An Intein-Mediated Split-nCas9 System for Base Editing in Plants

Guoliang Yuan; Haiwei Lu; Kuntal De; Md Mahmudul Hassan; Yang Liu; Yi Li; Wellington Muchero; Paul E. Abraham; Gerald A. Tuskan; Xiaohan Yang

doi:10.1021/acssynbio.1c00507

An Intein-Mediated Split-nCas9 System for Base Editing in Plants

Guoliang Yuan, Haiwei Lu, Kuntal De, Md Mahmudul Hassan, Yang Liu, Yi Li, Wellington Muchero, Paul E. Abraham, Gerald A. Tuskan, Xiaohan Yang

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

9 Scopus citations

Abstract

Virus-assisted delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system represents a promising approach for editing plant genomes. Among the CRISPR/Cas systems, CRISPR/Cas9 is most widely used; however, to pack the relatively large size of the CRISPR/Cas9 system into viral vectors with confined packaging capacity is challenging. To address this technical challenge, we developed a strategy based on split inteins that splits the required CRISPR/Cas9 components across a dual-vector system. The CRISPR/Cas reassembles into an active form following co-infection to achieve targeted genome editing in plant cells. An intein-mediated split system was adapted and optimized in plant cells by a successful demonstration of split-eYGFPuv expression. Using a plant-based biosensor, we demonstrated for the first time that the split-nCas9 can induce efficient base editing in plant cells. We identified several split sites for future biodesign strategies. Overall, this strategy provides new opportunities to bridge different CRISPR/Cas9 tools including base editor, prime editor, and CRISPR activation with virus-mediated gene editing.

Original language	English
Pages (from-to)	2513-2517
Number of pages	5
Journal	ACS Synthetic Biology
Volume	11
Issue number	7
DOIs	https://doi.org/10.1021/acssynbio.1c00507
State	Published - Jul 15 2022

Funding

This manuscript has been authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan ( http://energy.gov/downloads/doe-public-access-plan ). The manuscript is supported by the Center for Bioenergy Innovation (CBI), a U.S. Department of Energy (DOE) Research Center and the Secure Ecosystem Engineering and Design (SEED) project funded by the Genomic Science Program of the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (BER) as part of the Secure Biosystems Design Science Focus Area (SFA). Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. Department of Energy under Contract Number DE-AC05-00OR22725.

Funders	Funder number
Research Center
Secure Biosystems Design Science Focus Area
Secure Ecosystem Engineering and Design
U.S. Department of Energy
Office of Science
Biological and Environmental Research
Oak Ridge National Laboratory	DE-AC05-00OR22725
Center for Bioenergy Innovation
Southern Finance Association

Keywords

CRISPR/Cas9
base editing
biosensor
eYGFPuv
split-SpnCas9
transient gene expression

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10.1021/acssynbio.1c00507

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title = "An Intein-Mediated Split-nCas9 System for Base Editing in Plants",

abstract = "Virus-assisted delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system represents a promising approach for editing plant genomes. Among the CRISPR/Cas systems, CRISPR/Cas9 is most widely used; however, to pack the relatively large size of the CRISPR/Cas9 system into viral vectors with confined packaging capacity is challenging. To address this technical challenge, we developed a strategy based on split inteins that splits the required CRISPR/Cas9 components across a dual-vector system. The CRISPR/Cas reassembles into an active form following co-infection to achieve targeted genome editing in plant cells. An intein-mediated split system was adapted and optimized in plant cells by a successful demonstration of split-eYGFPuv expression. Using a plant-based biosensor, we demonstrated for the first time that the split-nCas9 can induce efficient base editing in plant cells. We identified several split sites for future biodesign strategies. Overall, this strategy provides new opportunities to bridge different CRISPR/Cas9 tools including base editor, prime editor, and CRISPR activation with virus-mediated gene editing.",

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author = "Guoliang Yuan and Haiwei Lu and Kuntal De and Hassan, {Md Mahmudul} and Yang Liu and Yi Li and Wellington Muchero and Abraham, {Paul E.} and Tuskan, {Gerald A.} and Xiaohan Yang",

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doi = "10.1021/acssynbio.1c00507",

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AU - Lu, Haiwei

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AU - Hassan, Md Mahmudul

AU - Liu, Yang

AU - Li, Yi

AU - Muchero, Wellington

AU - Abraham, Paul E.

AU - Tuskan, Gerald A.

AU - Yang, Xiaohan

PY - 2022/7/15

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AB - Virus-assisted delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system represents a promising approach for editing plant genomes. Among the CRISPR/Cas systems, CRISPR/Cas9 is most widely used; however, to pack the relatively large size of the CRISPR/Cas9 system into viral vectors with confined packaging capacity is challenging. To address this technical challenge, we developed a strategy based on split inteins that splits the required CRISPR/Cas9 components across a dual-vector system. The CRISPR/Cas reassembles into an active form following co-infection to achieve targeted genome editing in plant cells. An intein-mediated split system was adapted and optimized in plant cells by a successful demonstration of split-eYGFPuv expression. Using a plant-based biosensor, we demonstrated for the first time that the split-nCas9 can induce efficient base editing in plant cells. We identified several split sites for future biodesign strategies. Overall, this strategy provides new opportunities to bridge different CRISPR/Cas9 tools including base editor, prime editor, and CRISPR activation with virus-mediated gene editing.

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An Intein-Mediated Split-nCas9 System for Base Editing in Plants

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