CN117384942A - Split-Cas9 system suitable for plant genome editing and application thereof - Google Patents

Split-Cas9 system suitable for plant genome editing and application thereof Download PDF

Info

Publication number
CN117384942A
CN117384942A CN202311373061.8A CN202311373061A CN117384942A CN 117384942 A CN117384942 A CN 117384942A CN 202311373061 A CN202311373061 A CN 202311373061A CN 117384942 A CN117384942 A CN 117384942A
Authority
CN
China
Prior art keywords
intein
cas
expression element
promoter
terminator
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202311373061.8A
Other languages
Chinese (zh)
Inventor
孙永伟
胡利喆
胡丹玲
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inner Mongolia University
Original Assignee
Inner Mongolia University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Inner Mongolia University filed Critical Inner Mongolia University
Priority to CN202311373061.8A priority Critical patent/CN117384942A/en
Publication of CN117384942A publication Critical patent/CN117384942A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Saccharide Compounds (AREA)

Abstract

The invention belongs to the technical field of plant molecular biology, and particularly relates to a Split-Cas9 system suitable for editing a plant genome and application thereof. The invention aims to provide a new choice for plant genome editing technology. The technical scheme of the invention is a Split-Cas9 system suitable for editing plant genome, which splits Intein into Intin N And Intin C Splitting NCas9 and CCas9 by Cas9, and reconstructing the NCas9 and the CCas9 into an expression vector; specifically comprises a gRNA expression element and an intein-Cas 9 expression element aiming at a target gene; the intein-Cas 9 expression element comprises two units, each unit consisting of: promoter-structural sequence region-terminator; the structural sequence region includes Intelin N Or Intin C And NCas9 or CCas9, and the elements comprised in the structural sequence regions of the two units may not be identical. The Split-Cas9 has less influence on genome editing activity, effectively reduces the size of a single transcription unit, and provides more possibility for virus-mediated genome editing technology.

