Comprehensive deletion scan of anti-CRISPR AcrIIA4 reveals essential and dispensable domains for Cas9 inhibition.

Autor:	Iturralde AB; Systems Biology and Genome Engineering Section, Center for Genomics and Data Science Research, National Human Genome Research Institute, NIH, Bethesda, MD., Weller CA; Systems Biology and Genome Engineering Section, Center for Genomics and Data Science Research, National Human Genome Research Institute, NIH, Bethesda, MD., Giovanetti SM; Systems Biology and Genome Engineering Section, Center for Genomics and Data Science Research, National Human Genome Research Institute, NIH, Bethesda, MD., Sadhu MJ; Systems Biology and Genome Engineering Section, Center for Genomics and Data Science Research, National Human Genome Research Institute, NIH, Bethesda, MD.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2024 Nov 26; Vol. 121 (48), pp. e2413743121. Date of Electronic Publication: 2024 Nov 21.
DOI:	10.1073/pnas.2413743121
Abstrakt:	Delineating a protein's essential and dispensable domains provides critical insight into how it carries out its function. Here, we developed a high-throughput method to synthesize and test the functionality of all possible in-frame and continuous deletions in a gene of interest, enabling rapid and unbiased determination of protein domain importance. Our approach generates precise deletions using a CRISPR library framework that is free from constraints of gRNA target site availability and efficacy. We applied our method to AcrIIA4, a phage-encoded anti-CRISPR protein that robustly inhibits SpCas9. Extensive structural characterization has shown that AcrIIA4 physically occupies the DNA-binding interfaces of several SpCas9 domains; nonetheless, the importance of each AcrIIA4 interaction for SpCas9 inhibition is unknown. We used our approach to determine the essential and dispensable regions of AcrIIA4. Surprisingly, not all contacts with SpCas9 were required, and in particular, we found that the AcrIIA4 loop that inserts into SpCas9's RuvC catalytic domain can be deleted. Our results show that AcrIIA4 inhibits SpCas9 primarily by blocking PAM binding and that its interaction with the SpCas9 catalytic domain is inessential. Competing Interests: Competing interests statement:The authors declare no competing interest.
Databáze:	MEDLINE
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