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. 2022 Mar 8:28:202-218.
doi: 10.1016/j.omtn.2022.03.003. eCollection 2022 Jun 14.

Improved loss-of-function CRISPR-Cas9 genome editing in human cells concomitant with inhibition of TGF-β signaling

Tarun Mishra  1 , Vipin Bhardwaj  1 , Neha Ahuja  2 , Pallavi Gadgil  1 , Pavitra Ramdas  1 , Sanjeev Shukla  2 , Ajit Chande  1
Affiliations

Improved loss-of-function CRISPR-Cas9 genome editing in human cells concomitant with inhibition of TGF-β signaling

Tarun Mishra et al. Mol Ther Nucleic Acids. .

Abstract

Strategies to modulate cellular DNA repair pathways hold immense potential to enhance the efficiency of CRISPR-Cas9 genome editing platform. In the absence of a repair template, CRISPR-Cas9-induced DNA double-strand breaks are repaired by the endogenous cellular DNA repair pathways to generate loss-of-function edits. Here, we describe a reporter-based assay for expeditious measurement of loss-of-function editing by CRISPR-Cas9. An unbiased chemical screen performed using this assay enabled the identification of small molecules that promote loss-of-function editing. Iterative rounds of screens reveal Repsox, a TGF-β signaling inhibitor, as a CRISPR-Cas9 editing efficiency enhancer. Repsox invariably increased CRISPR-Cas9 editing in a panel of commonly used cell lines in biomedical research and primary cells. Furthermore, Repsox-mediated editing enhancement in primary human CD4+ T cells enabled the generation of HIV-1-resistant cells with high efficiency. This study demonstrates the potential of transiently targeting cellular pathways by small molecules to improve genome editing for research applications and is expected to benefit gene therapy efforts.

Keywords: CRISPR-Cas9; Cas9 VLPs; DNA repair; Repsox; TGF-β signaling; editing in primary T cells; genome editing; small molecules.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
A rapid screening assay to identify loss-of-function editing enhancers (A) Rapid loss of mGFP compared with GFP by CRISPR-Cas9 editing as shown in representative images (scale bar, 200 μm) and corresponding intensity plots. (B) Representative images of a screen-ready assay indicating an internal RFP reporter (scale bar, 100 μm). (C) Schematics of the chemical screen setup and the robustness evaluated from three randomly selected plates (D). (E) Classes of small molecules used in the screen. (F) A scatterplot depicting a primary screen produced by plotting an RFP/GFP ratio with a cut-off line. (G) Secondary validation screen (n = 3, ±SD) and hit selection (green dot indicates Repsox) on the basis of the robustness of the assay as obtained from three plates (H).
Figure 2
Figure 2
Repsox promotes loss-of-function editing (A) Dose-dependent effects of Repsox on the loss of GFP, indicated by RFP/GFP ratio, in HEK293T cells transfected with gGFP, Cas9, GFP, and RFP and the structure of Repsox in the inset (left panel). Alamar blue assay to measure cell growth for corresponding concentrations of the Repsox (right panel); (n = 3, ±SD). (B) Representative images for mGFP editing at 10 μM concentration of the Repsox (scale bar, 100 μm). (C) The effect of the Repsox challenge on gene editing assessed using flow cytometry (representative histogram panels) and the corresponding fold enhancement (right panel; n = 3, ±SD) from the lentivirally integrated mGFP cassette in the HEK293T cells (C) and Jurkat cells (D). Editing of engineered luciferase locus obtained by lentiviral integration into cell lines of various tissue origins as indicated (E). The lower panel shows fold enhancement of editing for corresponding cell lines (n = 3, ±SD). (F) Comparison of GFP editing using two guide RNAs simultaneously targeting the GFP locus (n = 3, ±SD). (G) Representative flow cytometry histograms of JTAg cells stained for CXCR4 under various indicated treatment conditions.
Figure 3
Figure 3
Targeted deep sequencing analysis Editing percentage in Repsox-treated JTAg and HEK293T cells expressed as sequence percentage of total reads. Next-generation sequencing (NGS) was performed for five loci (one on-target and four off-targets [ESRRG, SCARB1, AKT2, and MAMLD1]).
Figure 4
Figure 4
Effects of Repsox on cell cycle and genome integrity (A) Flow cytometry profiles of HEK293T, Jurkat TAg (JTAg), and K562 cells treated either with DMSO or Repsox (10 μM) for 48 h and observed for cell cycle phases. (B) Representative images of comets in the indicated conditions (upper panel). Graph representing the length of DNA tail observed from HEK293T cells when treated with DMSO, Repsox (10 μM), and doxorubicin (20 μM) (lower panel) (n = 50, ±SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, nonsignificant.
Figure 5
Figure 5
Repsox orchestrates editing enhancement effect through TGF-β receptor (A) MCF7 cells seeded in 24-well plate and transfected with mGFP along with Cas9, gGFP-expressing plasmids further treated with DMSO and 10 μM Repsox for 48 h and analyzed by FACS for residual GFP positivity. (B) Fold change in GFP expression from DMSO- and Repsox-treated experimental sets. (C) SMAD inhibition by Repsox (left) and increase in the editing of GFP plotted as luciferase units and GFP count, respectively (right) (n = 3, ±SD). (D) Cas9 protein expression by western blotting after treatment of Cas9 expressing HEK293T cells with DMSO or Repsox (5 and 10 μM). Actin served as a loading control. (E) Western blot depicting expression of TGF-βRI in wild-type (WT) and knockout (K/O) HEK293T and HeLa cells. Actin served as a loading control. Effect of TGF-βRI knockout on the genome editing enhancement and sensitivity to Repsox in (F and G) HEK293T (n = 4, ±SD) and (H and I) HeLa cells (n = 3, ±SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, nonsignificant.
Figure 6
Figure 6
Cas9 VLP-mediated genome editing and enhancement by Repsox (A) Schematics of Cas9 RNP complex packaging in VLPs and delivery to target cells. (B) Western blot of analysis of Cas9 from the producer cells and from VLP under indicated conditions. (C) Cas9 intracellular levels from Cas9-VLPs transduced (left) or Cas9-expressing plasmid transfected cells (right) at indicated time points. The reported time points correspond to the time of analysis following transduction or transfection. (D) Editing in HEK293T mGFP stable cells by Cas9 VLPs and fold increase in GFP-editing with Repsox. Luciferase editing in stable cell lines with Cas9 VLPs with a fold increase in editing using Repsox in (E) HEK293T (n = 4, ±SD) and (F) K562 (n = 4, ±SD). (G) Representative flow cytometry histograms depicting Repsox-mediated enhancement genome editing using Cas9 VLPs targeting CXCR4 in primary human CD4+ cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, nonsignificant.
Figure 7
Figure 7
Repsox enhances editing in primary cells Representative flow cytometry profiles indicate Repsox-enhanced loss of CXCR4 expression in human primary CD4+ T cells from three different donors.
Figure 8
Figure 8
Generation of HIV-1-resistant CD4+ T cells by small molecule-assisted editing CXCR4 loss-of-function editing and concomitant resistance of the CD4+ T cells to X4-tropic HIV-1 infection as assessed by HIV-1 capsid (p24) staining in the CXCR4-edited cells that received either DMSO or Repsox.
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