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CN-121991950-A - Efficient and accurate plant gene knockout method

CN121991950ACN 121991950 ACN121991950 ACN 121991950ACN-121991950-A

Abstract

The invention belongs to the field of genetic engineering, and discloses a high-efficiency and accurate plant gene knockout method. According to the invention, the sequence containing the termination codon cluster is inserted into the genome through double pegRNA, so that the termination codon cluster sequence capable of knocking out the target gene efficiently and accurately is screened. By combining the method, the invention utilizes a guide editing system and a plurality of pegRNA programs to realize that only one guide editing protein is needed, and can carry out efficient and accurate knockout, base substitution, insertion, deletion, substitution and other genome modification on one or more target genes.

Inventors

  • GAO CAIXIA
  • LI HONGCHAO
  • CHEN KUNLING

Assignees

  • 中国科学院遗传与发育生物学研究所

Dates

Publication Date
20260508
Application Date
20251107
Priority Date
20241107

Claims (20)

  1. 1. A guide editing guide RNA (pegRNA) consisting of a first pegRNA and a second pegRNA, the first pegRNA or second pegRNA comprising a guide sequence, a scaffold (scaffold) sequence, a reverse transcription template (RT) sequence, a Primer Binding Site (PBS) sequence, Wherein the scaffold sequence of the first pegRNA can complex with a CRISPR nuclease and nick a first target sequence of the sense strand of a target double-stranded DNA sequence and the reverse transcription template (RT) sequence of the first pegRNA sequence has the sequence of a Stop Codon Cluster (SCC); the SCC comprises at least one, at least two, or at least three stop codons; Alternatively, the stop codon is selected from TAA, TAG and TGA, preferably from TAA and TAG; Wherein the reverse transcription template (RT) sequence of the second pegRNA has a partial or complete complementarity to the RT sequence of the first pegRNA, preferably, at least 13bp to about 40bp overlap; The second pegRNA can complex with a CRISPR nuclease and create a nick in a second target sequence of the antisense strand of the target double-stranded DNA sequence.
  2. 2. The guided editing guide RNA of claim 1, wherein the SCC has a sequence length of 13-38 nt.
  3. 3. The guided editing guide RNA of claim 1 or 2, wherein the SCC comprises at least one of the following features (a) - (b): (a) The RNA secondary structure of the SCC comprises a stem loop structure; (b) The gibbs free energy of the RNA secondary structure of the SCC is in the range of-0.2 to-0.05 kcal/mol/base.
  4. 4. The guide editing guide RNA according to any one of claims 1 to 3, wherein the 3' end of the SCC is base C.
  5. 5. The guided editing guide RNA of any one of claims 1-4, wherein the SCC comprises a sequence as set forth in any one of SEQ ID NOs 39-72.
  6. 6. The guide editing guide RNA according to any one of claims 1 to 5, wherein the gap between the nicks of the first and second target sequences is not less than 20bp, preferably not less than 30bp, more preferably 30bp to 100bp.
  7. 7. The guided editing guide RNA of any one of claims 1 to 6, wherein the scaffold sequence of the first pegRNA or the second pegRNA is shown in SEQ ID No. 7.
  8. 8. A genome editing system, comprising: i) a) a CRISPR nuclease and/or an expression construct comprising a nucleotide sequence encoding said CRISPR nuclease, and a reverse transcriptase and/or an expression construct comprising a nucleotide sequence encoding said reverse transcriptase, or B) A directed editing fusion protein and/or an expression construct comprising a nucleotide sequence encoding said directed editing fusion protein, wherein said directed editing fusion protein comprises a CRISPR nuclease and a reverse transcriptase, and Ii) at least one pegRNA and/or an expression construct containing a nucleotide sequence encoding the at least one pegRNA, the at least one pegRNA comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 or more pegRNA of any one of claims 1 to 7, and pegRNA of any one of claims 1 to 7 consisting of a first pegRNA and a second pegRNA.
  9. 9. A genome editing system according to claim 8 wherein the fusion protein of i) -b) further comprises a recombinase, preferably Cre, bxb1, phiC31 recombinase, more preferably Cre.
