgtgaa atcgctaata aggtcacgtg aaatcgctaa tcaaaaaggc acgtgagaac gctaatagcc ctttcagatc aacagcttgc aaacacccct cgctccggca agtagttaca gcaagtagta tgttcaatta gcttttcaat tatgaatata tatatcaatt attggtcgcc cttggcttgt ggacaatgcg ctacgcgcac cggctccgcc cgtggacaac cgcaagcggt tgcccaccgt cgagcgccag cgcctttgcc

120

180

240

300

360

420

Page 14

PL235 163 Β1

SEQLTXT.txt cacaacccgg cggccggccg caacagatcg ttttataaat tttttttttt gaaaaagaaa 480 aagcccgaaa ggcggcaacc tctcgggctt ctggatttcc gatccccgga attagatctt 54Θ ggcaggatat attgtggtgt aacgtatcac aagtttgtac aaaaaagcag gctccgcggc 600 cgcccccttc acctagactc gacgcgtcct agagatccgt caacatggtg gagcacgaca 660 ctctcgtcta ctccaagaat atcaaagata cagtctcaga agaccaaagg gctattgaga 720 cttttcaaca aagggtaata tcgggaaacc tcctcggatt ccattgccca gctatctgtc 780 acttcatcaa aaggacagta gaaaaggaag gtggcaccta caaatgccat cattgcgata 840 aaggaaaggc tatcgttcaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac 900 ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt 960 gatgtgatat ctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc 102® cttcctctat ataaggaagt tcatttcatt tggagaggac gaccccgata tgaaaaagcc 1080 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga 114Θ cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg 12ΘΘ tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta 126 © tcg gcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag 1320 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc 1380 tgaaaccgaa ctgcccgctg ttctgcaggt aaatttctag tttttctcct tcattttctt 1440 ggttaggacc cttttctctt tttatttttt tgagctttga tctttcttta aactgatcta 1560 ttttttaatt gattggttat ggtgtaaata ttacatagct ttaactgata atctgattac 1560 tttatttcgt gtgtctatga tgatgatgat aactgcagcc ggtcgcggag gccatggatg 1620 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa 1680 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc 1740 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc 180Θ tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct 1860 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga 1920 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt 1980

Page 15

PL235 163 Β1

SEQLTXT.txt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc 2040 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg 2100 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag 216Θ ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct 2220 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg 228Θ aatagagtag atgccgaccg ggatccggag agctcgaatt tccccgatcg ttcaaacatt 2340 tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa 2400 tttctgttga attscgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg 2460 agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 2520 atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg 2580 gaattcatcg atgatatcag atcaagggtg ggcgcgccga accagctttc ttgtacaaag 2640 tggtgatccc cctcgaatcg aattccgtgc agcgtgaccc ggtcgtgccc ctctctagag 2760 ataatgagca ttgcatgtct aagttataaa aaattaccac atattttttt tgtcacactt 276Θ gtttgaagtg cagtttatct atctttatac atatatttaa actttactct acgaataata 2820 taatctatag tactacaata atatcagtgt tttagagaat catataaatg aacagttaga 2880 catggtctaa aggacaattg agtattttga caacaggact ctacagtttt atctttttag 2940 tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc tatataatac ttcatccatt 3000 ttattagtac atccatttag ggtttagggt taatggtttt tatagactaa tttttttagt 3Θ6Θ acatctattt tattctattt tagcctctaa attaagaaaa ctaaaactct attttagttt 3120 ttttatttaa taatttagat ataaaataga ataaaataaa gtgactaaaa attaaacaaa 318Θ taccctttaa gaaattaaaa aaactaagga aacatttttc ttgtttcgag tagataatgc 3240 cagcctgtta aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg 3300 cgtcgggcca agcgaagcag acggcacggc atctctgtcg ctgcctctgg acccctctcg 3360 agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt gcgtggcgga 3420 gcggcagacg tgagccggca cggcaggcgg cctcctcctc ctctcacggc acggcagcta 348Θ cgggggattc ctttcccacc gctccttcgc tttcccttcc tcgcccgccg taataaatag 3540

