Intron-Based Single Transcript Unit CRISPR Systems for Plant Genome Editing
Zhaohui Zhong,
Shishi Liu,
Xiaopei Liu,
Binglin Liu,
Xu Tang,
Qiurong Ren,
Jianping Zhou,
Xuelian Zheng,
Yiping Qi,
Yong Zhang
Affiliations
Zhaohui Zhong
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Shishi Liu
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Xiaopei Liu
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Binglin Liu
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Xu Tang
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Qiurong Ren
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Jianping Zhou
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Xuelian Zheng
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Yiping Qi
Department of Plant Science and Landscape Architecture, University of Maryland
Yong Zhang
Department of Biotechnology, School of Life Sciences and Technology, Center for Informational Biology, University of Electronic Science and Technology of China
Abstract Background Expression of either Cas9 or Cas12a and guide RNAs by a single Polymerase II (Pol II) promoter represents a compact CRISPR expression system and has many advantages for different applications. In order to make this system routine in plant biology, engineering efforts are needed for developing and optimizing such single transcript unit (STU) systems for plant genome editing. Results To develop novel intron-based STU (iSTU) CRISPR system (STU CRISPR 3.0), we first evaluated three introns from three plant species for carrying guide RNAs by using an enhanced green fluorescence protein (eGFP) system in rice. After validation of proper intron slicing, we inserted these gRNA-containing introns into the open reading frames (ORFs) of Cas9 and Cas12a for testing their genome editing capability. Different guide RNA processing strategies have been tested for Cas9 and Cas12a. We demonstrated singular genome editing and multiplexed genome editing with these iSTU-Cas9 and iSTU-Cas12a systems. Conclusion We developed multiple iSTU-CRISPR/Cas9 and Cas12a systems for plant genome editing. Our results shed light on potential directions for further improvement of the iSTU systems.
