Gene editing systems and applications thereof

Abstract

The present application relates to a gene editing system and application thereof. The gene editing system is a prime editing system, which comprises an editing element and a pegRNA expression frame. The editing element comprises a promoter, a CSY4 nuclease, a self-cleaving peptide, a first nuclear localization signal sequence, a Cas9 protein variant, a linker, a reverse transcriptase, a second nuclear localization signal sequence and a terminator. The pegRNA expression frame comprises a CmYLCV promoter, a first CSY4 nuclease recognition site, an epegRNA, a second CSY4 nuclease recognition site, an sgRNA, a third CSY4 nuclease recognition site and a CaMV poly(A). The present application establishes the first efficient and universal prime editing platform suitable for soybean.

Claims

1. A guided editing system, characterized in that, The guided editing system includes editing elements and a pegRNA expression cassette. The editing elements include a promoter, a CSY4 nuclease, a self-cleaving peptide, a first nuclear localization signal sequence, a Cas9 protein variant, an adapter, a reverse transcriptase, a second nuclear localization signal sequence, and a terminator. The pegRNA expression cassette includes a CmYLCV promoter, a first CSY4 nuclease recognition site, an epigRNA, a second CSY4 nuclease recognition site, sgRNA, a third CSY4 nuclease recognition site, and a CaMV poly(A).

2. The guided editing system according to claim 1, characterized in that, The promoter is selected from CaMV 35S promoter, Nos promoter, ubiquitin promoter, actin promoter, and alcohol dehydrogenase promoter, and the terminator is selected from nos terminator, 35S terminator, CYC1 terminator, ADH1 terminator, TEF1 terminator, TEF2 terminator, PGK1 terminator, and ENO1 terminator.

3. The guided editing system according to claim 1 or 2, characterized in that, The Cas9 protein variant is the SpCas9 protein variant.

4. The guided editing system according to claim 1 or 2, characterized in that, The reverse transcriptase is selected from any of the sequences shown in SEQ ID NO: 6-9.

5. A nucleic acid molecule, expression vector, or host cell, characterized in that, The nucleic acid molecule, expression vector, or host cell includes the guided editing system according to any one of claims 1 to 4.

6. A gene editing method for legumes, characterized in that, The method is performed using the guided editing system according to any one of claims 1 to 4.

7. A method for preparing leguminous plant mutants, characterized in that, The method is performed using the guided editing system according to any one of claims 1 to 4.

8. The use of the guided editing system according to any one of claims 1 to 4 in any of the following: 1) Editing of legume genome sequences; 2) Preparation of products for editing the genome sequence of leguminous plants; 3) Improve the efficiency of legume genome sequence editing; 4) Prepare products that improve the efficiency of genome sequence editing in legumes.

9. The method according to claim 6 or 7, or the use according to claim 8, characterized in that, The legumes mentioned are selected from plants of the genera *Glycine*, *Vicia*, *Chickpea*, and *Alfalfa*.

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