Page 16

PL235 163 Β1

SEQLTXT.txt acaccccctc cacaccctct ttccccaacc ccagatctcc cccaaatcca cccgtcggca cccccccccc cctctctacc ttctctagat agttctactt ctgttcatgt ttgtgttaga cgttcgtaca cggatgcgac ctgtacgtca ttctctttgg ggaatcctgg gatggctcta ttttttttgt ttcgttgcat agggtttggt gtgcacttgt ttgtcgggtc atcttttcat gtctggttgg gcggtcgttc tsgatcggag ttattaattt tggatctgta tgtgtgtgcc atggatggaa atatcgatct aggataggta atacagagat gctttttgtt cgcttggttg attcgttcta gatcggagta gaatactgtt gaactgtatg tgtgtgtcat acatcttcat gatctaggat aggtatacat gttgatgtgg tgcagcatct attcatatgc tctaaccttg ttttataatt attttgatct tgatatactt gattttttta gccctgcctt catacgctat tgctcaccct gttgtttggt gttacttcgc aagaggaagg tgggcatgga caagaagtac gtaggctggg cggtgatcac ggacgagtat ggaaacaccg accgtcacag catcaagaag ggtaaatttc tgtgttcctt attctctcaa atttcgtata tgttctttgg tttagattct gtttgatcgt tagatatcat cttaattctc gttcaaataa tttgagtttt gtcgaataat tcgtgttgtt cggagcgcac acacacacaa cctccgcttc aaggtacgcc gctcgtcctc cggcgttccg gtccatggtt agggcccggt tccgtgtttg tgttagatcc gtgctgcta g gacacgttct gattgctaac ttgccagtgt gccgttccgc agacgggatc gatttcatga ttgccctttt cctttatttc aatatatgcc gctttttttt gtcttggttg tgatgatgtg tagaattctg tttcaaacta cctggtggat atacatattc atagttacga attgaagatg tacatgttga tgcgggtttt actgatgcat tgatgatgtg gtgtggttgg gcggtcgttc tcaaactacc tggtgtattt attaattttg agttacgagt ttaagatgga tggaaatatc gttttactga tgcatataca tgatggcata agtacctatc tattataata aacaagtatg ggatgatggc atatgcagca gctatatgtg ttatttgctt ggtactgttt cttttgtcga cccccgggat ccgccatggc cccgaagaag tccatcggcc tggacatcgg gaccaactct aaggtgccct cgaagaagtt caaggtcctg aacctgatag gggccctcct gttcgactca aatcttcgat tttgttttcg ttcgatccca gttaatctta gatcgaagac gattttctgg gattagggtt tcatagatat catccgattt tactcttcga tttgtagattt

3600

3660

3720

3780

3840

3900

3960

4020

4080

4140

4200

4260

4320

4380

4440

4500

4560

4620

4680

4740

4800

4860

4920

4980

5040

5100

Page 17

PL235 163 Β1

SEQLTXT.txt ctggtgttag tttctagttt gtgcgatcga atttgtcgat taatctgagt ttttctgatt 516Θ aacaggtgag actgctgagg cgaccaggct gaagcgtact gcaagaagac gttacacgag 5220 gcggaagaac aggatctgct acctccaaga gatcttcagc aacgagatgg cgaaggtcga 5280 cgactcgttc ttccaccgtc tcgaggagag cttcctggtc gaggaagaca agaagcacga 5340 gaggcacccg atcttcggga acatcgtgga cgaagtggcg taccacgaga agtaccccac 5400 aatctaccac ctccggaaga agctggttga ctccactgac aaggctgacc tgcgtctcat 546Θ ctacctggcc ctcgcccaca tgatcaagtt caggggtcac ttcctcatag agggggacct 5520 gaaccctgac aatagcgacg tcgacaagct gttcatccag ctggtgcaaa cctacaacca 5580 gctcttcgag gagaacccca tcaacgctag tggagttgac gccaaggcta tcctgtctgc 564Θ taggctgtcc aaaagccgac gcttggagaa cttaatcgct cagcttccgg gggagaagaa 5700 gaacggactc ftcgggaacc tgattgccct gagtctgggc ctgaccccga acttcaaaag 5760 caacttcgac ctcgccgagg acgcgaaact ccagctgtca aaggacacat acgacgacga 5820 cctggacaac ctgttagcac agatcgggga ccagtacgcc gacctctttc tggcagctaa 588Θ gaacctgtct gacgccatcc tcctgtccga catcctccga gtcaacactg agatcaccaa 5940 ggccccactg agcgcctcta tgataaaacg ctacgacgag caccaccagg acctgactct 6000 cctgaaggct ctcgtacgtc agcagctgcc tgagaagtac aaggagatct tcttcgacca 6060 gtcgaagaac ggctacgccg ggtacattga tggcggggct tctcaagagg agttctacaa 612Θ gttcatcaag cccatcctcg agaagatgga cggcaccgag gagctgctcg ttaagttgaa 618Θ cagagaggac ctgctccgaa agcagaggac ctttgataac gggtcgatcc cgcaccagat 624Θ ccatctcggt gagctccatg ctatcctgcg acgccaagag gacttctacc ccttcctcaa 6300 ggacaaccgc gagaagattg agaagatcct gacattccga atcccgtact acgtcgggcc 6360 tcttgcaagg ggcaacagcc ggttcgcttg gatgacccgc aagtcggagg agactatcac 6420 accctggaac tttgaggagg tcgtggacaa gggcgcatct gcccagagtt tcatcgagag 648Θ gatgaccaac ttcgataaga acctcccgaa cgagaaggtg ctcccgaagc actctctgct 654Θ ctacgagtac ttcaccgtgt acaacgagct gacgaaggtc aagtacgtga ccgagggtat 6600 gcggaagcct gcgttcctga gcggggagca aaagaaggcc atcgtcgatc tcctgttcaa 6660

