Improving integration efficiency and precision of CRISPR/Cas9-mediated genome editing

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) – CRISPR-associated protein 9 (Cas9) system has revolutionized the field of genome engineering, providing a cost-effective and fast tool for targeted gene modifications. Endogenous repair pathways, including error-prone non-homologous end joining and alternative end joining, or precise homology-directed repair (HDR), mend Cas9-induced DNA double-strand breaks. End joining repair can result in imprecise DNA insertions and deletions that may be used to disrupt gene function. At the same time, HDR enables accurate genomic alterations, such as gene knock-ins, by harnessing an exogenous DNA repair template carrying the desired genomic modification. Despite the potential benefits of the CRISPR-Cas9 system, two significant challenges can limit its application. As cells naturally favor end joining repair over HDR, the efficiency of precise genome engineering is often low. Furthermore, end joining repair pathways can cause deleterious on- and off-target effects. To gain a deeper understanding of the interplay between CRISPR/Cas9 and eukaryotic DNA damage repair pathways and to enhance the efficiency of precise modification and fidelity of CRISPR-centric genome editing, we devised several strategies: in Paper I, we developed a method to elucidate DNA repair mechanisms at Cas9-induced double strand breaks and identified a potent combination of compounds inhibiting the two major HDR-competing pathways. In Paper II, we advanced a Cas9 nuclease-based prime editing strategy, enabling the precise installation of small insertions at the cleavage site through a homology-independent mechanism. In Paper III, we established a co-selection strategy utilizing an endogenous selection marker to enrich edited cells with desired modifications. Lastly, in Paper IV, we discovered a natural, high-fidelity Cas9-orthologue that generates single-stranded DNA overhangs, facilitating non-homologous end joining- and HDR-mediated insertions at the target locus. These studies pave the way for drastically improved cell line generation protocols and potential applications for therapeutic gene editing.