Construction of CRISPR-Cas9 knockout vector of Fonsecaea monophora polyketide synthase gene

Abstract

Abstract: Objective To construct a Fonsecaea monophora polyketide synthase 1 (PKS1) gene knock-out vector by CRISPR/Cas9 system with pFC332 plasmid. Methods Primers of the gRNA skeleton fragment from pFC334 plasmid were designed, and then the gRNA skeleton fragment inserted with the AarⅠ restriction site was amplified by PCR. After that, the pFC334 plasmid was linearized with PstⅠ and MerI enzymes and a pFC334-AarⅠ vector was constructed. Subsequently, both pFC334-AarⅠ vector and pFC332 plasmid were digested with MreI and BglII. Digested products were ligated together in order to produce a novel recombinant vector called pFC332-gRNA-AarⅠ, with gRNA skeleton fragment. Finally, multiple gRNAs targeting PKS1 were designed and integrated into the pFC332-gRNA-AarⅠ vector, respectively. Results The obtained pFC332-gRNA-AarⅠ plasmid was 16533bp long, identified by sequencing. PCR, enzyme digestion and sequencing were applied to indicate that the PKS1 gene knock-out vector by CRISPR/Cas9 system was successfully constructed. Conclusion The Fonsecaea monophora polyketide synthase 1 (PKS1) gene knock-out vector by CRISPR/Cas9 system was successfully constructed, which laid a good foundation for the research of PKS1 gene and the biological functions of DHN-melanin in Fonsecaea monophora.

Key words: Fonsecaea monophora, polyketide synthase gene, CRISPR/Cas9, vector construction

CLC Number:

R379.9

LI Minying, HUANG Huan, LI Qian, LUO Mingfen, WANG Xiaoyue, LIU Hongfang, ZENG Weiying, XI Liyan. Construction of CRISPR-Cas9 knockout vector of Fonsecaea monophora polyketide synthase gene[J]. Chinese Journal of Mycology, 2021, 16(5): 303-307,313.

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