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-Structure paper
タイトル | Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity. |
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ジャーナル・号・ページ | Mol Cell, Vol. 82, Issue 6, Page 1199-11209.e6, Year 2022 |
掲載日 | 2022年3月17日 |
著者 | Connor A Tsuchida / Shouyue Zhang / Mohammad Saffari Doost / Yuqian Zhao / Jia Wang / Elizabeth O'Brien / Huan Fang / Cheng-Ping Li / Danyuan Li / Zhuo-Yan Hai / Jonathan Chuck / Julian Brötzmann / Araz Vartoumian / David Burstein / Xiao-Wei Chen / Eva Nogales / Jennifer A Doudna / Jun-Jie Gogo Liu / |
PubMed 要旨 | A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool. |
リンク | Mol Cell / PubMed:35219382 / PubMed Central |
手法 | EM (単粒子) |
解像度 | 2.9 - 3.7 Å |
構造データ | EMDB-32389, PDB-7way: EMDB-32390, PDB-7waz: EMDB-32391, PDB-7wb0: EMDB-32392, PDB-7wb1: |
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キーワード | RNA BINDING PROTEIN/RNA/DNA / CRISPR / CasX / sgRNA / R-loop complex / RNA BINDING PROTEIN-DNA-RNA complex / RNA BINDING PROTEIN / DNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex / RNA BINDING PROTEIN-DNA-RNA |