Page 1S

PL235 163 Β1 gaccaaccgc aaggtgaccg tcaagcagct cttcgactcc gtcgagatct cgggcgttga ccatgacctg ctcaagatta tcaaggacaa cattctagag gacatcgtgc tcacgctgac gcgcctcaag acctacgccc acctcttcga caggtatact ggatggggga ggctctcaag gagcggcaag acgatcctcg acttcctgaa gcagctcatc cacgacgact cgctcacgtt cggccagggt gattctctgc atgagcatat aaagggaatc ctccagaccg tcaaggtcgt caagcctgag aacatcgtga tcgagatggc gaagaactcg cgcgagcgca tgaagagaat gatcctcaag gagcacccag ttgagaacac ctacctgcag aacggcaggg acatgtacgt cgactacgac gttgaccata tcgtgcccca caaggtgctc acgaggtccg acaagaaccg ggtcgtgaag aagatgaaga actactggcg gcgcaagttc gacaacttga caaaggcaga gggcttcatt aagaggcagc tcgtcgagac cctggatagc aggatgaaca cgaagtacga ggtcatcacc ctcaagtcca agctggtgag ggtccgcgag atcaacaact accaccacgc cacggccctc atcaagaagt accctaagct ggtgtacgac gtgcgcaaga tgatcgcgaa caagtacttc ttctacagca acatcatgaa cggcgagatc agaaagaggc cgctcattga

SEQLTXT.txt caaggaggac tacttcaaga agatcgagtg ggacaggttc aacgcaagcc tggggaccta ggacttcctc gacaacgagg agaatgagga cctcttcgag gaccgcgaga tgatcgagga cgacaaggtg atgaagcaac tgaagaggcg gaagctgatc aacggcatca gggacaagca gtctgacggc ttcgctaacc gcaacttcat caaggaagac atccagaagg cgcaggtaag cgctaacctc gccggctccc ccgcaatcaa ggacgagctg gttaaagtga tgggcaggca gcgcgagaat cagacgactc agaagggcca tgaggagggt atcaaggaac tcggcagcca ccagctccag aatgagaagc tgtacctcta ggaccaagag ctggacatca acaggctgtc aagtttcctc aaggacgact cgatcgacaa cggcaagtct gacaacgtgc caagcgagga gcagctcctg aacgccaagc tcatcaccca gagaggaggc ctgtcggagc ttgataaggc taggcagatc acgaagcacg tggcgcagat cgagaacgac aagctcatcc gcgaggtgaa cgacttccgc aaggatttcc agttctacaa ccacgacgcc tacctgaacg cggtagttgg cgagagcgag ttcgtgtacg gcgactacaa gtcggagcag gagatcggca aggcgaccgc cttcttcaag accgagatca cgctggccaa gactaatggt gagactggcg agatcgtgtg

6720

6780

6840

6900

6960

7020

7080

7140

7200

7260

7320

7380

7440

7500

7560

7620

7680

7740

7800

7860

7920

7980

8040

8100

8160

8220

Page 19

PL235 163 Β1

SEQLTXT.txt ggataaggga agggacttcg cgtcaagaag accgaggtcc gaacagcgat aagctgattg cgatagccca accgtggctt caagaagctc aagtccgtga cgagaagaac ccaatcgact catcatcaag ctgccaaagt cgcttctgca ggagagttgc cttcctgtac ctcgccagcc gaagcagcta ttcgtcgagc cgagttcagc aagagggtga caacaagcac cgcgacaagc gctcacaaac ctgggtgcgc gcggtacacc tccactaagg tctctacgaa acaaggatcg gaattcgatg agtcaagcag tcctgttgcc ggtcttgcga aataattaac atgtaatgca caattataca tttaatacgc tcgcgcgcgg tgtcatctat acagttctgg tgctcaacac gagggaatcg aactaggaat ctctagctct cactgatctg gtctagcgct gggcttttag acgattgggg agagcaacgc agagttctga ccggtttata cgactgtgcg caaggttctg agaccggcgg cttcagcaag cgaggaagaa ggactgggac acagtgtgct ggtggtggcg aggagctgct cggcatcacc tcctcgaggc caagggctat actccctgtt cgagcttgag agaaaggcaa cgagcttgcg actacgagaa gctgaagggc agcacaagca ctacctggac tcctcgccga cgcgaacctg caattcgcga acaggccgaa cagcagcctt caagtacttc aggtgctcga cgcgaccctg acctgtctca gctcggaggt atcgttcaaa catttggcaa tgattatcat ataatttctg tgacgttatt tatgagatgg gatagaaaac aaaatatag c gttactagat cgaccggcat atttatattt atcaaggagc attgatcaga ggaactacga ggtcgcatag tgagatgcag gcccgcatga tcgggctttt agcagttcct cttagtttag aactcgcttg ctgcatcaga agtatgcctc aggtgaacat gagtcaatcc tcccgaagag ccaaagaagt acggcgggtt aaggtcgaga agggtaagtc atcatggagc ggtccagctt aaggaggtca agaaggacct aacggccgca agaggatgct ctcccgtcca agtacgtcaa tctccagagg acaatgagca gagatcatcg agcagatcag gataaggtgc tctccgccta aacattatac acctgtttac gataccacca tcgaccgcaa atccaccagt ctatcaccgg gattgagctc actagttcta taaagtttct taagattgaa ttgaattacg ttaagcatgt gttttttgat tagagtcccg gcgcaaacta ggataaatta ggaccaagcc cgttattctg acattgttac tcactgctag gagagctgaa gataactgcc cccacgtgag ttcagcaacg gtcgggtggt cgacgtgttc tcccacctttcagg cctgtttggg cctgtttggg

