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.

Figure 1.

Cas12a genome editing system. (A) FnCas12a and crRNA were expressed from a single plasmid with ura4 marker and ars1 replicating origin in fission yeast. As the first construct, FnCas12a and gRNA were expressed with adh1 and rrk1 promoter, respectively. (B) To test editing efficiency, we picked four gRNAs targeting the endogenous ade6⁺ CDS. One linear donor DNA was co-transformed to delete ade6 by homologous recombination. (C) The gRNA sequences used and their editing efficiency, with mean values ± S.E.M. The original colony counts are listed in Supplementary Table S6. (D) Colony PCR to check the ade6 deletion, in red colonies and white colonies from plates with PMG5–Ura w/ low adenine media. The 1 kb Plus DNA ladder from NEB (Catalog# N3200L) was used as the molecular weight standard. (E) Spot assay on plates to check adenine auxotroph.

Figure 2.

PAM preference for FnCas12a and genome editing at additional targets. (A) Yeast strains containing same gRNA targets but alternative PAMs with synonymous mutations were tested to measure the genome editing efficiency. (B) Transformants from parents strains with the PAM sequence CTTA compared to TTTA. (C) Cas12a editing efficiency targeting leu1, his3 and lys9, with mean values ± S.E.M. shown here. The original colony counts are listed in Supplementary Table S7.

Figure 3.

Expression of Cas12a crRNA using pol II promoters. (A) New constructs were designed using pol II promoters and terminators to express crRNA. (B) crRNA release from primary RNA transcripts. (C) New endogenous and exogenous pol II promoters tested and their editing efficiency targeting ade6⁺. The same gRNA sequence (gRNA-3) was used here. Error bars represent mean ± S.E.M. using three technical replicates. An unpaired t-test was used to assess the significance of higher efficiency using nmt1 or fba1 promoter compared to the rrk1 promoter (**P < 0.01, ***P < 0.001). The original colony counts were listed in Supplementary Table S8.

Figure 4.

Multiplex genome editing using crRNA arrays. (A) The crRNA arrays designed to express multiple crRNA on a single transcript. (B) Genome editing efficiency using rrk1 or fba1 promoter to drive the crRNA array expression, with their mean efficiency ± S.E.M. Gray: positive editing efficiency at ade6 alone using array-1. Orange: double positive editing efficiency at ade6 and leu1, using array-2. Blue: triple positive editing efficiency at ade6, leu1 and his3, using array-3. (C) The transformant count for ade6 or leu1 deletion from the group using array-2 driven by fba1 promoter.

Figure 5.

CRISPR interference using dCas12a. (A) The dCas12a with D917A mutation was used to reduce expression of ade6⁺. (B) One gRNA proximal to TSS was used to repress the ade6 expression. (C) Spot assay to check CRISPRi repression using dCas12a alone or dCas12a-Clr4. (D) CRISPRi repression of ade6 using dCas12a fused to the Clr4 catalytic domain or catalytic dead mutants.