Small molecules enhance CRISPR genome editing in pluripotent stem cells

Abstract

The bacterial CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ), but it remains inefficient for precise editing of genome sequences. Here we develop a reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR). Using our screening method, we have identified small molecules that can enhance CRISPR-mediated HDR efficiency, 3-fold for large fragment insertions and 9-fold for point mutations. Interestingly, we have also observed that a small molecule that inhibits HDR can enhance frame shift insertion and deletion (indel) mutations mediated by NHEJ. The identified small molecules function robustly in diverse cell types with minimal toxicity. The use of small molecules provides a simple and effective strategy to enhance precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells.

Figure 1. Establishment of a high-throughput chemical screening platform for modulating CRISPR-mediated HDR efficiency

(A) A fluorescence reporter system in E14 mouse ES cells to characterize the HDR efficiency. An sfGFP-encoding template is inserted at the Nanog locus. The PAM is labeled in green, the stop codon is shown in red, and the sgRNA target site is shaded in grey. The cutting site (scissors) is 3 bp downstream of CCA in this case. The binding sites of two sets of primers are shown by arrows. Primer set #1 binds to the sequences outside of the homology arms, and primer set #2 contains a forward primer binding to the sfGFP sequence and a reverse primer binding outside of the 3’ homology arm. (B) Fluorescence histograms of mouse ES cells transfected with different plasmid combinations using flow cytometry analysis. (C) Sequencing results of the Nanog locus in GFP-positive cells. (D) A scheme of the chemical screening platform and a waterfall plot of 3,918 small molecules screened for their activity of CRISPR-mediated gene insertion. Highlighted dots are validated compounds that showed increased or decreased insertion efficiency. The dotted line showed the mean value of all screened compounds. (E) Validation of two enhancing and two repressing compounds using flow cytometry analysis. (F) Efficiency of sfGFP insertion into the Nanog locus. Gel pictures showing sfGFP tagging using two sets of primers as shown in Figure 1A. The PCR products of primer set #1 were purified and cloned to a modified pUC19 backbone vector and sequenced. (G) Dose-dependent effects of four compounds for modulating CRISPR gene editing. All data are normalized to the knockin efficiency of DMSO-treated control cells (dotted lines). Error bars represent the standard deviation of three biological replicates.

Figure 2. Different identified small molecules could enhance HDR or NHEJ-mediated CRISPR genome editing

(A) A scheme of insertion strategy at the human ACTA2 locus (top). The PAM is labeled in green, and the sgRNA target site is shaded in grey. (B) Sequencing results of the ACTA2 locus in Venus-positive HeLa cells. (C) Efficiency of Venus insertion measured by flow cytometry analysis. The error bars indicate the standard deviation of three samples, and the p values are calculated using two-tailed student t-test. *, p < 0.05; **, p < 0.01. (D) The strategy for introducing the A4V point mutation at the human SOD1 locus in human iPS cells. The PAM is labeled in green, and the sgRNA target site is shaded in grey. The point mutation is labeled in red. (E) Sequencing results of the SOD1 locus. (F) Comparison of A4V allele mutant frequency and indel allele frequency in human iPS cells assayed by PCR cloning and bacterial colony sequencing with no template, DMSO or L755507. (G) Test of knockout efficiency using a clonal mouse ES cell line carrying a monoallelic sfGFP insertion at the Nanog locus in the presence of L755705 and AZT. The dot plots of cells transfected with a non-cognate sgRNA (sgGAL4) is shown on the top. The panel shows cells transfected with three different sgRNAs (their target sites shown in the scheme) in the presence of DMSO (left), L755507 (middle), and AZT (right).