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CRISPR RNA-guided autonomous delivery of Cas9

Abstract

Cas9 is an endonuclease that can be programed to autonomously deliver diverse effectors to specified genetic addresses. High-resolution structures of this protein and its associated CRISPR RNA guide explain the molecular mechanisms of CRISPR-RNA-guided DNA recognition and provide a molecular blueprint that has facilitated structure-guided functional remodeling. Here we retrace events that led from early efforts to understand the central role of Cas9 in CRISPR-mediated adaptive immunity to contemporary efforts aimed at developing and deploying this enzyme for programmable genetic editing.

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Fig. 1: The impact of sgRNA-loading and target DNA-binding on Cas9 structure.
Fig. 2: Engineering Cas9 to change PAM specificity.
Fig. 3: Cas9 for autonomous delivery of diverse effectors.
Fig. 4: Structure-guided design of Cas9 for enhanced target-specificity.

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Acknowledgements

Research in the Wiedenheft lab is supported by the National Institutes of Health (P20GM103500, P30GM110732, R01GM110270, R01GM108888, and R21 AI130670), the National Science Foundation EPSCoR (EPS-110134), the M. J. Murdock Charitable Trust, a young investigator award from Amgen, and the Montana State University Agricultural Experimental Station (USDA NIFA).

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Correspondence to Blake Wiedenheft.

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B.W. is the founder of SurGene LLC and is an inventor on patent applications related to CRISPR-Cas systems and applications thereof.

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Supplementary Information

Supplementary Video 1

Ligand-induced conformational changes of Cas9. The movie shows a series of conformational changes starting with a structure of Cas9 protein from Streptococcus pyogenes (SpCas9, PDB ID 4CMP) that transitions to the sgRNA-bound conformation (4ZT0), the ssDNA bound state (4008), PAM-bound state (4UN3), and finally to a complex that includes dsDNA + PAM (5F9R). Positions of the HNH and RuvC nuclease domains and their active sites are highlighted. The movie was created using ChimeraX (Protein Sci. 27, 14–25, 2018).

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Wilkinson, R.A., Martin, C., Nemudryi, A.A. et al. CRISPR RNA-guided autonomous delivery of Cas9. Nat Struct Mol Biol 26, 14–24 (2019). https://doi.org/10.1038/s41594-018-0173-y

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