8280

8340

8400

8460

8520

8580

8640

8700

8760

8820

8880

8940

9000

9060

9120

9180

9240

9300

9360

9420

9480

9540

9600

9660

9720

9780

Page 20

PL235 163 Β1

SEQLTXT.txt cgggttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 984Θ agtggcaccg agtcggtgct ttttttcaag ctgatccttg acaggatata ttggcgggta 9900 aactaagtcg ctgtatgtgt ttgtttgaga tctcatgtga gcaaaaggcc agcaaaaggc 9960 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 10020 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 10Θ80 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 10140 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 1Θ20Θ taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 10260 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 10320 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 10380 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 1Θ44Θ atttggtatc tgcgctctgc tgaagccagt taccttcgga agaagagttg gtagctcttg 10500 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 10560 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 19620 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 1Θ680 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgtgtaaca 10740 ttggtctagt gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc 10800 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc 10860 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac 10920 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc 16980 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg 11Θ40 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt 11100 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca 11160 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc 11220 tgaatcagga tattcttcta atacctggaa tgctgttttc cctgggatcg cagtggtgag 11280 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc 11340

Page 21

PL235 163 Β1

SEQLTXT.txt cgtcagccag tttagtctga ccatctcatc tgtaacaaca ttggcaacgc tacctttgcc 11400 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcggtaga ttgtcgcacc 11460 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga 1152Θ atttaatcgc ggccttgagc aagacgtttc ccgttgaata tggctcataa caccccttgt 11580 attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt tatcttgtgc 11640 aatgtaacat cagagatttt gagacacaac gtggctttgt tgaataaatc gaacttttgc 11760 tgagttgaag gatcagatca cgcatcttcc cgacaacgca gaccgttccg tggcaaagca 11760 aaagttcaaa atcaccaact ggtccaccta caacaaagct ctcatcaacc gtggctccct 11320 cactttctgg ctggatgatg gggcgattca ggcgatcccc atccaacagc cccaacagc ccgccgtcga 11880 <137> <b> <b> 12140> <b> <b> <b> <b> <b> <b> </b> <b> <b> <b> <b> <b> <b> <b> <b> <b> <b> <b> <b> <b> <p>

<223> Cas9 / sgRNA system for simultaneous editing of HvCKX1 and genes

HvCKX3 <460> 11 tttttatccc cggaagcctg tggatagagg gtagttatcc acgtgaaacc gctaatgccc 60 cgcaaagcct tgattcacgg ggctttccgg cccgctccaa aaactatcca cgtgaaatcg 12Θ ctaatcaggg tacgtgaaat cgctaatcgg agtacgtgaa atcgctaata aggtcacgtg 180 aaatcgctaa tcaaaaaggc acgtgagaac gctaatagcc ctttcagatc aacagcttgc 240 aaacacccct cgctccggca agtagttaca gcaagtagta tgttcaatta gcttttcaat 300 tatgaatata tatatcaatt attggtcgcc cttggcttgt ggacaatgcg ctacgcgcac 360 cggctccgcc cgtggacaac cgcaagcggt tgcccaccgt cgagcgccag cgcctttgcc 420 cacaacccgg cggccggccg caacagatcg ttttataaat tttttttttt gaaaaagaaa 480 aagcccgaaa ggcggcaacc tctcgggctt ctggatttcc gatccccgga attagatctt 54Θ ggcaggatat attgtggtgt aacgtatcac aagtttgtac aaaaaagcag gctccgcggc 6ΘΘ

Page 22

PL235 163 Β1 cgcccccttc acctagactc gacgcgtcct ctctcgtcta ctccaagaat atcaaagata cttttcaaca aagggtaata tcgggaaacc acttcatcaa aaggacagta gaaaaggaag aaggaaaggc tatcgttcaa gatgcctctg ccacgaggag catcgtggaa aaagaagacg gatgtgatat ctccactgac gtaagggatg cttcctctat ataaggaagt tcatttcatt tgaactcacc gcgacgtctg tcgagaagtt cctgatgcag ctctcggagg gcgaagaatc tggatatgtc ctgcgggtaa atagctgcgc tcggcacttt gcatcggccg cgctcccgat cgagagcctg acctattgca tctcccgccg tgaaaccgaa ctgcccgctg ttctgcaggt ggttaggacc cttttctctt tttatttttt ttttttaatt gattggttat ggtgtaaata tttatttcgt gtgtctatga tgatgatgat cgatcgctgc ggccgatctt agccagacga tcggtcaata cactacatgg cgtgatttca actggcaaac tgtgatggac gacaccgtca tgatgctttg ggccgaggac tgccccgaag ccaacaatgt cctgacggac aatggccgca tgttcgggga ttcccaatac gaggtcgcca gtatggagca gcagacgcgc tacttcgagc ggctccgggc gtatatgctc cgcattggtc gcaatttcga tgatgcagct tgggcgcagg

