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 - 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.
UR - http://www.scopus.com/inward/record.url?scp=85104854203&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85104854203&partnerID=8YFLogxK
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 -