Description

Split-Cas9 system suitable for plant genome editing and application thereof
Technical Field
The invention belongs to the technical field of plant molecular biology, and particularly relates to a Split-Cas9 system suitable for editing a plant genome and application thereof.
Background
The CRISPR/Cas system is a new genome site-directed editing technology which appears after ZFNs and TALENs technologies, and the CRISPR/Cas system after artificial modification mainly consists of two parts, namely Cas nuclease for shearing target DNA and gRNA for guiding the nuclease to the DNA target position, and different genes are edited by only replacing a nucleotide sequence of about 20bp on the original gRNA, so that the system has the characteristics of simplicity, high efficiency, universality, accuracy and the like, and has become an important research tool in modern plant molecular biology and agricultural breeding work. However, the use of CRISPR/Cas in plants is also largely dependent on genetic transformation systems, and the use of genome editing techniques for plant species or genotypes for which genetic transformation systems have not been established remains limited. The virus-mediated genome editing technology can deliver CRISPR/Cas main components at the individual level of plants, is less limited by genotypes, can obtain genome edited plants without any exogenous transgenic components without genetic transformation, regeneration and other steps, and is therefore receiving more and more attention from scientists. However, most viruses cannot express the SpCas9 (1368 aa) or SaCas9 (1053 aa) nuclease with larger volumes because the infection capacity of the virus is directly inversely related to the size of the exogenous gene to be expressed, and most of the studies reported at present are based on the realization of genome editing by using the virus to deliver the gRNA on the basis of transgenic plants which have obtained stably expressed the SpCas9 or SaCas9 genes. Although the expression of the corresponding nuclease by plant viruses has been reported, the modified viruses cannot be systematically expressed in plants because of the large nuclease or virus type limitation, and a regeneration system is still required to obtain genome editing plants which can be stably inherited. The virus-mediated genome editing technology has very wide application prospect, not only can break through the restriction of genetic transformation, but also can rapidly obtain non-transgenic genome editing plants, thereby being beneficial to accelerating the crop breeding process, reducing the supervision cost and promoting the industrialized application of genome editing crops. However, most plant viruses cannot express SpCas9 and other large exogenous proteins, so that the application range of the plant viruses is limited.
As CRISPR/Cas technology continues to mature, smaller nucleases are being discovered, such as Cas Φ (paush et al 2020), asCas12f1, un1Cas12f1, spCas12f1 and Cas λ of only 400 to 800 amino acids, where the earliest reports of Cas Φ, spCas12f1 and Cas λ all demonstrated that genome editing can be achieved in plants with editing efficiencies of: 0.85%, 14% -59% and 18%. Subsequent applications of these novel nucleases in plants have also been developed. However, the efficiency of editing these nucleases in plants is far lower than those of early-developed nucleases such as SpCas9, saCas9 and LbCas12a, and the like, while the efficiency of editing SpCas12f1 is high, the temperature treatment at 45 ℃ is required, and the working condition limits the application of the nucleases in plants. And whether these newly discovered mini nucleases have editing activity in wheat and leymus chinensis has not been reported. In view of the above, the newly discovered small nucleases can not be directly applied to virus-mediated plant genome editing technology, and a great deal of work is still required to optimize the editing efficiency.
In the medical field, adeno-associated virus (AAV) -mediated CRISPR/Cas delivery is considered one of the most promising systems for current gene therapy, and has now shown good efficacy in clinical trials for the treatment of various genetic diseases. However, also because of the limitation that viruses cannot express larger proteins, single-base editing or guided editing related elements cannot be delivered through adenoviruses, so that a scholars propose a scheme of dividing Cas9 into fragments with different sizes and then assembling the fragments into complete proteins through endosomes to play a role of nuclease, namely a Split-Cas9 technology.
Disclosure of Invention
The invention aims to provide a new choice for plant genome editing technology.
The technical scheme of the invention is a Split-Cas9 system suitable for editing plant genome, which splits Intein into Intin N And Intin C Splitting NCas9 and CCas9 by Cas9, and reconstructing the NCas9 and the CCas9 into an expression vector; concrete bagIncludes a gRNA expression element for a target gene, an intein-Cas 9 expression element 1 and an intein-Cas 9 expression element 2; constructing a gRNA expression element, an intein-Cas 9 expression element 1 and an intein-Cas 9 expression element 2 aiming at a target gene on the same expression vector or a plurality of expression vectors; the composition of intein-Cas 9 expression element 1 and intein-Cas 9 expression element 2 is one of the following:
a. the composition of the Intein-Cas 9 expression element 1 is a promoter-NCas 9-Intin N -terminator, intein-Cas 9 expression element 2 consisting of promoter-CCas 9-intelin c -a terminator;
b. the composition of the Intein-Cas 9 expression element 1 is a promoter-NCas 9-Intin N -terminator, intein-Cas 9 expression element 2 consisting of promoter-intelin c -a CCas 9-terminator;
c. the composition of the Intein-Cas 9 expression element 1 is a promoter-Intin N -NCas 9-terminator, intein-Cas 9 expression element 2 consisting of promoter-CCas 9-intelin c -a terminator;
d. the composition of the Intein-Cas 9 expression element 1 is a promoter-Intin N -NCas 9-terminator, intein-Cas 9 expression element 2 consisting of promoter-intelin c -a CCas 9-terminator;
e. the composition of the Intein-Cas 9 expression element 1 is a promoter-NCas 9-Intin C -terminator, intein-Cas 9 expression element 2 consisting of promoter-CCas 9-intelin N -a terminator;
f. the composition of the Intein-Cas 9 expression element 1 is a promoter-NCas 9-Intin C -terminator, intein-Cas 9 expression element 2 consisting of promoter-intelin N -a CCas 9-terminator;
g. the composition of the Intein-Cas 9 expression element 1 is a promoter-Intin C -NCas 9-terminator, intein-Cas 9 expression element 2 consisting of promoter-intelin N -a CCas 9-terminator;
