Background: Anti-CRISPR proteins are expressed by phages as a reaction to the bacterial CRISPR-Cas defense system. Recently, the structures of anti-CRISPR proteins have been determined, and their diverse functions have been clarified. Anti-CRISPR proteins such as LmAcrIIA2 and LmAcrIIA4 interact with the SpCas9:gRNA system and occlude the protospacer adjacent motif (PAM) recognition site, thereby preventing SpCas9:gRNA from binding to the DNA. Hence, anti-CRISPR proteins represent a powerful means to control and modulate the activity of SpCas9 and its nuclease-deficient version dSpCas9. LmAcrIIA2 and LmAcrIIA4 have been shown to be efficient inhibitors of SpCas9 in Escherichia coli, Saccharomyces cerevisiae, and mammalian cells. To date, there have been no reports of anti-CRISPR-based synthetic gene circuits engineered into yeast cells.

Results: We constructed in the yeast S. cerevisiae synthetic biosensors based on the anti-CRISPR-dSpCas9:gRNA interaction. Upon induction with galactose or β-estradiol, anti-CRISPR proteins (LmAcrIIA4, LmAcrIIA2, and StAcrIIA5) produced an enhancement in fluorescence expression by preventing the dSpCas9-Mxi1:gRNA complex from binding to the DNA. We found that LmAcrIIA2 performed as well as LmAcrIIA4 in S. cerevisiae, whereas StAcrIIA5, which had previously been tested in bacteria only, had non-negligible negative effects on yeast cell growth. The efficiency of anti-CRISPR-based biosensors was strongly dependent on the means by which the guide RNAs were produced. The best performance, as measured by the increase in fluorescence, was achieved using a "ribozyme-gRNA-ribozyme" expression cassette under the control of the yeast constitutive ADH1 promoter.

Conclusions: This work demonstrates that anti-CRISPR proteins are effective dSpCas9 suppressors in yeast cells. In particular, LmAcrIIA2 and LmAcrIIA4 could be employed as new components of yeast synthetic gene circuits.

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Journal Article

Authors

Li J, Xu Z, Chupalov A, Marchisio MA

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