2021
DOI: 10.1093/nar/gkab529
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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 fea… Show more

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Cited by 32 publications
(38 citation statements)
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“…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.…”
Section: Resultsmentioning
confidence: 99%
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“…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.…”
Section: Resultsmentioning
confidence: 99%
“…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.…”
Section: Discussionmentioning
confidence: 99%
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“…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.…”
Section: Resultsmentioning
confidence: 99%
“…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].…”
Section: Crispr-based Genetic Switches In Translation Levelmentioning
confidence: 99%