h. the composition of the Intein-Cas 9 expression element 1 is a promoter-Intin C -NCas 9-terminator, intein-Cas 9 expression element 2 consisting of promoter-CCas 9-intelin N -a terminator.
Further, the intein-Cas 9 expression element 1 and/or the promoter and terminator of the intein-Cas 9 expression element 2 further comprise an NLS.
Specifically, the composition of the Intein-Cas 9 expression element 1 and the Intein-Cas 9 expression element 2 is a promoter-NCas 9-Intin N -terminator-promoter-Intin C -CCas 9-NLS-terminator.
In particular, the Cas9 is SaCas9, cas12a, cas12j or Cas12f.
Specifically, the intein is Ssp DnaE, sce VMA, ssp DnaB, mtu RecA or GBD Pol-1 (cis).
Furthermore, the amino acid sequence of NSaCas9 is shown in positions 1-533 of SEQ ID No. 3.
Specifically, the amino acid sequence of CSaCas9 is shown in 534 th to 1054 th positions of SEQ ID No. 3.
Further, the Intin N The amino acid sequence of (2) is shown as SEQ ID No. 1.
Further, the Intin C The amino acid sequence of (2) is shown as SEQ ID No. 2.
Specifically, the expression vector is a plant expression vector.
Still further, the plant expression vector is a pCambia series vector.
Wherein the promoter is a plant promoter.
Further, the plant promoter is an action, 35S or a Ubiquitin.
In particular, the terminator is a Nos, TA3-polyA or CaMV 3' UTR.
Further, the target genes are OsPDS, osNYC1 and OsNYC4.
The invention also provides application of the Split-Cas9 system suitable for editing the plant genome in editing the plant genome.
The invention has the beneficial effects that: in order to expand the application range of virus-mediated genome editing technology in plants, the invention utilizes intein-mediated protein resolution and recombination technology in plants to divide Cas9 into NCas9 and CCas9 which are fused with the N-terminal and C-terminal of intein respectively, and the results show that: intein-mediated Split-Cas9 has less effect on genome editing activity, effectively reduces the size of a single transcription unit, and provides more possibilities for virus-mediated genome editing technology.
Drawings
FIG. 1, working schematic diagram of intein after artificial transformation; pro: a promoter; gene N And Gene C Respectively dividing target gene fragments; intin N And Intin C Nucleotides corresponding to the N-terminal amino acid sequence and the C-terminal amino acid sequence of the intein are respectively included; ter: a terminator; transcription and Translation: transcription and translation; N-Protein: gene N A post-transcriptional translated product; C-Protein: gene C A post-transcriptional translated product; trans-spalling: shearing proteins; full-length protein: the whole protein obtained by the expression of the target gene.
FIG. 2, the respective cassette structure for genome editing; ubi-Pro: the Ubiquitin promoter, d35S: double 35S promoter, N-SaCas9: nucleotide sequence corresponding to the amino acid sequence of the N end of the SaCas9 protein, C-SaCas9: a nucleotide sequence corresponding to the amino acid sequence of the C end of the SaCas9 protein; intin N And Intin C Nucleotides corresponding to the N-terminal amino acid sequence and the C-terminal amino acid sequence of the intein are respectively included; ter: a terminator; osU3 rice U3 promoter; gRNA: a targeting sequence.
FIG. 3, partial plant gene sequencing results; wild type: wild type; gene: a gene; 2 nd exon: a second exon; 1 st exon first exon.
FIG. 4, mutant plant phenotype; wild type: wild type; day 0: day 0; day 3: day 3.
Figure 5, potential sites for intein-mediated SaCas9 partitioning (shaded sites).
Detailed Description
Inteins (inteins), i.e., protein introns, are a stretch of amino acid sequences present in a precursor protein, and the principle of their operation is shown in FIG. 1. In the process of converting the precursor protein into the mature protein, the intein is released from the precursor protein by self-shearing, and peptide chains at two ends are connected by peptide bonds, the process is protein splicing, and the process does not need a specific cell environment and any auxiliary factors, and can even be performed in vitro. At present, no report is yet made on intein-mediated split-Cas9 systems in plants.
The rice material used for rice transformation in the following examples was Zhonghua 11 (Hao M, yan S Y, fu C Y, et al identification and segregation of character of mutants induced from Zhonghua (Oryza sativa L. Subsp. Japonica) by T-DNA insertion [ J ]. Southwest China Journal of Agricultural Sciences,2006,19 (5): 777-781 ]) supplied by the university of inner Mongolian student' S university of life sciences. The endoenzymes, kits, and PCR enzymes used in the experiments were purchased from all gold corporation. Other reagents are all of domestic analytical purity. Primer, DNA synthesis and sequencing were all done by Shanghai.
Table 1 primers used in the examples
EXAMPLE 1 construction of expression vectors
The split site of SaCas9 has 105 (FIG. 5), the structural characteristics of SaCas9 are further analyzed and combined with the splicing principle of the Npu DnaE intein, saCas9 is divided into two sections of 520 amino acids and 533 amino acids, and corresponding plant vector expression cassettes Ubi-NSaCas 9-Intin N-nos and d35 s-Intec-CSaCas 9-NLS-nos are respectively designed and synthesized according to related nucleotide sequences. After the expression cassette is obtained by synthesis, the Ubi-NSaCas 9-intelin n-nos are firstly cloned into the SmaI site of a pCXUN vector (Sun Y, zhang X, wu C, et al engineering thermo-sphere-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase [ J ]. Molecular plant,2016,9 (4): 628-631) by a homologous recombination back method to obtain pCXUN-NSaCas9, and then the d35 s-intelin C-csaacas 9-NLS-nos expression cassette is cloned into the Kpn I site of the pCXUN-NSaCas9 to obtain the pCXUN-NSaCas 9-csaacas 9 vector.
Genome clipping targets are designed according to sequences of OsPDS (genebank accession number: AF 049356.1), osNYC1 (genebank accession number: AB 255025.1) and OsNYC4 (genebank accession number: AB 255025.1) genes respectively, and corresponding gRNA expression cassettes OsU3-PDS1, osU3-NYC1 and OsU-NYC 4 are obtained by an overlapping PCR method. OsU3-PDS1, osU-NYC 1 and OsU-NYC 4 were cloned into pCXUN-NSaCas9-CSaCas9 at the Pme I site, respectively, using pCXUN-NSaCas9-CSaCas9 as base vector. Three sequences of gRNA-PDS TGCCTCAAGCAATATGGGTTT (SEQ ID No. 4), gRNA-NYC1: CGAGCCCCTGAATCGCCGCCT (SEQ ID No. 5) and gRNA-NYC4: CCATGAGTTGGTCATAGACGG (SEQ ID No. 6) were constructed on a base vector to obtain vectors pCXUN-NSaCas9-CSaCas9-PDS, pCXUN-NSaCas9-CSaCas9-NYC1 and pCXUN-NSaCas9-CSaCas9-NYC4 (FIG. 2). And respectively transferring the three plasmids into EHA105 agrobacterium for later use by adopting an electrotransformation method.
