Abstract
We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000–100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30–50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16–18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.
Highlights
Gene-editing technologies, which are applicable in science as well as medicine[1], include the use of zinc-finger nucleases (ZFNs2–4), transcription activator-like effector nucleases (TALENs4–7) and the RNA-guided CRISPR/ Cas[9] system[1,8,9,10]
Complete bi-allelic inactivation of DPH1 in MCF7 cells prevents the synthesis of the toxin target diphthamide, which renders cells resistant to DT25
In combination with a donor plasmid containing a promoter-less expression cassette encoding the enzyme puromycin N-acetyltransferase (Pac) flanked by DPH1 homology arms, DPH1 gene inactivation can result from the homology-directed repair of DNA double-strand breaks
Summary
Gene-editing technologies, which are applicable in science as well as medicine[1], include the use of zinc-finger nucleases (ZFNs2–4), transcription activator-like effector nucleases (TALENs4–7) and the RNA-guided CRISPR/ Cas[9] system[1,8,9,10]. Effective and robust methods for the characterization and comparison of editing technologies are essential for applications in R&D and the development of editing-based therapies Such evaluations comprise analyses and comparisons of the efficacy as well as the specificity of target gene destruction and productive transgene integration. A prerequisite for optimizing gene editing is the reliable and robust detection and differentiation of mono- and bi-allelic gene inactivation as well as nonspecific and targeted integration events. Existing methods, such as the determination of phenotypes caused by insertions (e.g., drug resistance) or a lack of phenotypes (gene inactivation) or sequencing approaches, frequently do not differentiate mono- and bi-allelic inactivation. The simplicity and robustness of the method facilitate the optimization of gene-editing procedures as well as the identification and comparison of gene-editing modulators
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