Efficient multiplexed gene regulation inSaccharomyces cerevisiaeusing dCas12a

Abstract

CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for Saccharomyces cerevisiae and systematically evaluate its design features. This includes the role of the NLS position, use of repression domains, and the position of the gRNA target. Our optimal system is comprised of dCas12a E925A with a single C-terminal NLS and a Mxi1 or a MIG1 repression domain, which enables up to 97% downregulation of a reporter gene. We also extend this system to allow for inducible regulation via an RNAP II-controlled promoter, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous β-carotene pathway. Together these findings offer valuable insights into the design constraints of dCas12a-based CRISPRi and enable new avenues for flexible and efficient gene regulation in S. cerevisiae.

CRISPR-mediated protein-tagging signal amplification systems for efficient transcriptional activation and repression inSaccharomyces cerevisiae

“…To develop an orthogonal bifunctional CRISPR-mediated transcriptional regulation system, nuclease-deficient CRISPR proteins that can recognize different PAM sequences are required. dCpf1 from Lachnospiraceae bacterium ND2006, favoring 5′-TTTV-3′ PAM sequences (dCpf1), has been characterized ( 37 ) and used for CRISPR regulation or genome editing in S. cerevisiae ( 38 , 39 ). We thus chose dCpf1 to construct an orthogonal bifunctional CRISPR-mediated transcriptional regulation system.…”

“…Previously, the activation efficiency based on dCas9-VPR and the inhibition efficiency based on dCpf1-Mxi1 can reach to 4.5∼12-fold and 75%∼95%, respectively, for different promoter in S. cerevisiae ( 7 , 38 ). Here, we compared the regulation efficiency of transcription factors and coupled the transcription factors with SPY and Sun Tag systems.…”

“…Ciurkot et al. recently demonstrated that Mxi1 and MIG1 enabled better downregulation relative to other repression domains ( 37 , 38 ). Our result was consistent with their finding, as even we found that the repression efficiency of MIG1 and Mxi1 was better than that of TUP1.…”

Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems

“…Switching the position of spacer sequences in the CRISPR array has shown to have varying effects on targeting efficiencies. 48 This could be a potential approach to optimize the CRISPR arrays and improve the multiplexed targeting efficiencies. Moreover, using a truncated repeat sequence (19 nts) in place of the full-length 36 nts long repeats could reduce the complexity of the long CRISPR array transcript.…”

CRISPR-Based Genetic Switches and Other Complex Circuits: Research and Application

“…By simultaneously processing multiple gRNAs with different target sequences for directing dCas9, Csy4, along with other similar endoribonuclease such as Cas5d, Cas6a and Cse3, can build complex logic gates for precisely regulating gene expression [56] (Figure 2D). Moreover, Cas12a, with the dual functionality of cleaving dsDNA and processing its own gRNA, is also valuable in multiplexed gene regulation because it allows simultaneous control of multiple genes using a single enzyme pairing with different gRNAs, which is much simpler than the Csy4/dCas9 systems [34][35][36][37]55] (Figure 2E). Besides RNA endoribonuclease, another category of CRISPR enzyme, the RNA-guided RNA endonuclease, have also emerged in recent years with the potential as genetic switches at translational level, namely the Cas13a and Cas7-11 endonuclease [78][79][80].…”