SEQ ID No.1Intein N Amino acid sequence:
CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN*
SEQ ID No.2Intein C amino acid sequence: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN
SEQ ID No.3SACas9 amino acid sequence, NSaCas9 underlined 533aa, CSaCas9 remaining 520aa
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVK KLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKA LEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSP FGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTL KQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLN SELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSP VVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKI KLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG*
SEQ ID No.25 vector sequence: no. 43-292 bp is a Nos terminator, no. 325-1986 bp is C-SaCas9+3NLS, no. 1987-2094 bp is intein C The 2115-2841 bp is d35S promoter, the 2994-3246 bp is Nos terminator, and the 3262-3570 bp is intein N 3571 to 5169bp is N-SaCas9, 5182 to 7172bp is ubiquitin promoter, 7406 to 7525bp is gRNA scaffold (wherein the underlined region is replaced by gRNA-PDS, gRNA-NYC1 and gRNA-NYC4, respectively), 7399 to 7405bp is Ploy A sequence, 7555 to 7991OsU promoter;
gaattcgagctccaggaaacagctatgaccatgattacgaatccgatctagtaacatagatgacaccgcgcgcgataatttatcctagtttgcgcgctatattttgttttctatcgcgtattaaatgtataattgcgggactctaatcataaaaacccatctcataaataacgtcatgcattacatgttaattattacatgcttaacgtaattcaacagaaattatatgataatcatcgcaagaccggcaacaggattcaatcttaagaaactttattgccaaatgtttgaacgatctgcagccgggcggccgctttaagatctTCACACCTTCCTCTTCTTCTTCGGGGAGCCGCCGCTCACCTTGCGCTTCTTCTTAGGGGAGCCGCCGCTCACCTTGCGCTTCTTCTTCGGGCTGCCGCCGGAGCCCTTCTTGATGATCTGCGGGTGCTTCTTGGACTTCACCTCGTAGAGGTTGCCGAGGATGTCGGTGGAGTACTTCTTGATGGACTGGGTCTTGCTGGCGATGGTCTTGATGATGCGCGGCGGCCTCTTGTCGTTCATGTTCTCGAGGTACTCGCGGTAGGTGATGTCGATCATGTTCACCTCGATGCGGTTGAGGAGATCGTTGTTGACGCCGATCACGCGGTAGAGCTCGCCGTTGATCTTGATGAGATCGTTGTTGTAGAAGCTCGCGATGAACTCGGCCTGGTTGCTGATCTTCTTCAGCTTCTTGGCCTCCTCGTAGCACTTGCTGTTCACCTCGTAGTAGTTCTCCTTCTTGATCACATCAAGGTTCTTCACGGTCACGAACTTGTACACGCCATTATCGAGGTAAACGTCGAACCTGTACGGCTTCAGGCTCAGCTTGACCACCTTGTTCCGGGAGTTCGGGTAATCGTCGGTGATGTCGAGGTGGGCGTTGAGCTTATTGCCGTAGTACTTGATCTTCTTGATCACTGGGCCGTTGTCCTTCTTAGAGTACTTGGTGAGATAGTTGCCGGTCTCCTCGTAGTACTTGTAGAGTGGGTTCTTCTCATCGCCGTACTGCTCCATGATGAGCTTGAGCTTCTGGTAGGTCTGCGGATCGTGGTGGTACATGAGGAGCTTCTCCGGGGACTTGTTGATGAGCTTCTTGAGCTTGTCGTTGTCCTTGTCGTAGAGGCCGTTGAGGTTGTTCACGATGAGGGTGTTGCCCTTGTCGTCCTTGCGTGTGGAGTACAGGGTATCGTTGATCAGCTCGCGATTCGGCTTCTTATCCACCCTGTGGGAGTACTTGTAATCCTTGAAGTCCTTGATGTGCTTGATCTGGTGCGGGGTGATGAAGATCTCCTTGTACTCCTGCTCGGTCTCGATCTCAGGCATTGACTCGGCCTGCTTCTCCTCGAACATCTGATTCTCCATCACTTTCTTGGCCTTGTCCAGCTTCTTCCACTCCTTGAAGATGAAGTCGGCGTTCGCGATGATCAGGGCATCCTCGGCGTGGTGCTTGTAGCCCTTGTTGCGCTCCTTCTTGAACTTCCACTTCCGGCGGAGGAAGCTGGTGAAGCCGCCGTTGATGGACTTCACCTTCACATCCAGGTTGTTCACGCGGAAGTAGGACCTGAGGAGGTTCATCAAGCCCCTGGTCGCGTACCGGGTGTCCACCAGGTTGCGGTTAATGAAATCCTTCTGCACGCTGAACCTGTTGATGTCCCTCTCCTCCAGGAGGTACTCCTTCTTGGTCTTAGAGATCCTGCCCTTGCCCTTGGCGAGGTTGAGGATGTGCTTCTTGAAGGTCTCGTATGAGATCTTGGAGTCGGAGCTGGAGAGGTACTGGAACGGGGTCCTGTTGCCCTTCTTGCTATTCTCCTCCTGCTTCACGAGAACCTTGTTGTTGAAAGAGTTGTCGAAGGACACGGAGCGTGGGATGATGTGGTCCACCTCGTAGTTGAACGGGTTGTTGAGCAGATCCTCGAGCGGGATGGCCTCGAGGCTGTAGAGGCAGTTGGAGGCGATGAAGCCATTCTTGAGGGCGAAGTTGTGGTCGCGCTCCACGCCGATATCGTACACGTTCTGCTTGCCGAGGTACTTCCTTGTGGCGATCTTGATCATactagtgatctgggctgtcctctccaaatgaaatgaacttccttatatagaggaagggtcttgcgaaggatagtgggattgtgcgtcatcccttacgtcagtggagatgtcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttgaatgatagcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttctactgtcctttcgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgaaattaccctttgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgacatttttggagtaggggtgggattgtgcgtcatcccttacgtcagtggagatgtcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttgaatgatagcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttctactgtcctttcgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgaaattaccctttgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgacatttttggagtaggggtagggagctcgaattggtaatcaagcttggcactggccgtcgttttacaacccctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattcccgatctagtaacatagatgacaccgcgcgcgataatttatcctagtttgcgcgctatattttgttttctatcgcgtattaaatgtataattgcgggactctaatcataaaaacccatctcataaataacgtcatgcattacatgttaattattacatgcttaacgtaattcaacagaaattatatgataatcatcgcaagaccggcaacaggattcaatcttaagaaactttattgccaaatgtttgaacgatcggggaaattcggATCTCAGTTCGGGAGGTTATCCACCCTCATGAGGTCCAGCTCCCTCTCGAAGATCTCATCGATTGGGAGCATCTGGCCGTCCACGGTCATGAACTTGTGGTCCTTTGTGGCGCGGATGAGTGAGCCGTCCTCGAGGCAGTACTCGAACACCTCCTGCTCGCCGCGATCATGCCACTGGGCCACCGGCTGGGTGTAGATGTTGCCGTTGTTATCGACGCTGTACACGGTGCACTCGATCCGCTTCTCCACGATCTTGCCGATCGGGAGGAGGCCGTACTCCACTGTGAGGATCTCGGTCTCGTAGGAGAGGCACTTGCCCTCCTGCATATCATGGAGCTTGATCTTCTCGATGAGGTACTTCGCGTTCTCCTTGCCGGTGGTCCTGATGATCTCCTCGATCCGCTCGTTGGTCTGGCGGTTGCGCTTCTGCATCTCATTGATCATCTTCTGGGCATCCTTGGAATTCTTCTCCCTGGCCAGCTCGATGATGATGTCGTTTGGGAGGCCGTACTTCTTGATGATCGCGTTAATCACTTTGATAGACTGGATGAAGCTGCGCTTCACCACCGGGCTGAGGATGAAGTCATCAACGAGGGTGGTCGGAATCTCCTTCTGCTGGCTCAGGTCCACCTTCTTCGGGACCAGCTTGAGCCTGTTGAAGATAGCGATCTGGTTATCATTGGTGTGCCACAACTCGTCGAGGATCAAGTTGATGGCCTTGAGAGAGAGGTTATGCGTGCCCGTGTAGCCCTTCAGGTTTGAAATCTGCTCAATCTCCTCCTGCGTGAGCTCAGAATTGAGGTTGGTCAGCTCCTCCTGGATATCCTCAGAGGACTGGTAGATTGTGAGGATCTTGGCGATCTGGTCGAGGAGCTCCGCATTCTCAATGATCTCCTTCCTCGCGGTGATATCCTTGATGTCATGGTACACCTTGAGGTTAGTGAACTCTGGCTTGCCGGTGCTGGTGACCCGATAGCCCTTGATATCCTCCTCGTTCACCAGGATCTCCTTAGCGATTTGCTTCAGAGTCGGCTTCTTCTTCTGCTTGAACACATTCTCGATGATCTGGAACTTCTCGTAGTACTCGAGCTTCTCGTTCTCATCCCTTGTGATCACGAGGTTGTTCAAGTCATTGAGGGCGTTGTACAGGTCGGCATTGTAGGCGTACTTCACGCTCCTGAGCTCCTCCGGGAAGTAAGTGCAGTGGCCCATCAGCATCTCGTACCACTCCTTGATGTCCTTCCAGCCGAACGGGCTGCCCTCGCCTGGTCCCTCGTAGTAGGTCCTCCTTGTCTCGAGGAGATCAATGTAGGTATCAATGAAGGACTGATCGAGCTGATGGTAGGCCTTCTGCACCTTCAGCAGCTGCTTGGCCTCCTTCACGTAGTCGGATGTCTTGAACCTATTGATGCTGCCGCGCACCTCGCCGTCCTTCTTCAGCCGCTCGAGCTGCAGCTCCGCCACGTATTTCTCCTCGAGAGCCTTGCTGTTGCGGGAGATTTGCTCCTTTGTGCTGAGCTCGTTGCCAGTGTCCTCCTCCACCTCATTCACGTTGTGCACGCCCCTCCTCTTCGCGAGGTGCAGCAGGGCGGCGGAAAACTCCTCCTCGGAGAGCTTCTGGGAGAGGCCCTTGACCCTGGCCTCGTACGGATTGATGCCAGAGAGCTCAGAGTGGTCAGTGAGCAGGTTGTAGTCGAACAGCAGCTTCTTGACCCTCTGGATCCTGTGCCTCCTCCTCCTCTTCAGGCGGCGGGCGCCCCTCTTGCTGCGGCGGCCCTCGTTATTCTCCACGTTGGCCTCCTTGAAGAGCCTCACGCCGGCGTCGATCACGTCCCTGGTCTCGTAATCAATGATGCCGTAGCCCACGGAGGTGATGCCGATATCGAGGCCGAGGATGTAGTTGCGCTTCATggatcccccgggctgcagaagtaacaccaaacaacagggtgagcatcgacaaaagaaacagtaccaagcaaataaatagcgtatgaaggcagggctaaaaaaatccacatatagctgctgcatatgccatcatccaagtatatcaagatcaaaataattataaaacatacttgtttattataatagataggtactcaaggttagagcatatgaatagatgctgcatatgccatcatgtatatgcatcagtaaaacccacatcaacatgtatacctatcctagatcgatatttccatccatcttaaactcgtaactatgaagatgtatgacacacacatacagttccaaaattaataaatacaccaggtagtttgaaacagtattctactccgatctagaacgaatgaacgaccgcccaaccacaccacatcatcacaaccaagcgaacaaaaagcatctctgtatatgcatcagtaaaacccgcatcaacatgtatacctatcctagatcgatatttccatccatcatcttcaattcgtaactatgaatatgtatggcacacacatacagatccaaaattaataaatccaccaggtagtttgaaacagaattctactccgatctagaacgaccgcccaaccagaccacatcatcacaaccaagacaaaaaaaagcatgaaaagatgacccgacaaacaagtgcacggcatatattgaaataaaggaaaagggcaaaccaaaccctatgcaacgaaacaaaaaaaatcatgaaatcgatcccgtctgcggaacggctagagccatcccaggattccccaaagagaaacactggcaagttagcaatcagaacgtgtctgacgtacaggtcgcatccgtgtacgaacgctagcagcacggatctaacacaaacacggatctaacacaaacatgaacagaagtagaactaccgggccctaaccatggaccggaacgccgatctagagaaggtagagagggggggggggggaggacgagcggcgtaccttgaagcggaggtgccgacgggtggatttgggggagatctggttgtgtgtgtgtgcgctccgaacaacacgaggttggggaaagagggtgtggagggggtgtctatttattacggcgggcgaggaagggaaagcgaaggagcggtgggaaaggaatcccccgtagctgccgtgccgtgagaggaggaggaggccgcctgccgtgccggctcacgtctgccgctccgccacgcatttctggatgccgacagcggagcaagtccaacggtggagcggaactctcgagaggggtccagaggcagcgacagagatgccgtgccgtctgcttcgcttggcccgacgcgacgctgctggttcgctggttggtgtccgttagactcgtcgacggcgtttaacaggctggcattatctactcgaaacaagaaaaatgtttccttagtttttttaatttcttaaagggtatttgtttaatttttagtcactttattttattctattttatatctaaattattaaataaaaaaactaaaatagagttttagttttcttaatttagaggctaaaatagaataaaatagatgtactaaaaaaattagtctataaaaaccattaaccctaaaccctaaatggatgtactaataaaatggatgaagtattatataggtgaagctatttgcaaaaaaaaaggagaacacatgcacactaaaaagataaaactgtagagtcctgttgtcaaaatactcaattgtcctttagaccatgtctaactgttcatttatatgattctctaaaacactgatattattgtagtactatagattatattattcgtagagtaaagtttaaatatatgtataaagatagataaactgcacttcaaacaagtgtgacaaaaaaaatatgtggtaattttttataacttagacatgcaatgctcattatctctagagaggggcacgaccgggtcacgctgcactgcaggaattcgatatcaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgctagagcagcttgagcttggatcagattgtcgtttcccgccttcagtttAAAAAAATCTCGCCAACAAGTTGACGAGATAAACACGGCATTTTGCCTTGTTTTAGTAGATTCTGTAATTTTAGGTATAAGTACAATTTTCGTCTACCTCATACCTAAAATTACAGAGTACTAAAACGTGTGCAGGTGTTGTGTTCACCTGCGAGCtgccacggatcatctgcacaactcttttaaatcagctttgatctatgtggatagccgaggtggtactaatactagtctttgttgtcgtccaattgcgtaatgggccggcccatactgcaatacatgtcctgaaaggcttcatggcccactacgaaatgcttttctcctacagtttatcttacttcttcacatcacgtggtttccaacgtacccagtgttcccggcttccagcatttgctggtagcaccagtagaagacgcctgtcttgtgctatggtccctgactgcacatctgattcctccaagatccatgcatgcctgataactttaagttgcttcagaagaactttaagtgatctgttcgtatgtttaaagattccttgataaaataagttgcagttctgaaaatcctagaacttggagacctggatgaattacaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttattagaataacggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtgcatgccaaccacagggttcccctcgggatcaaagtactttgatccaacccctccgctgctatagtgcagtcggcttctgacgttcagtgcagccgtcttctgaaaacgacatgtcgcacaagtcctaagttacgcgacaggctgccgccctgcccttttcctggcgttttcttgtcgcgtgttttagtcgcataaagtagaatacttgcgactagaaccggagacattacgccatgaacaagagcgccgccgctggcctgctgggctatgcccgcgtcagcaccgacgaccaggacttgaccaaccaacgggccgaactgcacgcggccggctgcaccaagctgttttccgagaagatcaccggcaccaggcgcgaccgcccggagctggccaggatgcttgaccacctagccctggcgacgttgtgacagtgaccaggctagaccgcctggcccgcagcacccgcgacctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggcagagccgtgggccgacaccaccacgccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaatcatcgaccgcacccggagcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcaccccggcacagatcgcgca
cgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcgacc
ctgtaccgcgcacttgagcgcagcgaggaagtgacgcccaccgaggccaggcggcgcggtgccttccgtgaggacg
cattgaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcaccaggac
ggccaggacgaaccgtttttcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagccgc
ccgcgcacgtctcaaccgtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggc
cagcttggccgctgaagaaaccgagcgccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcat
gcggtcgctgcgtatatgatgcgatgagtaaataaacaaatacgcaaggggaacgcatgaaggttatcgctgtact
taaccagaaaggcgggtcaggcaagacgaccatcgcaacccatctagcccgcgccctgcaactcgccggggccgat
gttctgttagtcgattccgatccccagggcagtgcccgcgattgggcggccgtgcgggaagatcaaccgctaaccg
ttgtcggcatcgaccgcccgacgattgaccgcgacgtgaaggccatcggccggcgcgacttcgtagtgatcgacgg
agcgccccaggcggcggacttggctgtgtccgcgatcaaggcagccgacttcgtgctgattccggtgcagccaagc
ccttacgacatatgggcaaccgccgacctggtggagctggttaagcagcgcattgaggtcacggatggaaggctac
aagcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgcatcggcggtgaggttgccgaggcgctggccgggta
cgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcacaaccgtt
cttgaatcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcattt
gagttaatgaggtaaagagaaaatgagcaaaagcacaaacacgctaagtgccggccgtccgagcgcacgcagcagc
aaggctgcaacgttggccagcctggcagacacgccagccatgaagcgggtcaactttcagttgccggcggaggatc
acaccaagctgaagatgtacgcggtacgccaaggcaagaccattaccgagctgctatctgaatacatcgcgcagct
accagagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaaggaggcggcatggaaaatcaaga
acaaccaggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttggccaggcgtaagcggctgggtt
gtctgccggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgacggtcgcaaaccatccggcccg
gtacaaatcggcgcggcgctgggtgatgacctggtggagaagttgaaggccgcgcaggccgcccagcggcaacgca
tcgaggcagaagcacgccccggtgaatcgtggcaagcggccgctgatcgaatccgcaaagaatcccggcaaccgcc
ggcagccggtgcgccgtcgattaggaagccgcccaagggcgacgagcaaccagattttttcgttccgatgctctat
gacgtgggcacccgcgatagtcgcagcatcatggacgtggccgttttccgtctgtcgaagcgtgaccgacgagctg
gcgaggtgatccgctacgagcttccagacgggcacgtagaggtttccgcagggccggccggcatggccagtgtgtg
ggattacgacctggtactgatggcggtttcccatctaaccgaatccatgaaccgataccgggaagggaagggagac
aagcccggccgcgtgttccgtccacacgttgcggacgtactcaagttctgccggcgagccgatggcggaaagcaga
aagacgacctggtagaaacctgcattcggttaaacaccacgcacgttgccatgcagcgtacgaagaaggccaagaa
cggccgcctggtgacggtatccgagggtgaagccttgattagccgctacaagatcgtaaagagcgaaaccgggcgg
ccggagtacatcgagatcgagctagctgattggatgtaccgcgagatcacagaaggcaagaacccggacgtgctga
cggttcaccccgattactttttgatcgatcccggcatcggccgttttctctaccgcctggcacgccgcgccgcagg
caaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggagagttcaagaagttctgt
ttcaccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggcggggcaggctggcc
cgatcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaatgtacggagcagatgct
agggcaaattgccctagcaggggaaaaaggtcgaaaaggtctctttcctgtggatagcacgtacattgggaaccca
aagccgtacattgggaaccggaacccgtacattgggaacccaaagccgtacattgggaaccggtcacacatgtaag
tgactgatataaaagagaaaaaaggcgatttttccgcctaaaactctttaaaacttattaaaactcttaaaacccg
cctggcctgtgcataactgtctggccagcgcacagccgaagagctgcaaaaagcgcctacccttcggtcgctgcgc
tccctacgccccgccgcttcgcgtcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccaggca
atctaccagggcgcggacaagccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgc
gtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgc
cgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgt
agcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtg
tgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctg
cgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcagggg
ataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtt
tttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacagga
ctataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggat
acctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgta
ggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactat
cgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcga
ggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtat
ctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggt
agcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctttt
ctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcattctaggtactaaaacaa
ttcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgaca
tactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgc
ttctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtggga
aaagacaagttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtct
tcttcccagttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaagcggctgtcta
agctattcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatacagctcgataat
cttttcagggctttgttcatcttcatactcttccgagcaaaggacgccatcggcctcactcatgagcagattgctc
cagccatcatgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatcatgtcctttt
cccgttcaacatcataggtggtccctttataccggctgtccgtcatttttaaatataggttttcattttctcccac
cagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtatttttcgatcagttttttcaat
tccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaagaagc
taattataacaagacgaactccaattcactgttccttgcattctaaaaccttaaataccagaaaacagctttttca
aagttgttttcaaagttggcgtataacatagtatcgacggagccgattttgaaaccgcggtgatcacaggcagcaa
cgctctgtcatcgttacaatcaacatgctaccctccgcgagatcatccgtgtttcaaacccggcagcttagttgcc
gttcttccgaatagcatcggtaacatgagcaaagtctgccgccttacaacggctctcccgctgacgccgtcccgga
ctgatgggctgcctgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcag
gatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgtactgaatt
aacgccgaattaattcgggggatctggattttagtactggattttggttttaggaattagaaattttattgataga
agtattttacaaatacaaatacatactaagggtttcttatatgctcaacacatgagcgaaaccctataggaaccct
aattcccttatctgggaactactcacacattattatggagaaactcgagcttgtcgatcgacagatccggtcggca
tctactctatttctttgccctcggacgagtgctggggcgtcggtttccactatcggcgagtacttctacacagcca
tcggtccagacggccgcgcttctgcgggcgatttgtgtacgcccgacagtcccggctccggatcggacgattgcgt
cgcatcgaccctgcgcccaagctgcatcatcgaaattgccgtcaaccaagctctgatagagttggtcaagaccaat
gcggagcatatacgcccggagtcgtggcgatcctgcaagctccggatgcctccgctcgaagtagcgcgtctgctgc
tccatacaagccaaccacggcctccagaagaagatgttggcgacctcgtattgggaatccccgaacatcgcctcgc
tccagtcaatgaccgctgttatgcggccattgtccgtcaggacattgttggagccgaaatccgcgtgcacgaggtg
ccggacttcggggcagtcctcggcccaaagcatcagctcatcgagagcctgcgcgacggacgcactgacggtgtcg
tccatcacagtttgccagtgatacacatggggatcagcaatcgcgcatatgaaatcacgccatgtagtgtattgac
cgattccttgcggtccgaatgggccgaacccgctcgtctggctaagatcggccgcagcgatcgcatccatagcctc
cgcgaccggttgtagaacagcgggcagttcggtttcaggcaggtcttgcaacgtgacaccctgtgcacggcgggag
atgcaataggtcaggctctcgctaaactccccaatgtcaagcacttccggaatcgggagcgcggccgatgcaaagt
gccgataaacataacgatctttgtagaaaccatcggcgcagctatttacccgcaggacatatccacgccctcctac
atcgaagctgaaagcacgagattcttcgccctccgagagctgcatcaggtcggagacgctgtcgaacttttcgatc
agaaacttctcgacagacgtcgcggtgagttcaggctttttcatatctcattgccccccggatctgcgaaagctcg
agagagatagatttgtagagagagactggtgatttcagcgtgtcctctccaaatgaaatgaacttccttatataga
ggaaggtcttgcgaaggatagtgggattgtgcgtcatcccttacgtcagtggagatatcacatcaatccacttgct
ttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtc
ggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgtaggtgccaccttccttttctact
gtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgatattaccctttgttgaaaag
tctcaatagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccat
gttatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtggg
ggtccatctttgggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgt
aggtgccaccttccttttctactgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcc
cgatattaccctttgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgatattcttggagtag
acgagagtgtcgtgctccaccatgttggcaagctgctctagccaatacgcaaaccgcctctccccgcgcgttggcc
gattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagt
tagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggat
aacaatttcacacaggaaacagctatgaccatgattac
example 2 obtaining transgenic Rice
Selecting rice grains without mildew spots and normal bud openings, sterilizing with 75% alcohol for 1min, and cleaning with sterilized water for 1 min/time; sterilizing with 15% sodium hypochlorite for 20min, and cleaning with sterilized water for 3 times and 1 min/time; the sterilized rice grains were inoculated in an induction medium and cultured at 26℃for 20 days under light. And (3) selecting agrobacterium tumefaciens in the invasion solution, preparing agrobacterium tumefaciens heavy suspension with OD600 = 0.2, selecting the callus in a triangular flask, adding the agrobacterium tumefaciens heavy suspension, infecting for 10-15 min, discarding bacteria solution, inoculating the callus in a co-culture medium, and co-culturing for 48-72 h at 20 ℃. Inoculating the callus obtained in the previous step to a screening culture medium, and culturing in dark at 28 ℃ for 20-30 days; inoculating positive calli to a secondary screening culture medium, and taking monoclonal calli in the calli taking process, and carrying out dark culture at 28 ℃ for 7-10 days; inoculating positive callus to a differentiation medium, culturing for 15-20 days at 28 ℃ under illumination, inoculating to a rooting medium after 2-5 cm buds are differentiated, and culturing for 7-10 days at 30 ℃ under illumination.
Genotyping of T0 generation regenerated plants: the CTAB method is adopted to extract rice genome DNA, PCR detection primers are respectively designed according to target positions of three genes of OsPDS, osNYC1 and OsNYC4, corresponding transgenic plants are respectively amplified by the primers, PCR products are sent to a company for sequencing, and genome editing types are determined.
Sequencing results show that 10 transgenic plants of the pCXUN-NSaCas9-CSaCas9-PDS vector are homologous or bi-allelic mutant, 7 heterozygotes and 4 wild type plants. Of the 18 plants transformed with the pCXUN-NSaCas9-CSaCas9-NYC1 vector, 9 were homologous or bi-allelic, 2 for heterozygotes, 2 for chimeras and 5 for wild type. Of the 26 plants transformed with the pCXUN-NSaCas 9-csaacas 9-NYC4 vector, 18 were homologous or bi-allelic, 3 heterozygotes, 4 chimeras, and 1 wild type (table 2), with the gene mutations being of the main type, both base insertions and deletions (fig. 3). Analysis of transgenic plants with homologous mutations showed that the OsPDS gene mutants exhibited albino phenotype and that the OsNYC1 and OsNYC4 gene mutants exhibited a stay-green phenotype under dark induction (FIG. 4).
TABLE 2 Gene mutation type statistics
The experiment proves that intein-mediated SaCas9 splitting and recombination are realized in plants, and the influence on genome editing efficiency is small, and the method provides a new thought for virus-mediated plant genome editing technology and has important influence on the field of plant genome editing.

