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

doi:10.1016/j.molcel.2022.02.002

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

The Shmunis School of Biomedicine and Cancer Research

Research output: Contribution to journal › Article › peer-review

Abstract

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 language	English
Pages (from-to)	1199-1209.e6
Journal	Molecular Cell
Volume	82
Issue number	6
DOIs	https://doi.org/10.1016/j.molcel.2022.02.002
State	Published - 17 Mar 2022

Keywords

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

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10.1016/j.molcel.2022.02.002

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Tsuchida, C. A., Zhang, S., Doost, M. S., Zhao, Y., Wang, J., O'Brien, E., Fang, H., Li, C. P., Li, D., Hai, Z. Y., Chuck, J., Brötzmann, J., Vartoumian, A., Burstein, D., Chen, X. W., Nogales, E., Doudna, J. A., & Liu, J. J. G. (2022). Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity. Molecular Cell, 82(6), 1199-1209.e6. https://doi.org/10.1016/j.molcel.2022.02.002

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title = "Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity",

abstract = "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.",

keywords = "CRISPR, Cas12e, CasX, DNA cleavage, RNA-guided DNA nuclease, cryo-EM, genome editing, nucleic acid manipulation, sgRNA, structural engineering",

author = "Tsuchida, {Connor A.} and Shouyue Zhang and Doost, {Mohammad Saffari} and Yuqian Zhao and Jia Wang and Elizabeth O'Brien and Huan Fang and Li, {Cheng Ping} and Danyuan Li and Hai, {Zhuo Yan} and Jonathan Chuck and Julian Br{\"o}tzmann and Araz Vartoumian and David Burstein and Chen, {Xiao Wei} and Eva Nogales and Doudna, {Jennifer A.} and Liu, {Jun Jie Gogo}",

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year = "2022",

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doi = "10.1016/j.molcel.2022.02.002",

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Tsuchida, CA, Zhang, S, Doost, MS, Zhao, Y, Wang, J, O'Brien, E, Fang, H, Li, CP, Li, D, Hai, ZY, Chuck, J, Brötzmann, J, Vartoumian, A, Burstein, D, Chen, XW, Nogales, E, Doudna, JA & Liu, JJG 2022, 'Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity', Molecular Cell, vol. 82, no. 6, pp. 1199-1209.e6. https://doi.org/10.1016/j.molcel.2022.02.002

TY - JOUR

T1 - Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity

AU - Tsuchida, Connor A.

AU - Zhang, Shouyue

AU - Doost, Mohammad Saffari

AU - Zhao, Yuqian

AU - Wang, Jia

AU - O'Brien, Elizabeth

AU - Fang, Huan

AU - Li, Cheng Ping

AU - Li, Danyuan

AU - Hai, Zhuo Yan

AU - Chuck, Jonathan

AU - Brötzmann, Julian

AU - Vartoumian, Araz

AU - Burstein, David

AU - Chen, Xiao Wei

AU - Nogales, Eva

AU - Doudna, Jennifer A.

AU - Liu, Jun Jie Gogo

PY - 2022/3/17

Y1 - 2022/3/17

N2 - 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.

AB - 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.

KW - CRISPR

KW - Cas12e

KW - CasX

KW - DNA cleavage

KW - RNA-guided DNA nuclease

KW - cryo-EM

KW - genome editing

KW - nucleic acid manipulation

KW - sgRNA

KW - structural engineering

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AN - SCOPUS:85126272718

SN - 1097-2765

VL - 82

SP - 1199-1209.e6

JO - Molecular Cell

JF - Molecular Cell

IS - 6

ER -

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

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Keywords

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