Programming PAM antennae for efficient CRISPR-Cas9 DNA editing
- 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
- 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.
- Research Organization:
- Univ. 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, Vol. 6, Issue 19; ISSN 2375-2548
- Publisher:
- AAASCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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