Identification of functional sgRNA mutants lacking canonical secondary structure using high-throughput FACS screening
Zeyu Liang,
Chaoyong Huang,
Yan Xia,
Zhaojin Ye,
Shunhua Fan,
Junwei Zeng,
Shuyuan Guo,
Xiaoyan Ma,
Lichao Sun,
Yi-Xin Huo
Affiliations
Zeyu Liang
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
Chaoyong Huang
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
Yan Xia
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
Zhaojin Ye
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
Shunhua Fan
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
Junwei Zeng
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
Shuyuan Guo
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
Xiaoyan Ma
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China; Beijing Institute of Technology (Tangshan) Translational Research Center, Hebei 063611, China
Lichao Sun
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China; Beijing Institute of Technology (Tangshan) Translational Research Center, Hebei 063611, China
Yi-Xin Huo
Key Laboratory of Molecular Medicine and Biotherapy, Aerospace Center Hospital, School of Life Science, Beijing Institute of Technology, Beijing 100081, China; Beijing Institute of Technology (Tangshan) Translational Research Center, Hebei 063611, China; Corresponding author
Summary: Coexpressing multiple identical single guide RNAs (sgRNAs) in CRISPR-dependent engineering triggers genetic instability and phenotype loss. To provide sgRNA derivatives for efficient DNA digestion, we design a high-throughput digestion-activity-dependent positive screening strategy and astonishingly obtain functional nonrepetitive sgRNA mutants with up to 48 out of the 61 nucleotides mutated, and these nonrepetitive mutants completely lose canonical secondary sgRNA structure in simulation. Cas9-sgRNA complexes containing these noncanonical sgRNAs maintain wild-type level of digestion activities in vivo, indicating that the Cas9 protein is compatible with or is able to adjust the secondary structure of sgRNAs. Using these noncanonical sgRNAs, we achieve multiplex genetic engineering for gene knockout and base editing in microbial cell factories. Libraries of strains with rewired metabolism are constructed, and overproducers of isobutanol or 1,3-propanediol are identified by biosensor-based fluorescence-activated cell sorting (FACS). This work sheds light on the remarkable flexibility of the secondary structure of functional sgRNA.