SEQLTXT.txt agagatccgt caacatggtg gagcacgaca cagtctcaga agaccaaagg gctattgaga tcctcggatt ccattgccca gctatctgtc gtggcaccta caaatgccat cattgcgata ccgacagtgg tcccaaagat ggacccccac ttccaaccac gtcttcaaag caagtggatt acgcacaatc ccactatcct tcgcaagacc tggagaggac gaccccgata tgaaaaagcc tctgatcgaa aagttcgaca gcgtctccga tcgtgctttc agcttcgatg taggagggcg cgatggtttc tacaaagatc gttatgttta tccggaagtg cttgacattg gggaattcag tgcacagggt gtcacgttgc aagacctgcc aaatttctag tttttctcct tcattttctt tgagctttga tctttcttta aactgatcta ttacatagct ttaactgata atctgattac aactgcagcc ggtcgcggag gccatggatg gcgggttcgg cccattcgga ccgcaaggaa tatgcgcgat tgctgatccc catgtgtatc gtgcgtccgt cgcgcaggct ctcgatgagc tccggcacct cgtgcacgcg gatttcggct taacagcggt cattgactgg agcgaggcga acatcttctt ctggaggccg tggttggctt ggaggcatcc ggagcttgca ggatcgccgc ttgaccaact ctatcagagc ttggttgacg gtcgatgcga cgcaatcgtc cgatccggag

660

72Θ

78Θ

84Θ

900

960

1020

1080

1140

1200

1260

1320

1380

1440

1500

1560

1620

1680

1740

1800

1860

1920

1980

2040

2100

2160

Page 23

PL235 163 Β1 ccgggactgt cgggcgtaca caaatcgccc gtgtagaagt actcgccgat agtggaaacc aatagagtag atgccgaccg ggatccggag tggcaataaa gtttcttaag attgaatcct tttctgttga attacgttaa gcatgtaata agatgggttt ttatgattag agtcccgcaa atatagcgcg caaactagga taaattatcg gaattcatcg atgatatcag atcaagggtg tggtgatccc cctcgaatcg aattccgtgc ataatgagca ttgcatgtct aagttataaa gtttgaagtg cagtttatct atctttatac taatctatag tactacaata atatcagtgt catggtctaa aggacaattg agtattttga tgtgcatgtg ttctcctttt tttttgcaaa ttattagtac atccatttag ggtttagggt acatctattt tattctattt tagcctctaa ttttatttaa taatttagat ataaaataga taccctttaa gaaattaaaa aaactaagga cagcctgtta aacgccgtcg acgagtctaa cgtcgggcca agcgaagcag acggcacggc agagttccgc tccaccgttg gacttgctcc gcggcagacg tgagccggca cggcaggcgg cgggggattc ctttcccacc gctccttcgc acaccccctc cacaccctct ttccccaacc ccagatctcc cccaaatcca cccgtcggca cccccccccc cctctctacc ttctctagat

SEQLTXT.txt gcagaagcgc ggccgtctgg accgatggct gacgccccag cactcgtccg agggcaaagg agctcgaatt tccccgatcg ttcaaacatt gttgccggtc ttgcgatgat tatcatataa attaacatgt aatgcatgac gttatttatg ttatacattt aatacgcgat agaaaacaaa cgcgcg & tgt catctatgtt actagatcgg ggcgcgccga accagctttc ttgtacaaag agcgtgaccc ggtcgtgccc ctctctagag aaattaccac atattttttt tgtcacactt atatatttaa actttactct acgaataata tttagagaat catataaatg aacagttaga caacaggact ctacagtttt atctttttag tagcttcacc tatataatac ttcatccatt taatggtttt tatagactaa tttttttagt attaagaaaa ctaaaactct attttagttt ataaaataaa gtgactaaaa attaaacaaa aacatttttc ttgtttcgag tagataatgc cggacaccaa ccagcgaacc agcagcgtcg atctctgtcg ctgcctctgg acccctctcg gctgtcggca tccagaaatt gcgtggcgga cctcctcctc ctctcacggc acggcagcta tttcccttcc tcgcccgccg taataaatag tcgtgttgtt cggagcgcac acacacacaa cctccgcttc aaggtacgcc gctcgtcctc cggcgttccg gtccatggtt agggcccggt

