citations

                    Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways
                

Authors:

Schimmel J, Muñoz-Subirana N, Kool H, van Schendel R, van der Vlies S, Kamp JA, de Vrij FMS, Kushner SA, Smith GCM, Boulton SJ, Tijsterman M.

In:

Source: Cell Rep
Publication Date: (2023)
Issue: 42(2): 112019

Research Area:

Cancer Research/Cell Biology

Stem Cells

Basic Research

Molecular Biology

Cells used in publication:

Induced Pluripotent Stem Cell (iPS), human: Species: human; Tissue Origin:

Platform:

4D-Nucleofector® X-Unit

Experiment

For the HDR experiment in human iPS cells, a 60–70% confluent well of a 6-well plate of undifferentiated hiPSC colonies was incubated with RevitaCell Supplement (Gibco) and indicated inhibitors or DMSO 1 hour prior to nucleofection. The ribonucleoprotein complex (RNP) was assembled by combining Alt-R S.p.Cas9-GFP V3 with sgRNA consisting of Alt-R crRNA and tracrRNA (all from IDT and according to the manufacturer’s protocol). Sequences of sgRNA and ssODN2 template for GFP to BFP conversion were described previously (also see Table S1). The RNP, 1 mM ssODN (IDT Alt-R HDR Donor Oligo) and 2 mM NU7441 and/or 10 mM ART588 were added to the nucleofection solution of the P3 Primary Cell 4D-Nucleofector X Kit (Lonza, V4XP-3024). hiPSCs were lifted from the plate using StemPro Accutase Cell Dissociation Reagent (Gibco) and nucleofected using program CA-137 of the Lonza 4D-Nucleofector X Unit. The nucleofected cells were transferred to one well of a 6-well plate per condition and expanded in presence of inhibitors or DMSO for one week. DNA was isolated using the DNeasy Blood and Tissue kit (Qiagen).

Abstract

Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Pol?) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.

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