Claims (10)

1. Split-Cas9 system suitable for plant genome editing, characterized in that: the system splits Intein into Intin N And Intin C Splitting NCas9 and CCas9 by Cas9, and reconstructing the NCas9 and the CCas9 into an expression vector; specifically comprises a gRNA expression element aiming at a target gene, an intein-Cas 9 expression element 1 and an intein-Cas 9 expression element 2; gRNA expression element, intein-Cas 9 expression element 1 and directed against a target geneThe intein-Cas 9 expression element 2 is constructed onto the same expression vector or multiple expression vectors; the composition of intein-Cas 9 expression element 1 and intein-Cas 9 expression element 2 is one of the following:
a. the composition of the Intein-Cas 9 expression element 1 is a promoter-NCas 9-Intin N -terminator, intein-Cas 9 expression element 2 consisting of promoter-CCas 9-intelin c -a terminator;
b. the composition of the Intein-Cas 9 expression element 1 is a promoter-NCas 9-Intin N -terminator, intein-Cas 9 expression element 2 consisting of promoter-intelin c -a CCas 9-terminator;
c. the composition of the Intein-Cas 9 expression element 1 is a promoter-Intin N -NCas 9-terminator, intein-Cas 9 expression element 2 consisting of promoter-CCas 9-intelin c -a terminator;
d. the composition of the Intein-Cas 9 expression element 1 is a promoter-Intin N -NCas 9-terminator, intein-Cas 9 expression element 2 consisting of promoter-intelin c -a CCas 9-terminator;
e. the composition of the Intein-Cas 9 expression element 1 is a promoter-NCas 9-Intin C -terminator, intein-Cas 9 expression element 2 consisting of promoter-CCas 9-intelin N -a terminator;
f. the composition of the Intein-Cas 9 expression element 1 is a promoter-NCas 9-Intin C -terminator, intein-Cas 9 expression element 2 consisting of promoter-intelin N -a CCas 9-terminator;
g. the composition of the Intein-Cas 9 expression element 1 is a promoter-Intin C -NCas 9-terminator, intein-Cas 9 expression element 2 consisting of promoter-intelin N -a CCas 9-terminator;
h. the composition of the Intein-Cas 9 expression element 1 is a promoter-Intin C -NCas 9-terminator, intein-Cas 9 expression element 2 consisting of promoter-CCas 9-intelin N -a terminator.
2. The Split-Cas9 system according to claim 1, wherein: the promoter and terminator of the intein-Cas 9 expression element 1 and/or the intein-Cas 9 expression element 2 further comprise an NLS.
3. The Split-Cas9 system according to claim 2, wherein: the composition of the Intein-Cas 9 expression element 1 and the Intein-Cas 9 expression element 2 is a promoter-NCas 9-Intin N -terminator-promoter-Intin C -CCas 9-NLS-terminator.
4. The Split-Cas9 system according to claim 1, wherein: the Cas9 is SaCas9, cas12a, cas12j or Cas12f.
5. The Split-Cas9 system according to claim 1, wherein: the intein is Ssp DnaE, sce VMA, ssp DnaB, mtu RecA or GBD Pol-1 (cis).
6. The Split-Cas9 system according to claim 5, wherein: the amino acid sequence of NSaCas9 is shown in positions 1-533 of SEQ ID No. 3; the amino acid sequence of the CSaCas9 is shown in 534 th to 1054 th positions of SEQ ID No. 3.
7. The Split-Cas9 system according to claim 1, wherein: the Intin N The amino acid sequence of (2) is shown as SEQ ID No. 1; the Intin C The amino acid sequence of (2) is shown as SEQ ID No. 2.
8. The Split-Cas9 system according to claim 1, wherein: the promoter is an action, 35S or a Ubiquitin; the terminator is Nos, TA3-polyA or CaMV 3' UTR.
9. The Split-Cas9 system according to claim 1, wherein: the target genes are OsPDS, osNYC1 and OsNYC4.
10. Use of the Split-Cas9 system of any one of claims 1 to 9 for plant genome editing.
CN202311373061.8A 2023-10-23 2023-10-23 Split-Cas9 system suitable for plant genome editing and application thereof Pending CN117384942A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311373061.8A CN117384942A (en) 2023-10-23 2023-10-23 Split-Cas9 system suitable for plant genome editing and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311373061.8A CN117384942A (en) 2023-10-23 2023-10-23 Split-Cas9 system suitable for plant genome editing and application thereof

