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Characterizing the Function of Anti‐CRISPR protein AcrIIA11
Author(s) -
del Banco Serena Bautista,
Kaiser Brett,
Forsberg Kevin,
Malik Harmit,
Stoddard Barry
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.09175
Subject(s) - crispr , cas9 , biology , function (biology) , dna , computational biology , bacteria , cleavage (geology) , genetics , microbiology and biotechnology , gene , paleontology , fracture (geology)
Bacteria and the viruses that infect them are engaged in an evolutionary arms race that has driven the development of bacterial defense mechanisms and phage countermeasures to those defense mechanisms. One instance of this evolutionary competition is the development of the CRISPR‐Cas system, which enables the recognition and destruction of phage DNA. In response to this defense, phages have evolved anti‐CRISPR proteins that inhibit CRISPR‐Cas activity and promote phage infection. It is thought that many anti‐CRISPR proteins exist in nature, but only a handful have been identified and their mechanism of inhibition characterized. The goal of this project is to understand the mechanism by which a recently identified anti‐CRISPR protein, AcrIIA11, inhibits Streptococcus pyogenes Cas9 (SpyCas9). To address this, we have performed in vitro DNA cleavage and protein binding assays with purified components. We have determined the following: AcrIIA11 effectively inhibits Cas9 cleavage at an AcrIIA11: Cas9 ratio of 8:1; AcrIIA11 interacts directly with the Cas9 protein; homologs of AcrIIA11 show a range of ability to inhibit Cas9 activity but do no inhibit as well as AcrIIA11; and the C‐terminus of AcrIIA11 is not required for binding to Cas9. In future studies, we will test AcrIIA11 interaction with a wider range of Cas9 orthologs. From these results, we have gained a broader understanding of how AcrIIA11 interacts with SpyCas9. Support or Funding Information Seattle University Summer Undergraduate Fellowship (Peach Foundation)