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

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

Abstract: 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.

Date: 2021
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Persistent link: https://EconPapers.repec.org/RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-22009-2

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DOI: 10.1038/s41467-021-22009-2

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