Publications (1)

Publication Number Publication Date
CN117384942A true CN117384942A (en) 2024-01-12

Family

ID=89462606

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311373061.8A Pending CN117384942A (en) 2023-10-23 2023-10-23 Split-Cas9 system suitable for plant genome editing and application thereof

Country Status (1)

Country Link
CN (1) CN117384942A (en)

Similar Documents

Publication Publication Date Title
US11767536B2 (en) Method for obtaining glyphosate-resistant rice by site-directed nucleotide substitution
US12098374B2 (en) Optimized plant CRISPR/CPF1 systems
CN108130342B (en) Cpf 1-based plant genome fixed-point editing method
CN110526993B (en) Nucleic acid construct for gene editing
CN103013954B (en) Rice gene BADH2 site-directed knockout system and application thereof
US20210130838A1 (en) SYSTEMS AND METHODS FOR PLANT GENOME EDITING USING CAS 12a ORTHOLOGS
WO2021169925A1 (en) Fusion protein and application thereof
Lee et al. RETRACTED: Production of transgenic orchardgrass via Agrobacterium-mediated transformation of seed-derived callus tissues
CN111116725B (en) Gene Os11g0682000 and application of protein coded by same in regulation and control of bacterial leaf blight resistance of rice
Yang et al. An efficient transient gene expression system for protein subcellular localization assay and genome editing in citrus protoplasts
CN111057654B (en) Cas9 gene knockout vector applicable to morinda officinalis endophytic fungus A761 and construction method and application thereof
WO2023092731A1 (en) Mad7-nls fusion protein, and nucleic acid construct for site-directed editing of plant genome and application thereof
CN114480391A (en) Promoter for improving CRISPR/Cas9 system gene editing efficiency and application thereof
CN116286742B (en) CasD protein, CRISPR/CasD gene editing system and application thereof in plant gene editing
KR20190122595A (en) Gene Construct for Base Editing in Plant, Vector Comprising the Same and Method for Base Editing Using the Same
CN113528484B (en) nCas3 single-stranded endonuclease and application thereof
CN117384942A (en) Split-Cas9 system suitable for plant genome editing and application thereof
CN102477091A (en) Rice male sterile protein and its coding gene and application
Chopy et al. Genome editing by CRISPR-Cas9 technology in Petunia hybrida
CN112522299A (en) Method for obtaining rice with increased tillering by using OsTNC1 gene mutation
CN115595330B (en) CRISPR-Cas3 system and application thereof in aspect of resisting plant viruses
CN115976086B (en) Method for editing bacteria CRISPR-Cas9 gene and application thereof
CN118406698B (en) Application of ZmWIND gene in improving corn genetic transformation efficiency
CN113528409B (en) Engineering bacterium containing nCas3 single-stranded endonuclease, preparation method and application
CN114181943B (en) Method for creating early maturing corn germplasm and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination