Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells

Greta Bigelyte, Joshua K. Young (), Tautvydas Karvelis (), Karolina Budre, Rimante Zedaveinyte, Vesna Djukanovic, Elizabeth Ginkel, Sushmitha Paulraj, Stephen Gasior, Spencer Jones, Lanie Feigenbutz, Grace St. Clair, Pierluigi Barone, Jennifer Bohn, Ananta Acharya, Gina Zastrow-Hayes, Selgar Henkel-Heinecke, Arunas Silanskas, Ralf Seidel and Virginijus Siksnys ()
Additional contact information
Greta Bigelyte: Vilnius University
Joshua K. Young: Corteva Agriscience™
Tautvydas Karvelis: Vilnius University
Karolina Budre: Vilnius University
Rimante Zedaveinyte: Vilnius University
Vesna Djukanovic: Corteva Agriscience™
Elizabeth Ginkel: Corteva Agriscience™
Sushmitha Paulraj: Corteva Agriscience™
Stephen Gasior: Corteva Agriscience™
Spencer Jones: Corteva Agriscience™
Lanie Feigenbutz: Corteva Agriscience™
Grace St. Clair: Corteva Agriscience™
Pierluigi Barone: Corteva Agriscience™
Jennifer Bohn: Corteva Agriscience™
Ananta Acharya: Corteva Agriscience™
Gina Zastrow-Hayes: Corteva Agriscience™
Selgar Henkel-Heinecke: Leipzig University
Arunas Silanskas: Vilnius University
Ralf Seidel: Leipzig University
Virginijus Siksnys: Vilnius University

Nature Communications, 2021, vol. 12, issue 1, 1-8

Abstract: Abstract Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools.

Date: 2021
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DOI: 10.1038/s41467-021-26469-4

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