2220

2280

2340

2400

2460

2520

2580

2640

2700

2760

2820

2880

2940

3000

3060

3120

3180

3240

3300

3360

3420

3480

3540

3600

3660

3720

Page 24

PL235 163 Β1

SEQLTXT.txt agttctactt ctgttcatgt ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag 3780 cgttcgtaca cggatgcgac ctgtacgtca gacacgttct gattgctaac ttgccagtgt 3840 ttctctttgg ggaatcctgg gatggctcta gccgttccgc agacgggatc gatttcatga 390O ttttttttgt ttcgttgcat agggtttggt ttgccctttt cctttatttc aatatatgcc 3960 gtgcacttgt ttgtcgggtc atcttttcat gctttttttt gtcttggttg tgatgatgtg 4020 gtctggttgg gcggtcgttc tagatcggag tagaattctg fttcaaacta cctggtggat 4Θ8Θ ttattaattt tggatctgta tgtgtgtgcc atacatattc atagttacga attgaagatg 414Θ atggatggaa atatcgatct aggataggta tacatgttga tgcgggtttt actgatgcat 42Θ0 atacagagat gctttttgtt cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc 426Θ attcgttcta gatcggagta gaatactgtt tcaaactacc tggtgtattt attaattttg 432Θ gaactgtatg tgtgtgtcat acatcttcat agttacgagt ttaagatgga tggaaatatc 4388 gatctaggat aggtatacat gttgatgtgg gttttactga tgcatataca tgatggcata 4440 tgcagcatct attcatatgc tctaaccttg agtacctatc tattataata aacaagtatg 4500 ttttataatt attttgatct tgatatactt ggatgatggc atatgcagca gctatatgtg 4560 gattttttta gccctgcctt catacgctat ttatttgctt ggtactgttt cttttgtcga 4620 tgctcaccct gttgtttggt gttacttcgc cccccgggat ccgccatggc cccgaagaag 4680 aagaggaagg tgggcatgga caagaagtac tccatcggcc tggacatcgg gaccaactct 4740 gtaggctggg cggtgatcac ggacgagtat aaggtgccct cgaagaagtt caaggtcctg 4800 ggaaacaccg accgtcacag catcaagaag aacctgatag gggccctcct gttcgactca 4860 ggtaaatttc tgtgttcctt attctctcaa aatcttcgat tttgttttcg ttcgatccca 4920 atttcgtata tgttctttgg tttagattct gttaatctta gatcgaagac gattttctgg 4980 gtttgatcgt tagatatcat cttaattctc gattagggtt tcatagatat catccgattt 5040 gttcaaataa tttgagtttt gtcgaataat tactcttcga tttgtgattt ctatctagat 5100 ctggtgttag tttctagttt gtgcgatcga atttgtcgat taatctgagt ttttctgatt 5160 aacaggtgag actgctgagg cgaccaggct gaagcgtact gcaagaagac gttacacgag 5220 gcggaagaac aggatctgct acctccaaga gatcttcagc aacgagatgg cgaaggtcga 5280

Page 25

PL235 163 Β1

SEQLTXT.txt cgactcgttc ttccaccgtc tcgaggagag cttcctggtc gaggaagaca agaagcacga 5340 gaggcacccg atcttcggga acatcgtgga cgaagtggcg taccacgaga agtaccccac 5400 aatctaccac ctccggaaga agctggttga ctccactgac aaggctgacc tgcgtctcat 5460 ctacctggcc ctcgcccaca tgatcaagtt caggggtcac ttcctcatag agggggacct 5520 gaaccctgac aatagcgacg tcgacaagct gttcatccag ctggtgcaaa cctacaacca 5580 gctcftcgag gagaacccca tcaacgctag tggagttgac gccaaggcta tcctgtctgc 5640 taggctgtcc aaaagccgac gcttggagaa cttaatcgct cagcttccgg gggagaagaa 5700 gaacggactc ttcgggaacc tgattgccct gagtctgggc ctgaccccga acttcaaaag 5760 caacttcgac ctcgccgagg acgcgaaact ccagctgtca aaggacacat acgacgacga 5820 cctggacaac ctgttagcac agatcgggga ccagtacgcc gacctctttc tggcagctaa 5880 gaacctgtct gacgccatcc tcctgtccga catcctccga gtcaacactg agatcaccaa 5940 ggccccactg agcgcctcta tgataaaacg ctacgacgag caccaccagg acctgactct 60Θ0 cctgaaggct ctcgtacgtc agcagctgcc tgagaagtac aaggagatct tcttcgacca 6060 gtcgaagaac ggctacgccg ggtacattga tggcggggct tctcaagagg agttctacaa 6120 gttcatcaag cccatcctcg agaagatgga cggcaccgag gagctgctcg ttaagttgaa 6180 cagagaggac ctgctccgaa agcagaggac ctttgataac gggtcgatcc cgcaccagat 6240 ccatctcggt gagctccatg ctatcctgcg acgccaagag gacttctacc ccttcctcaa 6360 ggacaaccgc gagaagattg agaagatcct gacattccga atcccgtact acgtcgggcc 6360 tcttgcaagg ggcaacagcc ggttcgcttg gatgacccgc aagtcggagg agactatcac 6420 accctggaac tttgaggagg tcgtggacaa gggcgcatct gcccagagtt tcatcgagag 6480 gatgaccaac ttcgataaga acctcccgaa cgagaaggtg ctcccgaagc actctctgct 6540 ctacgagtac ttcaccgtgt acaacgagct gacgaaggtc aagtacgtga ccgagggtat 660Θ gcggaagcct gcgttcctga gcggggagca aaagaaggcc atcgtcgatc tcctgttcaa 6660 gaccaaccgc aaggtgaccg tcaagcagct caaggaggac tacttcaaga agatcgagtg 6720 cttcgactcc gtcgagatct cgggcgttga ggacaggttc aacgcaagcc tggggaccta 6780 ccatgacctg ctcaagatta tcaaggacaa ggacttcctc gacaacgagg agaatgagga 6840

