Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene

Abstract

During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7-gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene (GMNN). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9-GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9-GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.

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

“…While SCR7 was shown to increase HDR mediated integration of dsDNA into fetal porcine fibroblasts [55], the efficiency to introduce a point mutation by ssODN could not be increased [56]. Estimation of NHEJ and HDR by an extrachromosomal fluorescent reporter construct in H1 cells did not show a significant difference in HDR or NHEJ efficiency between SCR7 treated and non-treated controls [57].…”

Methods for Enhancing Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Homology-Directed Repair Efficiency

“…Mechanistically, SCR7 blocks the NHEJ pathway by binding to DNA ligase IV, a key enzyme of the NHEJ pathway, in a concentration-dependent manner (Vartak and Raghavan, 2015). Specifically, it works by reducing the affinity of DNA ligase IV for DSBs (Gerlach et al, 2018). SCR7 binds to the DNA-binding domain of DNA ligase IV, thereby preventing DNA ligase IV from binding to the DNA ends, resulting in the elimination of the NHEJ pathway (Vartak and Raghavan, 2015).…”

“…Interestingly, experiments have shown that the addition of SCR7 does not improve HDR efficiency during genome editing. In contrast, coordinated expression of Cas9 in the HDR-dominant cell cycle is more efficient in inducing HDR than inhibition of NHEJ (Gerlach et al, 2018). Nonetheless, existing studies have shown that these manipulations may be difficult to perform, or the process of manipulation may cause greater damage to cells.…”

Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks

“…This engineered Cas9-geminin construct increased the HDR rate by 1.87-fold compared with wild-type Cas9 in HEK-293T cells [88]. The efficacy of the hCas9-geminin approach was subsequently confirmed in human pluripotent stem cells and porcine fibroblasts [120,121], making it the most promising tool for enhancing HDR ( Figure 6). Lomova et al, found that there was an additive effect between manipulation of Cas9 expression and cell cycle synchronization via RO-3306, which improved the HDR/NHEJ ratio by an average of 20-fold compared with the control condition in hPSCs [118].…”