An improved CRISPRi system in .

CRISPR interference (CRISPRi) has been developed and widely used for gene repression in various hosts. Here we report an improved CRISPRi system in by fusing dCas9 with endogenous transcriptional repressor domains. The CRISPRi system shows strong repression of , with the highest efficiency of 85%. Repression of native genes is demonstrated by targeting promoter. is efficiently repressed and the mutant strains show much slower growth in methanol medium. Effects of gRNA expression and processing on CRISPRi efficiency is also investigated. It is found that gRNA processing by HH/HDV ribozymes or Csy4 endoribonuclease generating clean gRNA is critical to achieve strong repression, and Csy4 cleavage shows higher repression efficiency. However, gRNA expression using native tRNA transcription and processing systems results in relatively weaker repression of . By expression of two gRNAs targeting promoters of and in an array together with Cys4 recognition sites, both genes can be repressed simultaneously. Cys4-mediated gRNA array processing is further applied to repress fatty acyl-CoA synthetase genes ( and ). Both genes are efficiently repressed, demonstrating that Cys4 endoribonuclease has the ability to cleave gRNAs array and can be can be used for multiplexed gene repression in .