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TitleChimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.
Journal, issue, pagesMol Cell, Vol. 82, Issue 6, Page 1199-11209.e6, Year 2022
Publish dateMar 17, 2022
AuthorsConnor 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 AbstractA 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.
External linksMol Cell / PubMed:35219382 / PubMed Central
MethodsEM (single particle)
Resolution2.9 - 3.7 Å
Structure data

32389

7way

EMDB-32389, PDB-7way:
PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state
Method: EM (single particle) / Resolution: 2.9 Å

32390

7waz

EMDB-32390, PDB-7waz:
PlmCasX-sgRNAv1-dsDNA ternary complex at ts loading state
Method: EM (single particle) / Resolution: 3.4 Å

32391

7wb0

EMDB-32391, PDB-7wb0:
PlmCasX-sgRNAv1-dsDNA ternary complex at nts loading state with flexible H2 domain
Method: EM (single particle) / Resolution: 3.2 Å

32392

7wb1

EMDB-32392, PDB-7wb1:
PlmCasX-sgRNAv2-dsDNA ternary complex at nts loading state
Method: EM (single particle) / Resolution: 3.7 Å

Source
  • Planctomycetes (bacteria)
  • planctomycetes bacterium (bacteria)
KeywordsRNA 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

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