  10. 10. The genome editing system of claim 8 or 9, wherein the at least one pegRNA is driven to transcribe by a composite promoter comprising a 35S enhancer, a CmYLCV promoter and/or a U6 promoter; alternatively, the U6 promoter is a truncated U6 promoter variant.
  11. 11. The genome editing system according to any one of claim 8 to 10, wherein, The first pegRNA and/or the second pegRNA of the at least one pegRNA are each in a different expression construct, or At least two first pegRNA, at least two second pegRNA, or at least one first pegRNA and at least one second pegRNA of the at least one pegRNA are in the same expression construct, or The first pegRNA and the second pegRNA of the at least one pegRNA are both in the same expression construct.
  12. 12. The genome editing system according to any of claims 8 to 11 wherein the first pegRNA and/or the second pegRNA further comprise a tevopre sequence 3' to the PBS sequence, and/or The first pegRNA and/or second pegRNA further comprise a polyT sequence at the 3' end.
  13. 13. The genome editing system of any of claims 8-12 wherein the 5 'end of a first pegRNA and/or a second pegRNA is linked to a first ribozyme or tRNA designed to cleave the first pegRNA and/or second pegRNA, or to cleave a fusion of the first pegRNA and/or second pegRNA, tevopre sequence and/or a polyT sequence, at the 5' end of the first pegRNA and/or second pegRNA, and/or the 3 'end of the first pegRNA and/or second pegRNA is linked to a second ribozyme or tRNA designed to cleave the first pegRNA and/or second pegRNA, or to cleave a fusion of the first pegRNA and/or second pegRNA, tevopre sequence and/or a polyT sequence, at the 3' end of the first pegRNA and/or second pegRNA; Optionally, the tRNA is selected from at least one of tRNAGly, tRNAOsAsp and tRNAZmIle; optionally, the ribozyme comprises an HDV ribozyme.
  14. 14. The genome editing system of claim 13 wherein when the first pegRNA and the second pegRNA of the at least one pegRNA are both in the same expression construct and the total number of first pegRNA and second pegRNA of the at least one pegRNA is 6 or less, the tRNA is tRNAGly, or When the first pegRNA and the second pegRNA of the at least one pegRNA are both in the same expression construct and the total number of first pegRNA and second pegRNA of the at least one pegRNA is greater than 6, the tRNA is selected from at least two, preferably three, of tRNAGly, tRNAOsAsp and tRNAZmIle.
  15. 15. Genome editing system according to any of claims 8-14, wherein the first and second target sequences in pegRNA are associated with a plant trait, such as an agronomic trait, whereby the plant is caused to have an altered (preferably improved) trait, such as an agronomic trait, relative to a wild type plant by the genome editing system.
  16. 16. The genome editing system of any of claims 8-15, wherein the at least one pegRNA is capable of forming a complex with the CRISPR nuclease or fusion protein and targeting the CRISPR nuclease or fusion protein to a target sequence in a genome resulting in a nick on a target strand within the target sequence.
  17. 17. The genome editing system of any of claims 8-16, wherein the CRISPR nuclease is a Cas9 nuclease or a variant thereof.
  18. 18. The genome editing system according to any of claims 8-17, wherein the CRISPR nuclease is a CRISPR nickase, such as a Cas9 nickase or a variant thereof, such as the Cas9 nickase or a variant thereof comprising a sequence selected from the group consisting of SEQ ID NOs 1 or 2; optionally, the CRISPR nuclease, such as Cas9 nickase, and the reverse transcriptase are linked by a linker.
  19. 19. The genome editing system of any of claims 8-18, wherein the reverse transcriptase is an M-MLV reverse transcriptase or a functional variant thereof; Alternatively, the RNase H domain of the reverse transcriptase, e.g., M-MLV reverse transcriptase or a functional variant thereof, is deleted and comprises the sequence as set forth in SEQ ID NO. 5.
  20. 20. Genome editing system according to any of claims 8-19, wherein a reverse transcriptase, such as M-MLV reverse transcriptase or a functional variant thereof, is fused at the N-or C-terminus to nucleocapsid proteins (NC) directly or through a linker.

