CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells

Mitchell G. Kluesner, Walker S. Lahr, Cara lin Lonetree, Branden A. Smeester, Xiaohong Qiu, Nicholas J. Slipek, Patricia N. Claudio Vázquez, Samuel P. Pitzen, Emily J. Pomeroy, Madison J. Vignes, Samantha C. Lee, Samuel P. Bingea, Aneesha A. Andrew, Beau R. Webber, Branden S. Moriarity

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

CRISPR-Cas9 cytidine and adenosine base editors (CBEs and ABEs) can disrupt genes without introducing double-stranded breaks by inactivating splice sites (BE-splice) or by introducing premature stop (pmSTOP) codons. However, no in-depth comparison of these methods or a modular tool for designing BE-splice sgRNAs exists. To address these needs, we develop SpliceR (http://z.umn.edu/spliceR) to design and rank BE-splice sgRNAs for any Ensembl annotated genome, and compared disruption approaches in T cells using a screen against the TCR-CD3 MHC Class I immune synapse. Among the targeted genes, we find that targeting splice-donors is the most reliable disruption method, followed by targeting splice-acceptors, and introducing pmSTOPs. Further, the CBE BE4 is more effective for disruption than the ABE ABE7.10, however this disparity is eliminated by employing ABE8e. Collectively, we demonstrate a robust method for gene disruption, accompanied by a modular design tool that is of use to basic and translational researchers alike.

Original languageEnglish (US)
Article number2437
JournalNature communications
Volume12
Issue number1
DOIs
StatePublished - Apr 23 2021

Bibliographical note

Funding Information:
We thank Dr. Matthew Johnson at the University of Minnesota for discussions surrounding flow cytometry. We thank Dr. Kevin Holden at Synthego for insight about chemically modified sgRNAs. This work was funded by the Children's Cancer Research Fund,NIH grant R03AI144840, and the University of Minnesota Academic Investment Research Program.

Publisher Copyright:
© 2021, The Author(s).

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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