Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells

Abstract

CRISPR/Cas9 is an efficient customizable nuclease to generate double-strand breaks (DSBs) in the genome. This process results in knockout of the targeted gene or knock-in of a specific DNA fragment at the targeted locus in the genome of various species. However, efficiency of knock-in mediated by homology-directed repair (HDR) pathway is substantially lower compared with the efficiency of knockout mediated by the nonhomologous end-joining (NHEJ) pathway. Suppressing NHEJ pathway or enhancing HDR pathway has been proven to enhance the nuclease-mediated knock-in efficiency in cultured cells and model organisms. We here investigated the effect of small molecules, Scr7, L755507 and resveratrol, on promoting HDR efficiency in porcine fetal fibroblasts. Results from eGFP reporter assay showed that these small molecules could increase the HDR efficiency by 2–3-fold in porcine fetal fibroblasts. When transfecting with the homologous template DNA and CRISPR/Cas9 plasmid and treating with small molecules, the rate of knock-in porcine fetal fibroblast cell lines with large DNA fragment integration could reach more than 50% of the screened cell colonies, compared with 26.1% knock-in cell lines in the DMSO-treated group. The application of small molecules offers a beneficial approach to improve the frequency of precise genetic modifications in primary somatic cells.

Unlocking HDR-mediated Nucleotide Editing by identifying high-efficiency target sites using machine learning

“…The The distribution of efficiencies is shown in Figure 1, clearly demonstrating the 3-fold lower efficiency for HDR (median = 0.199) compared to NHEJ (median = 0.606) when choosing targets traditionally. These values are comparable to previous work (21)(22)(23).…”

Clinical applications of CRISPR‐based genome editing and diagnostics

“…At this stage, genetic knockouts are facile, but replacing sequences by HDR remains inefficient and technically challenging, although great strides are being made to improve the technique. [65][66][67][68][69][70][71][72] CRISPR systems have generated an extraordinary level of excitement for the clinical potential for curing human disease. In this nascent stage of CRISPR in the clinic, initial applications are treating diseases with the most accessible cells (immune cells and HSPCs edited ex vivo), which avoids the ongoing challenge of tissue-specific delivery in vivo.…”

“…65 The likelihood of successful insertion can be improved using a few different strategies, but the general inefficiency of this gene replacement approach represents a substantial challenge in the field. [65][66][67][68][69][70][71][72] As our ability to carefully manipulate DNA grows, so will the clinical potential of CRISPR technologies. 17 CURRENT AND NEAR-TERM THERAPEUTIC APPLICATIONS Genetic therapies offer hope for treating and effectively curing monogenic diseases by reversing the underlying genetic cause.…”

CRISPR/Cas9 therapeutics for liver diseases

“…While these studies showed promising results in improving CRISPR/Cas9 activity, it was recently reported that successful knock‐in in mouse ES cells and zygotes using the targeted insertion of large DNA fragments (7.4 kb and 5.8 kb) was achieved without any NHEJ inhibitors . Also, HDR can be facilitated with greater target specificity by using a Cas9 mutant Cas9n that can cleave only a single DNA strand; and that the CRISPR/Cas9‐mediated HDR can be enhanced by adding small molecules, such as resveratrol, Scr7, and L755507 . Addition of these molecules to the cell culture medium boosted HDR efficiency two‐ to three‐fold in porcine fetal fibroblast cells after they were transfected with the homologous template DNA and a CRISPR/Cas9 plasmid.…”