Description

Efficient and accurate plant gene knockout method Technical Field The invention belongs to the field of genetic engineering. Specifically, the invention comprises a design and high-efficiency insertion method of a protein translation termination codon cluster sequence so as to realize high-efficiency and accurate knockout of plant genes. The DNA sequence can be reversely transcribed by utilizing a plant-guided editing system (PE), and the DNA sequence containing a termination codon cluster (stop codon cluster, SCC) is efficiently inserted into a target gene by combining the capability of efficiently inserting double pegs into a heterologous sequence, so that the efficient and accurate knockout of the plant gene is realized. Based on the method, a plurality of different pegRNA are combined to construct an all-in-one system for modifying the plant genome, namely, the multi-gene and multi-type genome modification such as knockout, base substitution, small fragment insertion, deletion, substitution, long fragment deletion and replication of the plant gene can be realized by only guiding and editing PE protein. Background Genomic variation is the basis for creating new varieties of plants. Plant breeding has undergone a lengthy history. From ancient times, humans have been selecting natural variations at low frequencies to domesticate plants. With the development of life science and technology, human beings autonomously create genomic variations by hybridization, artificial mutagenesis and other techniques to improve plants. However, the genomic variations obtained by these techniques are random and inefficient. The development of targeted genome editing technology, in particular the development of a simple and efficient CRISPR/Cas system, enables people to accurately and efficiently reconstruct plant genomes, and greatly accelerates the plant breeding process. CRISPR/Cas is a defense system of bacteria and archaea against viral infection, and Cas nuclease is guided to cleave target sequences by guide RNAs (grnas). Based on this principle, CRISPR/Cas9 systems were developed that could target cleavage of eukaryotic genomes resulting in small genomic fragment insertion or deletion mutations. In addition, by fusing Cas9 protein and other effector such as deaminase and reverse transcriptase, a series of genome editing tools such as base editor and guide editor were developed. These rich editing tools not only can realize conventional gene knockout, but also can carry out precise base substitution, substitution of small fragments, insertion or deletion on the genome. CRISPR/Cas technology is continually evolving towards more precision, efficiency and versatility. Gene knockout is a genetic engineering technique that selectively inactivates a particular gene. Gene knockout has important application value in biological research, biological breeding, disease treatment and the like. Conventional gene knockout methods use grnas to guide Cas9 nucleases to cleave the target genomic duplex. Cas9 proteins contain both HNH and RuvC domains, cleaving the targeting and non-targeting strands of the gRNA, respectively, resulting in a Double Strand Break (DSB) of the target genome. Subsequently, cells repair DSBs by means of non-homologous end joining (NHEJ), possibly introducing small base insertion or deletion (index) mutations at the nicks. If indel is not a multiple of 3, the target gene is subjected to frame shift mutation to generate a premature stop codon (premature stop codon), so that the function of the target protein is lost, and the target gene knockout is realized. Cas 9-mediated gene knockout is simple to operate, but suffers from a number of drawbacks. If (1) the off-target efficiency is high. When regions similar to the target sequence are present in the genome, non-targeted DNA cleavage is likely to occur, resulting in unintended and non-specific genetic modifications. (2) imprecise protein translation termination position. Since Cas 9-mediated gene knockout is the creation of indels by cellular NHEJ repair, non-3 fold indels will result in frameshift mutation of the protein of interest until a stop codon is found in the downstream sequence. The sequence content and the size of the indels are uncertain, so that the termination position of protein translation is uncertain, and precise termination is difficult to achieve. (3) the efficiency of gene knockout is lower than the efficiency of genome mutation. Based on Indel production, there is a ratio of Indel mutations that produce a 3N fold, and only in the wild type protein, from the addition or absence of a few amino acid residues, premature stop codons cannot be produced, resulting in lower actual gene knockout efficiency than Indel mutations. In addition, using the cytosine base editor, specific codons, including forward-chain CGA (Arg, R), CAG (Gln, Q) and CAA (Gln, Q) and reverse-chain TGG (Trp, W) codons, are targeted such that cytosine (C) is converted to thym