Page 26

PL235 163 Β1

SEQLTXT.txt cattctagag gacatcgtgc tcacgctgac cctcttcgag gaccgcgaga tgatcgagga gcgcctcaag acctacgccc acctcttcga cgacaaggtg atgaagcaac tgaagaggcg caggtatact ggatggggga ggctctcaag gaagctgatc aacggcatca gggacaagca gagcggcaag acgatcctcg acttcctgaa gtctgacggc ttcgctaacc gcaacttcat gcagctcatc cacgacgact cgctcacgtt caaggaagac atccagaagg cgcaggtaag cggccagggt gattctctgc atgagcatat cgctaacctc gccggctccc ccgcaatcaa aaagggaatc ctccagaccg tcaaggtcgt ggacgagctg gttaaagtga tgggcaggca caagcctgag aacatcgtga tcgagatggc gcgcgagaat cagacgactc agaagggcca gaagaactcg cgcgagcgca tgaagagaat tgaggagggt atcaaggaac tcggcagcca gatcctcaag gagcacccag ttgagaacac ccagctccag aatgagaagc tgtacctcta ctacctgcag aacggcaggg acatgtacgt ggaccaagag ctggacatca acaggctgtc cgactacgac gttgaccata tcgtgcccca aagtttcctc aaggacgact cgatcgacaa caaggtgctc acgaggtccg acaagaaccg cggcaagtct gacaacgtgc caagcgagga ggtcgtgaag aagatgaaga actactggcg gcagctcctg aacgccaagc tcatcaccca gcgcaagttc gacaacttga caaaggcaga gagaggaggc ctgtcggagc ttgataagg c gggcttcatt aagaggcagc tcgtcgagac taggcagatc acgaagcacg tggcgcagat cctggatagc aggatgaaca cgaagtacga cgagaacgac aagctcatcc gcgaggtgaa ggtcatcacc ctcaagtcca agctggtgag cgacttccgc aaggatttcc agttctacaa ggtccgcgag atcaacaact accaccacgc ccacgacgcc tacctgaacg cggtagttgg cacggccctc atcaagaagt accctaagct cgagagcgag ttcgtgtacg gcgactacaa ggtgtacgac gtgcgcaaga tgatcgcgaa gtcggagcag gagatcggca aggcgaccgc caagtacttc ttctacagca acatcatgaa cttcttcaag accgagatca cgctggccaa cggcgagatc agaaagaggc cgctcattga gactaatggt gagactggcg agatcgtgtg ggataaggga agggacttcg cgactgtgcg caaggttctg agtatgcctc aggtgaacat cgtcaagaag accgaggtcc agaccggcgg cttcagcaag gagtcaatcc tcccgaagag gaacagcgat aagctgattg cgaggaagaa ggactgggtac ccagagggtac

69ΘΘ

6960

7020

7080

7140

7200

7260

7320

7380

7440

7500

7560

7620

7680

7740

7800

7860

7920

7980

8040

8100

8160

8220

8280

8340

8400

Page 27

PL235 163 Β1

SEQLTXT.txt cgatagccca accgtggctt acagtgtgct ggtggtggcg aaggtcgaga agggtaagtc 8460 caagaagctc aagtccgtga aggagctgct cggcatcacc atcatggagc ggtccagctt 8520 cgagaagaac ccaatcgact tcctcgaggc caagggctat aaggaggtca agaaggacct 8580 catcatcaag ctgccaaagt actccctgtt cgagcttgag aacggccgca agaggatgct 8640 cgcttctgca ggagagttgc agaaaggcaa cgagcttgcg ctcccgtcca agtacgtcaa 8700 cttcctgtac ctcgccagcc actacgagaa gctgaagggc tctccagagg acaatgagca 876Θ gaagcagcta ttcgtcgagc agcacaagca ctacctggac gagatcatcg agcagatcag 8820 cgagttcagc aagagggtga tcctcgccga cgcgaacctg gataaggtgc tctccgccta 8880 caacaagcac cgcgacaagc caattcgcga acaggccgaa aacattatac acctgtttac 8940 gctcacaaac ctgggtgcgc cagcagcctt caagtacttc gataccacca tcgaccgcaa 90Θ0 gcggtacacc tccactaagg aggtgctcga cgcgaccctg atccaccagt ctatcaccgg 906Θ tctctacgaa acaaggatcg acctgtctca gctcggaggt gattgagctc actagttcta 9120 gaattcgatg agtcaagcag atcgttcaaa catttggcaa taaagtttct taagattgaa 9180 tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt 9240 aataattaac atgtaatgca tgacgttatt tatgagatgg gttttttgat tagagtcccg 93ΘΘ caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta 9360 tcgcgcgcgg tgtcatctat gttactagat cgaccggcat ggaccaagcc cgttattctg 9420 acagttctgg tgctcaacac atttatattt atcaaggagc acattgttac tcactgctag 9480 gagggaatcg aactaggaat attgatcaga ggaactacga gagagctgaa gataactgcc 9540 ctctagctct cactgatctg ggtcgcatag tgagatgcag cccacgtgag ttcagcaacg 96ΘΘ gtctagcgct gggcttttag gcccgcatga tcgggctttt gtcgggtggt cgacgtgttc 9660 acgattgggg agagcaacgc agcagttcct cttagtttag tcccacctcg cctgtccagc 972Θ agagttctga ccggtttata aactcgcttg ctgcatcaga cttgcaacaa agcgcatctg 9780 gtgtagtggt atcatagtac cctcccacgg tactgaccag ggttcgattc cctggatgcg 9849 cagatcaccg cggcgtctcc tagttttaga gctagaaata gcaagttaaa ataaggctag 9900 tccgttatca acttgaaaaa gtggcaccga gtcggtgcca acaaagcgca tctggtgtag 9960

