Saccharomyces cerevisiae Synthetic Transcriptional Networks Harnessing dCas12a and Type V-A anti-CRISPR Proteins

Abstract

Type V-A anti-CRISPR proteins (AcrVAs) represent the response from phages to the CRISPR-Cas12a prokaryotic immune system. CRISPR-Cas12a was repurposed, in high eukaryotes, to carry out gene editing and transcription regulation, the latter via a nuclease-dead Cas12a (dCas12a). Consequently, AcrVAs were adopted to regulate (d)­Cas12a activity. However, the usage of both dCas12a-based transcription factors and AcrVAs in the yeast Saccharomyces cerevisiae has not been explored. In this work, we show that, in the baker’s yeast, two dCas12a proteins (denAsCas12a and dLbCas12a) work both as activators (upon fusion to a strong activation domain) and repressors, whereas dMbCa12a is nonfunctional. The activation efficiency of dCas12a-ADs manifests a dependence on the number of crRNA binding sites, whereas it is not directly correlated to the amount of crRNA in the cells. Moreover, AcrVA1, AcrVA4, and AcrVA5 are able to inhibit dLbCa12a in yeast, and denAsCas12a is only inhibited by AcrVA1. However, AcrVA1 performs well at high concentration only. Coexpression of two or three AcrVAs does not enhance inhibition of dCas12a­(-AD), suggesting a competition between different AcrVAs. Further, AcrVA4 significantly limits gene editing by LbCas12a. Overall, our results indicate that dCas12a:crRNA and AcrVA proteins are highly performant components in S. cerevisiae synthetic transcriptional networks.

Strong and tunable anti‐CRISPR/Cas activities in plants

“…Previous studies in other organisms, including bacteria, yeast and mammalian cells, already demonstrated the potent anti-Cas9 effect of AcrIIA4 (Li et al, 2018;Nakamura et al, 2019;Rauch et al, 2017). Recently, Yu and Marchisio (2021) also reported the high anti-Cas12a inhibitory capacity of AcrVA1 in yeast.…”

“…Therefore, development of anti-Cas12a activities in planta can offer new possibilities for gene regulation. Anti-Cas12a protein AcrVA1 has been described to cleave the crRNA when bound to Cas12a preventing editing and CRISPRa in yeast and mammalian cells (Kempton et al, 2020;Yu and Marchisio, 2021;Figure 1e). Following the same procedure as described for AcrIIA4, we selected two targets previously characterized for Cas12a: XT1 and FT genes (Bernab e-Orts et al, 2019).…”

Novel S. cerevisiae Hybrid Synthetic Promoters Based on Foreign Core Promoter Sequences

“…From the third to the eighth group, the promoters are rather weak (see Tables S4–S6 ). The strongest of them almost matches a different synthetic promoter constructed in our lab (termed genCYC1t_pCYC1noTATA [ 26 ]) that we recently used to study the activity of type V anti-CRISPR proteins in S. cerevisiae [ 37 ].…”

“…The strongest MLPcore* expressed a 28.3-fold higher fluorescence than the weakest pTKcore* (see Figure 2 C). Interestingly, MLPcore* turned out to be 1.9-fold stronger than the constitutive TEF1 promoter—widely employed in yeast synthetic gene circuits—whereas the pTKcore* strength was comparable with that of the weakest synthetic promoter previously built in our lab and termed “truncated_pCYC1”, where we just shortened pCYC1 5′UTR down to 24 nt [ 37 ] (details about the number of TSSs activated by each of these promoters and the distance between TSSs and the TATA box are presented in Table S3 ).…”

Synthetic polycistronic sequences in eukaryotes

“…[ [113] , [114] , [115] ], The baker's yeast, moreover, is commonly treated as a cell factory to host heterologous pathways (such as that for β-carotene biosynthesis [ 60 , 112 ]) and as a chassis for synthetic networks that make use of the interactions between transcription factors derived from nuclease-deficient Cas protein and their antagonist anti -CRISPR macromolecules (see Fig. 4 ) [ 109 , 111 ]. Beekwilder and coworkers [ 112 ] introduced in S. cerevisiae a polycistronic vector that carried three β-carotene biosynthesis genes: crtI, crtE, and crtYB, to engineer a carotenoid pathway.…”