Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity

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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)1199-1209.e6
JournalMolecular Cell
Issue number6
StatePublished - 17 Mar 2022


  • Cas12e
  • CasX
  • DNA cleavage
  • RNA-guided DNA nuclease
  • cryo-EM
  • genome editing
  • nucleic acid manipulation
  • sgRNA
  • structural engineering


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