Programming PAM antennae for efficient CRISPR-Cas9 DNA editing

Wang, Fei; Hao, Yaya; Li, Qian; Li, Jiang; Zhang, Honglu; Zhang, Xueli; Wang, Lihua; Bustamante, Carlos; Fan, Chunhai

doi:10.1126/sciadv.aay9948

Programming PAM antennae for efficient CRISPR-Cas9 DNA editing

Journal Article · Fri May 08 00:00:00 EDT 2020 · Science Advances

DOI:https://doi.org/10.1126/sciadv.aay9948· OSTI ID:1816260

^[1]; ^[2]; Li, Qian ^[2]; ^[3]; Zhang, Honglu ^[4]; Zhang, Xueli ^[5]; Wang, Lihua ^[6]; ^[7]; ^[2]

Southern Medical Univ. Affiliated Fengxian Hospital, Shanghai (China). Shanghai Jiao Tong University Affiliated Sixth People's Hospital South Campus. Joint Research Center for Precision Medicine; Shanghai Jiao Tong Univ., Shanghai (China). Renji Hospital. School of Medicine. Frontiers Science Center of Transformative Molecules. Inst. of Molecular Medicine. School of Chemistry and Chemical Engineering; OSTI
Shanghai Jiao Tong Univ., Shanghai (China). Renji Hospital. School of Medicine. Frontiers Science Center of Transformative Molecules. Inst. of Molecular Medicine. School of Chemistry and Chemical Engineering
Chinese Academy of Sciences, Shanghai (China). Shanghai Inst. of Applied Physics. CAS Key Lab. of Interfacial Physics and Technology, Shanghai Advanced Research Inst. Shanghai Synchrotron Radiation Facility. Zhangjiang Lab.
Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst. Kavli Energy Nanosciences Inst. at Berkeley. Dept. of Molecular and Cell Biology. Dept. of Physics. Inst. for Quantitative Biosciences (QB3). Dept. of Chemistry
Joint Research Center for Precision Medicine, Shanghai Jiao Tong University Affiliated Sixth People's Hospital South Campus, Southern Medical University Affiliated Fengxian Hospital, Shanghai 201499, China.
Chinese Academy of Sciences, Shanghai (China). Shanghai Inst. of Applied Physics. CAS Key Lab. of Interfacial Physics and Technology; East China Normal Univ., Shanghai (China). School of Chemistry and Molecular Engineering. Shanghai Key Lab. of Green Chemistry and Chemical Processes
Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst. Kavli Energy Nanosciences Inst. at Berkeley. Dept. of Molecular and Cell Biology. Dept. of Physics. Inst. for Quantitative Biosciences (QB3). Dept. of Chemistry

Bacterial CRISPR-Cas9 nucleases have been repurposed as powerful genome editing tools. Whereas engineering guide RNAs or Cas nucleases have proven to improve the efficiency of CRISPR editing, modulation of protospacer-adjacent motif (PAM), indispensable for CRISPR, has been less explored. Here, we develop a DNA origami–based platform to program a PAM antenna microenvironment and address its performance at the single-molecule level with submolecular resolution. To mimic spatially controlled in vivo PAM distribution as may occur in chromatin, we investigate the effect of PAM antennae surrounding target DNA. We find that PAM antennae effectively sensitize the DNA cleavage by recruiting Cas9 molecules. Super-resolution tracking of single single-guide RNA/Cas9s reveals localized translocation of Cas9 among spatially proximal PAMs. We find that the introduction of the PAM antennae effectively modulates the microenvironment for enhanced target cleavage (up to ~50%). These results provide insight into factors that promote more efficient genome editing.

View Accepted Manuscript (DOE)

Research Organization:: University of California, Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1816260

Journal Information:: Science Advances, Journal Name: Science Advances Journal Issue: 19 Vol. 6; ISSN 2375-2548

Publisher:: AAASCopyright Statement

Country of Publication:: United States

Language:: English

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