Extended Data Fig. 4: Transient microhomology-mediated end-joining (MMEJ) inhibition by siRNAs.
From: Efficient high-precision homology-directed repair-dependent genome editing by HDRobust
(a) Editing efficiencies of the VCAN target using Cas9D10A double nicking in H9 hESCs carrying the K3753R mutation, and with additional POLQ mutations (V896* full knockout, DE2540AA polymerase knockout, 6x silent SNPs that do not change the encoding amino acids), transient POLQ inhibition with siRNAs, or transient RAD51 inhibition with the small molecule B02. The K3753R mutation will prevent backup NHEJ repair when MMEJ is inhibited. Frequencies of deletions are presented based on microhomology (MH) length. For HDR, replicates are depicted by dots. The mean outcome purities (percentage HDR of all editing events) are given. Independent biological replicates were performed (n = 2, except DNA-PKcs K3753R / DE2540AA / B02 and DNA-PKcs K3753R / 6x silent SNP n = 3, DNA-PKcs K3753R / DE2540AA n = 5, and DNA-PKcs K3753R n = 6) and error bars show the s.e.m. (b) A scheme of the different siRNAs targeting the POLQ mRNA to induce Ago2/RISC assisted cleavage as well as a site of silent mutations that do not change the encoded amino acids, and translated Polϴ is shown below. Polϴ motifs described to be detrimental for homologous recombination/HDR are colored rose, Polϴ inhibitory mutations are red. (c) Time course of POLQ mRNA knock down with siRNA 765. POLQ expression was normalized with GAPDH expression and is given relative to untreated cells. Independent biological replicates were performed (n = 2) and error bars show the s.e.m.
