CRISPR–Cas12a system in fission yeast for multiplex genomic editing and CRISPR interference

Abstract

The CRISPR–Cas12a is a class II, type V clustered regularly interspaced short palindromic repeat (CRISPR) system with both RNase and DNase activity. Compared to the CRISPR–Cas9 system, it recognizes T-rich PAM sequences and has the advantage of multiplex genomic editing. Here, in fission yeast Schizosaccharomyces pombe, we successfully implemented the CRISPR–Cas12a system for versatile genomic editing and manipulation. In addition to the rrk1 promoter, we used new pol II promoters from endogenous coding genes to express crRNA for Cas12a and obtained a much higher editing efficiency. This new design expands the promoter choices for potential applications in fission yeast and other organisms. In addition, we expressed a gRNA array using a strong constitutive pol II promoter. The array transcript is processed by Cas12a itself to release multiple mature crRNAs. With this construct, multiplex genomic editing of up to three loci was achieved from a single yeast transformation. We also built a CRISPR interference system using a DNase-dead Cas12a to significantly repress endogenous gene expression. Our study provides the first CRISPR-Cas12a toolkit for efficient and rapid genomic gene editing and regulation in fission yeast.

Efficient multiplexed gene regulation inSaccharomyces cerevisiaeusing dCas12a

“…The intrinsic RNase activity of Cas12a also allows for an additional design of RNAP II expressed gRNAs, whereby a spacer is flanked by two direct repeats. In this case, the two direct repeats are recognized by Cas12a and the pre-crRNA is processed into its mature form of a spacer and a single direct repeat ( 28 ). Promoters recognized by RNAP II are generally well-characterized, exhibit a wide range of strengths and many are able to dynamically respond to changes in environmental conditions or the presence of an inducer molecule allowing for gRNA expression to be dynamically controlled ( 54 ).…”

“…Promoters recognized by RNAP II are generally well-characterized, exhibit a wide range of strengths and many are able to dynamically respond to changes in environmental conditions or the presence of an inducer molecule allowing for gRNA expression to be dynamically controlled ( 54 ). A recent study on Cas12a-mediated genome editing showed that driving gRNA expression from a RNAP II promoter increased gRNA availability and improved editing efficiency ( 28 ).…”

“…This may be due to the known increased processivity of RNAP II over RNAP III ( 54 ). Numerous studies have shown that the availability of the gRNA is the limiting factor for CRISPR–Cas9 mediated genome editing in eukaryotic cells ( 28 , 56 , 57 ). Therefore, our results also suggest that despite using a multicopy plasmid in combination with a strong RNAP III promoter, the concentration of gRNA may still not be sufficient to fully saturate the available dCas12a pool.…”

Implementation of dCas9-mediated CRISPRi in the fission yeast Schizosaccharomyces pombe

“…The other approach to bypass the limitation of PAM is to employ another CRISPR-Cas system with different PAM specificity. Recently, it was reported that a type V CRISPR system, CRISPR-Cas12a, is applicable for CRISPRi in fission yeast ( Zhao and Boeke 2020 ). Since Cas12a has a T-rich PAM, 5′-TTTV-3′ (V is A, G, or C), Cas12a and Cas9, for which PAM is G-rich and short 5′-NGG-3′, complement each other to cover wide variety of target sites.…”

“…Recently, a CRISPRi method based on the CRISPR-Cas12a system was established in S. pombe ( Zhao and Boeke 2020 ). However, to our knowledge, CRISPRi mediated by dCas9 had not been implemented in this organism.…”