DNA targeting by compact Cas9d and its resurrected ancestor

Rodrigo Fregoso Ocampo, Jack P. K. Bravo, Tyler L. Dangerfield, Isabel Nocedal, Samatar A. Jirde, Lisa M. Alexander, Nicole C. Thomas, Anjali Das, Sarah Nielson, Kenneth A. Johnson, Christopher T. Brown, Cristina N. Butterfield, Daniela S. A. Goltsman and David W. Taylor (dtaylor@utexas.edu)
Additional contact information
Rodrigo Fregoso Ocampo: University of Texas at Austin
Jack P. K. Bravo: University of Texas at Austin
Tyler L. Dangerfield: University of Texas at Austin
Isabel Nocedal: Inc.
Samatar A. Jirde: Inc.
Lisa M. Alexander: Inc.
Nicole C. Thomas: Inc.
Anjali Das: University of Texas at Austin
Sarah Nielson: University of Texas at Austin
Kenneth A. Johnson: University of Texas at Austin
Christopher T. Brown: Inc.
Cristina N. Butterfield: Inc.
Daniela S. A. Goltsman: Inc.
David W. Taylor: University of Texas at Austin

Nature Communications, 2025, vol. 16, issue 1, 1-16

Abstract: Abstract Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains for DNA recognition. The sgRNA can be truncated by up to ~25% yet still retain activity in vivo. Using ancestral sequence reconstruction, we generated compact nucleases capable of efficient genome editing in mammalian cells. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for future re-engineering of minimal RNA-guided DNA endonucleases.

Date: 2025
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DOI: 10.1038/s41467-024-55573-4

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