Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion?

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing has become a standard method in molecular biology, for the establishment of genetically modified cellular and animal models, for the identification and validation of drug targets in animals, and is heavily tested for use in gene therapy of humans. While the efficiency of CRISPR mediated gene targeting is much higher than of classical targeted mutagenesis, the efficiency of CRISPR genome editing to introduce defined changes into the genome is still low. Overcoming this problem will have a great impact on the use of CRISPR genome editing in academic and industrial research and the clinic. This review will present efforts to achieve this goal by small molecules, which modify the DNA repair mechanisms to facilitate the precise alteration of the genome.

Improvements in Gene Editing Technology Boost Its Applications in Livestock

“…Transfection or injection of long DNA fragments containing a gene of interest has been used as a strategy to express foreign genes in cells in vitro ( Kohn et al, 1987 ; Bayna and Rosen, 1990 ) and for the production of GE animals. However, targeted integration has been a challenge due to the low rate of HDR in the cells and the high probability of random integration (reviewed by Bischoff et al, 2020 ). Different approaches to improve the integration of long fragments of DNA have been developed, including CRISPR/Cas9 mediating homologous recombination (HR), microhomology-mediated end-joining (MMEJ) targeted integration, homology-mediated end joining (HMEJ)-based targeted integration, and the NHEJ-mediated KI named homology-independent targeted integration (HITI) ( Suzuki et al, 2016 ; Wu et al, 2016 ; Yao et al, 2017a , b ).…”

Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models

“…In contrast to NHEJ, other repair pathways, i.e., HR, microhomology-mediated end joining (MMEJ), and single-strand annealing (SSA), depend on a DNA template and are predominant in S/G2 phases. To favor KI, different strategies with small molecules have been used to arrest cells at different phase of the cycle (Yeh et al, 2019;Bischoff et al, 2020) but these strategies are difficult to apply to embryos. To favor HDR pathways predominant in S/G2, Cas9 can be degraded by the proteasome in G1 phase (Figure 3B and Table 4) by fusion to geminin degron (Gutschner et al, 2016;Charpentier et al, 2018;Lomova et al, 2019).…”

“…To favor KI, small inhibitors of NHEJ or essential molecules carried to the DSB via gRNA, via Cas9 (Figure 3C and Table 4) have been used. NHEJ inhibitors have mainly been tested on cells (for reviews, see Yeh et al, 2019;Bischoff et al, 2020) and SCR7, an inhibitor of ligase IV, has led to KI increase in mouse (Maruyama et al, 2015;Singh et al, 2015) and rat embryos (Ma et al, 2016). Cas9 in fusion with a domain of CtIP has shown increased KI efficiency in human cells and rats (Charpentier et al, 2018;Tran et al, 2019).…”

Correction of a CFTR/G542X Mutation Using CRISPR/Cas9 Editing in Ovine-bovine Interspecies Embryos

“…Genome editing by CRISPR/Cas9 generates double-strand breaks (DSBs) that evoke the error-prone non-homologous end joining (NHEJ) DNA repair pathway, which might cause off-target mutagenesis 13 . During the S to G2 phases of cell circle, DSBs can also activate an alternative DNA repair pathway, the homology-directed repair (HDR), that repairs the DSB sites using a template from the homologous chromosome or an introduced exogenous DNA molecule, leading to the introduction of desired mutations 14 .…”