Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

Abstract

A now frequently used method to edit mammalian genomes uses the nucleases CRISPR/Cas9 and CRISPR/Cpf1 or the nickase CRISPR/Cas9n to introduce double-strand breaks which are then repaired by homology-directed repair using DNA donor molecules carrying desired mutations. Using a mixture of small molecules, the "CRISPY" mix, we achieve a 2.8- to 7.2-fold increase in precise genome editing with Cas9n, resulting in the introduction of the intended nucleotide substitutions in almost 50% of chromosomes or of gene encoding a blue fluorescent protein in 27% of cells, to our knowledge the highest editing efficiency in human induced pluripotent stem cells described to date. Furthermore, the CRISPY mix improves precise genome editing with Cpf1 2.3- to 4.0-fold, allowing almost 20% of chromosomes to be edited. The components of the CRISPY mix do not always increase the editing efficiency in the immortalized or primary cell lines tested, suggesting that employed repair pathways are cell-type specific.

Fig. 1

Small molecules described or anticipated to target key proteins of NHEJ and HDR. Proteins are labeled with black text and inhibitors and enhancing small molecules are marked red and green, respectively. STL127705, NU7026, or SCR7 have been described to inhibit Ku70/80, DNA-PK, or DNA ligase IV, respectively. MLN4924, RS-1, Trichostatin A, or Resveratrol have been described to enhance CtIP, RAD51, or ATM, respectively. NSC 15520 has been described to block the association of RPA to p53 and RAD9. AICAR is an inhibitor of RAD52 and B02 is an ihibitor of RAD51. For simplicity, some proteins and protein interactions are not depicted

Fig. 2

Effects of small molecules on targeted nucleotide substitution (TNS) efficiency in iCRISPR hiPSCs. Shown are TNS efficiencies in CALD1, KATNA1 and SLITRK1 with Cas9n (a) and Cas9 (b) in 409-B2 iCRISPR hiPSCs. TNS efficiency is given in relative units (RU) with the mean of controls set to 1 to account for varying efficiency in different loci. Shown are technical replicates of n independent experiments. Data from Fig. 3 and Supplementary Fig. 3 are included. Gray and black bars represent the mean of the control and the respective small molecule, respectively. Concentrations used were 20 µM NU7026, 0.01 µM Trichostatin A, 0.5 µM MLN4924, 1 µM NSC 19630, 5 µM NSC 15520, 20 µM AICAR, 1 µM RS-1, 1 µM Resveratrol, 1 µM SCR7, 5 µM L755507, 5 µM STL127685, and 20 µM B02. Mean absolute percentages of TNS and indels of all technical replicates are shown in Supplementary Table 4

Fig. 3

Impact of small-molecule combinations on targeted nucleotide substitution (TNS) efficiency in iPSCs and hESCs. Shown are TNS efficiencies in CALD1, KATNA1, and SLITRK1 with Cas9n and Cas9, and in HPRT and DNMT1 with Cpf1. Small molecules have an additive effect on TNS efficiency with Cas9n (a) but not with Cas9 (b) in the 409-B2 iCRISPR hiPSC lines. TNS of HPRT and DNMT1 in 409-B2 hiPSCs with recombinant Cpf1 was increased using the CRISPY mix as well (c). Using the CRISPY mix, TNS efficiency was also increased in SC102 A1 hiPSCs and H9 hESCs with plasmid-delivered Cas9n-2A-GFP (GFP-FACS enriched), and in chimpanzee SandraA ciPSCs with recombinant Cpf1 (d). Shown are TNS, TNS + indels, and indels with green, gray, or blue bars, respectively. Error bars show the SD of three technical replicates for a, b, and c, and two technical replicates for d. Concentrations used were 20 µM of NU7026, 0.01 µM of Trichostatin A, 0.5 µM MLN4924, 1 µM NSC 19630, 5 µM NSC 15520, 20 µM AICAR, and 1 µM RS-1. CRISPY mix indicates a small-molecule mix of NU7026, Trichostatin A, MLN4924, and NSC 15520. Statistical significances of TNS efficiency changes was determined using a two-way ANOVA and Tukey’s multiple comparison pooled across the three genes CALD1, KATNA1, and SLITRK1. Genes and treatments were treated as random and fixed effect, respectively. P-values are adjusted for multiple comparison (**P ≤ 0.01, ***P ≤ 0.001). Overall, there was a clear treatment effect (F(12, 24) = 32.954, P ≤ 0.001)

Fig. 4

Impact of the CRISPY mix on gene fragment insertion efficiency in iCRISPR hiPSCs. Shown is the insertion efficiency of a gene fragment coding for a blue fluorescent protein (BFP) in the heterozygous AAVS1 iCRISPR locus using a single-stranded DNA donor in 409-B2 iCRISPR-Cas9n hiPSCs. The design of the mtagBFP2 ssODN donor and the iCRISPR system is shown in a. We inserted a 871 nt (including 50 nt homology arms) sequence coding for a 2A-self cleaving peptide in front of a blue fluorescent protein (BFP), directly after the N-terminal nuclear localization signal sequence (NLS) of the Cas9n in the heterozygous AAVS1 iCRISPR locus. If the sequence is inserted, doxycycline will lead to expression of nucleus-imported BFP. Representative images of the mock, NU7026, and CRISPY mix treatment, after 7 days of BFP expression are shown in b as phase contrast (PC), propidium iodide nuclei staining (PI), mtagBFP2 expression (BFP), and merge of PI and BFP. Two images (× 50 magnification, white size bar 200 µm) from each of three technical replicates for the respective treatments were used to quantify the percentage of cells with BFP insertion using ImageJ (c)