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.
Highlights
Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas[9] 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
We present a bacterial-type II CRISPR-Cas[9] system as a novel genome mutagenesis technique in the serotype D strain of the basidiomycetous pathogenic yeast C. neoformans, which presents a low frequency of homologous recombination for gene disruption[17,22]
We found that optimizing the target sites by sgRNAcas[9] software and shortening the distance (
Summary
Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas[9] 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. Off-target editing by the CRISPR-Cas[9] system that triggers undesirable genetic alterations commonly occurs[10,11], and CRISPR-Cas[9] is cytotoxic in certain organisms, including Saccharomyces cerevisiae and Schizosaccharomyces pombe[12,13,14] Another pragmatic problem encountered in our study is that the persistence of the CRISPR-Cas[9] system in the cells precludes the restoration of the mutated gene in C. neoformans because the system targets the complement gene that contains the same target sequence of gRNA. We report the establishment of a practical and feasible CRISPR-Cas[9] system for gene targeting and restoration in C. neoformans
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