Types I and V Anti-CRISPR Proteins: From Phage Defense to Eukaryotic Synthetic Gene Circuits

Yu, Lifang; Marchisio, Mario Andrea

doi:10.3389/fbioe.2020.575393

Cited by 9 publications

(8 citation statements)

References 136 publications

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“…As a response to CRISPR-Cas, phages developed various anti-CRISPR (Acr) proteins able to block the immune function of CRISPR-Cas. , In particular, three type V-A anti-CRISPR proteins (AcrVA1, AcrVA4, and AcrVA5) were discovered by using a comprehensive bioinformatics and experimental screening approach in 2018 . Compared to AcrVA4 and AcrVA5, AcrVA1 inhibits a broader spectrum of Cas12a orthologues.…”

Section: Introductionmentioning

confidence: 99%

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

Marchisio

2021

ACS Synth. Biol.

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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.

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Section: Introductionmentioning

confidence: 99%

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

Marchisio

2021

ACS Synth. Biol.

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“…AcrVA5, in contrast, works as an acetyltransferase and the acetylated Cas12a is no longer able to interact with PAM. Moreover, AcrVA1 inhibits a broader range of Cas12a homologs compared to AcrVA4 and AcrVA5 ( Marino et al, 2018 ; Yu and Marchisio, 2020 ). In a previous work from our lab, these three AcrVAs have been shown to inhibit LbCas12a and denAsCas12a (AcrVA1 only) in budding yeast cells ( Yu and Marchisio, 2021 ).…”

Section: Resultsmentioning

confidence: 99%

“…The anti-CRISPR (Acr) proteins are a response, evolved by phages, to contrast the CRISPR-Cas immune systems ( Tickner et al, 2020 ). Three type V anti-CRISPR proteins (AcrVA1, AcrVA4, and AcrVA5) have been shown to inhibit strongly the activity of different (d)Cas12a proteins via distinct mechanisms ( Yu and Marchisio, 2020 ). In particular, AcrVA1 prevents the Cas12a:crRNA complex from binding the DNA by mimicking PAM and (sometimes) truncating the crRNA.…”

Section: Resultsmentioning

confidence: 99%

“…Similar to other type V Cas proteins, Cas12c appears more effective as an activator rather than a repressor. Moreover, its activity can be controlled by AcrVA1, whereas AcrVA4 and AcrVA5 ( Yu and Marchisio, 2020 ) have no effect on it. We tested Cas12c behavior as a signal carrier in synthetic gene hybrid gates—where translation was downregulated by up-to-two riboswitches responding to tetracycline ( Kötter et al, 2009 ).…”

Section: Introductionmentioning

confidence: 99%

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Hybrid Boolean gates show that Cas12c controls transcription activation effectively in the yeast S. cerevisiae

Liu,

Ge,

Marchisio

2023

Front. Bioeng. Biotechnol.

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Among CRISPR-Cas systems, type V CRISPR-Cas12c is of significant interest because Cas12c recognizes a very simple PAM (TN) and has the ability to silence gene expression without cleaving the DNA. We studied how new transcription factors for the yeast Saccharomyces cerevisiae can be built on Cas12c. We found that, upon fusion to a strong activation domain, Cas12c is an efficient activator. Its functionality was proved as a component of hybrid Boolean gates, i.e., logic circuits that mix transcriptional and translational control (the latter reached via tetracycline-responsive riboswitches). Moreover, Cas12c activity can be strongly inhibited by the anti-CRISPR AcrVA1 protein. Thus, Cas12c has the potential to be a new tool to control the activation of gene expression within yeast synthetic gene circuits.

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“…Another approach to optimizing the efficiency of genome editing experiments in eukaryotic systems is to limit off-target effects imposed by Cas9 ( Jiang et al, 2019 ; Davidson et al, 2020 ; Marino et al, 2020 ; Shivram et al, 2021 ; Zhang and Marchisio, 2021 ) or Cas12a ( Knott et al, 2019 ; Zhang H. et al, 2019 ; Davidson et al, 2020 ; Marino et al, 2020 ; Yu and Marchisio, 2020 ) by using small molecules called anti-CRISPRs. Importantly, both Cas9 and Cas12a genome editing technologies can be regulated by expression of anti-CRISPRs in S. cerevisiae ( Li J. et al, 2018 ; Yu and Marchisio, 2021 ; Zhang and Marchisio, 2022 ).…”

Section: Strategies To Reduce Off-target Binding and Cleavage By Cas ...mentioning

confidence: 99%

Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae

Antony

Hinz

Wyrick

2022

Front. Bioeng. Biotechnol.

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The versatility of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) genome editing makes it a popular tool for many research and biotechnology applications. Recent advancements in genome editing in eukaryotic organisms, like fungi, allow for precise manipulation of genetic information and fine-tuned control of gene expression. Here, we provide an overview of CRISPR genome editing technologies in yeast, with a particular focus on Saccharomyces cerevisiae. We describe the tools and methods that have been previously developed for genome editing in Saccharomyces cerevisiae and discuss tips and experimental tricks for promoting efficient, marker-free genome editing in this model organism. These include sgRNA design and expression, multiplexing genome editing, optimizing Cas9 expression, allele-specific editing in diploid cells, and understanding the impact of chromatin on genome editing. Finally, we summarize recent studies describing the potential pitfalls of using CRISPR genome targeting in yeast, including the induction of background mutations.

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Types I and V Anti-CRISPR Proteins: From Phage Defense to Eukaryotic Synthetic Gene Circuits

Cited by 9 publications

References 136 publications

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

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

Hybrid Boolean gates show that Cas12c controls transcription activation effectively in the yeast S. cerevisiae

Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae

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