Target DNA recognition and cleavage by a reconstituted Type I-G CRISPR-Cas immune effector complex

详细信息    查看全文

• 作者：Sonali Majumdar ; Marianne Ligon ; William Colby Skinner ; Rebecca M. Terns…
• 关键词：CRISPR ; Cas ; Cst ; Cas3 ; Type I ; G ; Pyrococcus furiosus
• 刊名：Extremophiles
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：21
• 期：1
• 页码：95-107
• 全文大小：2452KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Microbiology; Biotechnology; Biochemistry, general; Microbial Ecology;
• 出版者：Springer Japan
• ISSN：1433-4909
• 卷排序：21

文摘

CRISPR-Cas immune systems defend prokaryotes against viruses and plasmids. CRISPR RNAs (crRNAs) associate with various CRISPR-associated (Cas) protein modules to form structurally and functionally diverse (Type I-VI) crRNP immune effector complexes. Previously, we identified three, co-existing effector complexes in Pyrococcus furiosus -Type I-A (Csa), Type I-G (Cst), and Type III-B (Cmr)—and demonstrated that each complex functions in vivo to eliminate invader DNA. Here, we reconstitute functional Cst crRNP complexes in vitro from recombinant Cas proteins and synthetic crRNAs and investigate mechanisms of crRNP assembly and invader DNA recognition and destruction. All four known Cst-affiliated Cas proteins (Cas5t, Cst1, Cst2, and Cas3) are required for activity, but each subunit plays a distinct role. Cas5t and Cst2 comprise a minimal set of proteins that selectively interact with crRNA. Further addition of Cst1, enables the four subunit crRNP (Cas5t, Cst1, Cst2, crRNA) to specifically bind complementary, double-stranded DNA targets and to recruit the Cas3 effector nuclease, which catalyzes cleavages at specific sites within the displaced, non-target DNA strand. Our results indicate that Type I-G crRNPs selectively bind target DNA in a crRNA and, protospacer adjacent motif dependent manner to recruit a dedicated Cas3 nuclease for invader DNA destruction.