TY - JOUR
T1 - CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells
AU - Kluesner, Mitchell G.
AU - Lahr, Walker S.
AU - Lonetree, Cara lin
AU - Smeester, Branden A.
AU - Qiu, Xiaohong
AU - Slipek, Nicholas J.
AU - Claudio Vázquez, Patricia N.
AU - Pitzen, Samuel P.
AU - Pomeroy, Emily J.
AU - Vignes, Madison J.
AU - Lee, Samantha C.
AU - Bingea, Samuel P.
AU - Andrew, Aneesha A.
AU - Webber, Beau R.
AU - Moriarity, Branden S.
N1 - 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).
PY - 2021/12/1
Y1 - 2021/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41467-021-22009-2
DO - 10.1038/s41467-021-22009-2
M3 - Article
C2 - 33893286
AN - SCOPUS:85104854203
SN - 2041-1723
VL - 12
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 2437
ER -