An Intein-Mediated Split–nCas9 System for Base Editing in Plants
[1];
[1]
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States, The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States
- Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States, The Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States, Department of Genetics and Plant Breeding, Patuakhali Science and Technology University, Dumki, Patuakhali 8602, Bangladesh
- Department of Plant Science and Landscape Architecture, University of Connecticut, Storrs, Connecticut 06269, United States
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.
- Research Organization:
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Grant/Contract Number:
- AC05-00OR22725
- OSTI ID:
- 1876223
- Alternate ID(s):
- OSTI ID: 1876333
- Journal Information:
- ACS Synthetic Biology, Journal Name: ACS Synthetic Biology Vol. 11 Journal Issue: 7; ISSN 2161-5063
- Publisher:
- American Chemical SocietyCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Similar Records
CRISPR/Cas9-based gene activation and base editing in Populus
eYGFPuv-Assisted Transgenic Selection in Populus deltoides WV94 and Multiplex Genome Editing in Protoplasts of P. trichocarpa × P. deltoides Clone ‘52-225’