A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification.
Bio Protoc
; 11(7): e3971, 2021 Apr 05.
Article
en En
| MEDLINE
| ID: mdl-33889665
ABSTRACT
CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors ( Liu et al., 2020 ). In this approach, we have optimized the multiplex PCR to produce an overlapping PCR product in a single reaction to generate the sgRNA expression cassette. We also amplified two sgRNA expression cassettes through a single round of PCR. Then, the sgRNA expression cassette(s) is cloned into the binary vectors in a Gateway LR or Golden gate reaction. The system reported here provides a much more efficient and simpler procedure to construct expression clones for CRISPR/Cas9-mediated genome editing. In this protocol, we describe the detailed step-by-step instructions for using this system.
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Banco de datos:
MEDLINE
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En
Revista:
Bio Protoc
Año:
2021
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Article
País de afiliación:
China