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

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.

Base Editors for Citrus Gene Editing

“…Both ABEs and CBEs can modify cis -regulatory elements to fine-tune gene functions. They can also be utilized to mutate start codon ATG to interrupt protein translation ( Kluesner et al, 2021 ; Molla et al, 2021 ).…”

mRNA-mediated delivery of gene editing tools to human primary muscle stem cells

“…ABE-mediated disruption of splice donors has been shown to correlate well with protein loss. 25 Skipping of NCAM1 exon 7 from the mRNA should induce a frameshift with the subsequent introduction of a premature stop codon and nonsense-mediated mRNA decay. However, the mutation of this splice donor site resulted in only a minor reduction in the percentage of NCAM1-expressing cells.…”

The use of base editing technology to characterize single nucleotide variants

“…Base editing has been implemented to generate gene knockouts by introducing premature stop codons [9] , [50] or disrupting splice sites [43] . SNVs induced to cause premature stop codons can achieve gene knockouts with higher predictability and efficiency compared to NHEJ or HDR methods for gene knockouts [50] .…”