citations

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

Authors:

Kluesner MG, Lahr WS, Lonetree CL, Smeester BA, Qiu X, Slipek NJ, Claudio Vázquez PN, Pitzen SP, Pomeroy EJ, Vignes MJ, Lee SC, Bingea SP, Andrew AA, Webber BR, Moriarity BS

In:

Source: Nature
Publication Date: (2021)
Issue: 12(1): 2437

Cells used in publication:

K-562: Species: human; Tissue Origin: blood
T cell, human stim.: Species: human; Tissue Origin: blood

Platform:

4D-Nucleofector® X-Unit

Experiment

Forty-eight hours following T cell activation, Dynabeads were magnetically removed, cells were washed once with PBS, spun down, and resuspended in P3 Primary Cell Nucleofector TM Solution containing Supplement 1 (Lonza, Basel, Switzerland). Ten million T cells were combined with 1 µg of chemically modified sgRNA (Synthego, Menlo Park, CA) and 1.5 µg codon optimized BE4 mRNA (TriLink Biotechnologies, San Diego, CA), codon optimized ABE7.10 mRNA (TriLink Biotechnologies), or codon optimized SpCas9 mRNA (TriLink Biotechnologies). T cells were electroporated with the 4D-Nucleofector system (Lonza) using a P3 16-well Nucleocuvette kit, with 1 × 10^6 T cells per 20 µL cuvette using the program EO-115.

On the day of electroporation, 2 × 10^5 K562 cells were resuspended in SE buffer with Supplement 1 and combined with 1 µg of chemically modified sgRNA, and 1.5 µg of enzyme mRNA. Cells were electroporated with the 4D-Nucleofector system using a SE 16- well Nucleocuvette kit, with 2 × 10^5 cells per 20 µL cuvette using the program FF-120.

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.

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