Page 28

PL235 163 Β1

SEQLTXT.txt tggtatcata gtaccctccc acggtactga ccagggttcg attccctgga tgcgcacaag 10020 ttcatccaga gccccagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 10080 atcaacttga aaaagtggca ccgagtcggt gctttttttc aagctgatcc ttgacaggat 10140 atattggcgg gtaaactaag tcgctgtatg tgtttgtttg agatctcatg tgagcaaaag 18200 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 10260 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 1032Θ gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 16380 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 16440 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 16500 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 10560 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 10620 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 10680 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaagaagag 10740 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtt mp tttgtttgca 10800 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 10860 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 1Θ920 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 1Θ980 tatatgtgta acattggtct agtgattaga aaaactcatc gagcatcaaa tgaaactgca 11Θ40 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 111Θ0 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 11160 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 11220 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt 1128Θ ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa 11340 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa 11400 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa 11460 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttccctggga 11520

Page 29

PL235 163 Β1

5EQLTXT.txt tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca acattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcggt agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggccttg agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt tgttgaataa atcgaacttt tgctgagttg aaggatcaga tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa gcaaaagttc aaaatcacca actggtccac ctacaacaaa gctctcatca accgtggctc cctcactttc tggctggatg atggggcgat tcaggcgatc cccatccaac agccgcgccgtg

11580

11640

11700

11760

11820

11880

11940

12,000

12060

12120

12140

Claims (12)

Patent claims 1. A synthetic Cas9 endonuclease gene having the nucleotide sequence shown as SEQ. No. 1. 2. An empty sgRNA targeting cassette consisting of: a) a U6 promoter having the nucleotide sequence shown as SEQ. no.2 and b) a gRNA coding sequence having the nucleotide sequence shown as SEQ. No. 3 connected operationally with the one in between c) a sequence encoding a site for cloning a sequence complementary to the edited sequence, preferably containing a restriction enzyme recognition sequence. The empty sgRNA targeting cassette according to claim 1; The cassette of claim 2, wherein the sequence comprises two Bsal restriction sites for directional cloning of a sequence complementary to the edited sequence, the cassette preferably having the sequence shown as SEQ. no 4. 4. A filled sgRNA targeting cassette consisting of: a) a U6 promoter having the nucleotide sequence shown as SEQ. no.2 and b) a gRNA coding sequence having the nucleotide sequence shown as SEQ. No. 3 connected operationally with the one in between c) a sequence complementary to the edited sequence, preferably cloned into two sites for the restriction enzyme Bsal. 5. The filled sgRNA targeting cassette according to claim 5; 4. The method of claim 4, wherein as a sequence complementary to the edited sequence it contains a sequence selected from the group consisting of: a) a sequence comprising a 21-nucleotide sequence complementary to the edited target gene sequence (spacer), preferably in the form of a double-stranded DNA oligonucleotide with the following structure: 5'-CT TGNNNNNNNNNNNNNNNNNNNN-3 ' 3'-NNNNNNNNNNNNNNNNNNNNCAAA-5 'where N is any selected nucleotide, b) a PTG (polycistronic tRNA / gRNA) sequence with the structure shown in Fig. 4 containing several single sequences containing a 20 or 21 nucleotide sequence complementary to the edited target gene sequence (s) separated by short tRNA sequences. 6. The filled sgRNA targeting cassette according to claim 6; The spacer of the target gene (spacer) selected from Seq. 4 or 5, comprising a 21-nucleotide sequence complementary to the edited target gene sequence (spacer). no.5-7. 7. The filled sgRNA targeting cassette according to claim 7; The method of claim 5 or 6, characterized in that it comprises the PTG sequence having the nucleotide sequence shown as SEQ. no.8. A kit for the production of a Cas9 / sgRNA system for editing the genome of a monocotyledonous plant, in particular of barley, characterized in that it comprises the synthetic Cas9 endonuclease gene according to claim 1. 1 and an empty sgRNA targeting cassette as defined in claim 1; 2-3. 9. The Cas9 / sgRNA system for editing the genome of a monocotyledonous plant, in particular of barley, characterized in that it comprises the synthetic Cas9 endonuclease gene as defined in claim 1. 1 and a filled sgRNA targeting cassette as defined in claim 1, 4-7. 10. The kit according to p. 8 or the system according to claim 8 The method of claim 9, which is in the form of a plasmid vector. 11. Method for editing the genome of a monocotyledonous plant, in particular of barley, characterized in that the Cas9 / sgRNA system as defined in claim 1 is introduced into the plant cell. 10, the transformed cells are then isolated and the transgenic plants recovered therefrom. 12. The method according to p. The method of claim 11, characterized in that the Cas9 / sgRNA system is introduced into the cells of immature barley embryos by means of Agrobacterium tumefaciens.