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

Autor: Kluesner MG; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA., Lahr WS; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA., Lonetree CL; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA., Smeester BA; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA., Qiu X; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA., Slipek NJ; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA., Claudio Vázquez PN; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA.; Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA., Pitzen SP; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.; Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA., Pomeroy EJ; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA., Vignes MJ; College of Biological Sciences, University of Minnesota, Minneapolis, MN, USA., Lee SC; Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA.; College of Biological Sciences, University of Minnesota, Minneapolis, MN, USA., Bingea SP; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA., Andrew AA; Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA.; College of Biological Sciences, University of Minnesota, Minneapolis, MN, USA., Webber BR; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. webb0178@umn.edu.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. webb0178@umn.edu.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. webb0178@umn.edu.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA. webb0178@umn.edu., Moriarity BS; Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu.; Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu.; Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu.; Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2021 Apr 23; Vol. 12 (1), pp. 2437. Date of Electronic Publication: 2021 Apr 23.
DOI: 10.1038/s41467-021-22009-2
Abstrakt: 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.
Databáze: MEDLINE
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