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Jun-Jie Gogo Liu group and Jennifer Doudna group develop a highly efficient mini-CRISPR gene editing tool


  The programmability of CRISPR systems holds tremendous potential as transformative tools for genome or epic-genome editing. After years of effort, only a few types of CRISPR-Cas nucleases have been widely used for efficient genome editing, such as Cas9 and Cas12a. While efficient for genome editing, the large size of Cas9 and Cas12a (1,000-1,500 amino acids (aa)) precludes their ability to be delivered via adeno-associated virus (AAV), which is useful for therapeutic delivery but has a limited transgene size of just 4.7 kilobase pairs (kbp). Also, most Cas9 and Cas12a nucleases originate from pathogenic bacteria and show severe off-target effects while used as exogenous gene editing tool.


Figure 1, The three structural states within PlmCasX ternary complex.

  Gogo group has been focused on exploring the molecular mechanism and application development of different nuclease systems. In 2019, Gogo and collaborators reported a mini-CRISPR CasX (<1000aa) originating from non-pathogenic bacteria which works for mammalian genome editing (Liu J.J. et al., Nature, 2019). Via structural study, Gogo and collaborators also revealed that CasX uses a single nuclease domain for double stranded DNA cleavage. This DNA cleavage mechanism is highly different from Cas9 and Cas12a. While so far, all reported small Cas nucleases, including CasX, CasPhi and Cas12f, show relatively low editing efficacy.

Figure 2, Editing efficacy by CasX before and after engineering. The engineered CasX tool shows up to 90.5% editing in our fluorescent reporter assay (A) and 56.1% editing at an endogenous human gene (B).

  By collaboration with Jennifer Doudna group in UC Berkeley, Gogo group published their most recent result titled “Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity in Molecular Cell. In this work, Gogo group reveals that structural features of two CasX homologues 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. Notably, PlmCasX-R1-v2 shows up to 90.5% editing in our fluorescent reporter assay and 56.1% editing at an endogenous human gene. These results advance both the mechanistic understanding of CasX and its application as a genome editing tool.

  Jun-Jie Gogo Liu (Assistant professor, Tsinghua) and Jennifer Doudna (Professor, UC Berkeley) hold the corresponding credit for this work. Shouyue Zhang (Postdoc fellow in ICSB, Tsinghua), Yuqian Zhao (Postdoc scholar, Tsinghua), Connor Tsuchida (Ph.D student, UC Berkeley) and Mohammad Saffarri Doost ( M.D. student, UC Davis) are the co-first authors.  Eva Nogales (Professor, LBNL), Xiao-Wei Chen (Professor, Peking University), David Burstein (Assistant professor, Tel Aviv University), Yi Xue (Assistant professor, Tsinghua) and Xianyang Fang (Assistant professor, Tsinghua) essentially contributed to this work. EM data were collected at the Tsinghua Cryo-EM facility and the Cal-Cryo facility at UC Berkeley.  The data were analyzed using the Bio-Computation platform at the Tsinghua University Branch of the Chinese National Center for Protein Sciences (Beijing). This project was supported by NSFC (project no. 32150018) and Chunfeng Fund (project no. 2021Z99CFY020) and start-up funds from Tsinghua University, Beijing (J.J.G.L.)




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