title: A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.
abstract: Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This 'suicide' CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans.
authors: National Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University (DMNU), Tianjin 300071, China.
authors: Yu
authors: Y
authors: Wang
authors: National Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University (DMNU), Tianjin 300071, China.
authors: Dongsheng
authors: D
authors: Wei
authors: National Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University (DMNU), Tianjin 300071, China.
authors: Xiangyang
authors: X
authors: Zhu
authors: National Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University (DMNU), Tianjin 300071, China.
authors: Jiao
authors: J
authors: Pan
authors: National Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University (DMNU), Tianjin 300071, China.
authors: Ping
authors: P
authors: Zhang
authors: Beijing Key Laboratory of Genetic Engineering Drug and Biotechnology, Institute of Biochemistry and Biotechnology, College of Life Sciences, Beijing Normal University, Beijing 100875, China.
authors: Liang
authors: L
authors: Huo
authors: National Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University (DMNU), Tianjin 300071, China.|Beijing Key Laboratory of Genetic Engineering Drug and Biotechnology, Institute of Biochemistry and Biotechnology, College of Life Sciences, Beijing Normal University, Beijing 100875, China.
authors: Xudong
authors: X
authors: Zhu
mesh_terms: D064113:CRISPR-Cas Systems; D003455:Cryptococcus neoformans / Q000235:genetics*; D018274:Electroporation; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D016681:Genome, Fungal