Bioactive small molecules enhance CRISPR/Cas9 loss-of-function editing in human cells

In a recent Molecular Therapy: Nucleic Acids study, researchers demonstrate enhanced loss-of-function editing of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in human cells. This effect was accompanied by the hindrance of transforming growth factor β (TGFβ) signaling.

Study: Improved loss-of-function CRISPR/Cas9 genome editing in human cells concomitant with inhibition of TGFβ signaling. Image Credit: Yurchanka Siarhei /


CRISPR/Cas9 is a cutting-edge genome editing technology that has been used for several research purposes, including epigenetic alterations, endogenous gene tagging, live imaging of genome transcription and editing, as well as, most crucially, modifying genes for therapeutic purposes.

Approaches to modulate cellular deoxyribonucleic acid (DNA)-repair mechanisms have tremendous potential in improving the performance of the CRISPR/Cas9 genome editing platform. During the lack of a repair template, CRISPR/Cas9-induced DNA double-strand breaks (DSBs) are corrected by endogenous cellular DNA repair networks that result in loss-of-function edits.

Although CRISPR/Cas9 can alter the genome, improving its efficiency in human cells, particularly primary cells, remains a challenge. The inhibitory mechanisms or conflicting signals that work against CRISPR/Cas9 might be the reasons for this effect.

About the study

In the present study, researchers present a reporter-based technique for the quick quantification of CRISPR/Cas9 loss-of-function editing. To this end, a large-scale chemical screen using this technique identified small compounds that enhance CRISPR/Cas9 loss-of-function editing.

Cyclin destruction box (CDB) was fused to the N-terminus of the green fluorescent protein (GFP) gene resulting in modified GFP (mGFP) to allow for the quick assessment of GFP loss as a function of editing due to the capacity of CDB to yield a protein for breakdown. A time-course evaluation of mGFP versus unmodified GFP was conducted parallelly to evaluate the ability of genome editing efficiency measurement by the assay.

Further, the team developed a chemical screen employing the mGFP reporter to identify small compounds according to their capability to boost loss-of-function editing. The GFP fluorescence loss as a function of CRISPR/Cas9 editing was measured.

Study findings

The researchers found that mGFP has about six to 10 hours lower half-life than unmodified GFP, thereby indicating the rapid quantification of genome editing efficiency of the mGFP reporter-based assay developed here. This assay discovered that biologically active small molecules such as Repsox can be rearranged to enhance genome editing by suppressing competing signals that impede CRISPR/Cas9 activity.

Small compounds provide several benefits over genetic alterations including greater control and ease of use, as well as the ability to adjust small molecules' impacts by altering combinations and concentrations.

The quick mode of action, high efficiency, easy application, and reversibility of Repsox presents numerous benefits. For example, previous studies have demonstrated that Repsox has no negative impacts on primary human cells. Furthermore, various manufacturers offer Repsox, which can be used to stimulate on-target genome editing.

Taken together, experimental approaches that provide higher delivery and transfection efficiency of Cas9 ribonucleoprotein (RNP) combination with Repsox are likely to result in efficiency gains.

In the present study, the accentuating impact of Respox on CRISPR/Cas9 functionality was verified using various gene transfer mechanisms such as integrating lentiviruses, lipoplexes, electroporation, and calcium phosphate co-precipitation. Augmented Cas9-RNPs delivery by Repsox through virus-like particles (VLPs) was also confirmed.

The efficiencies of each of these modalities were based on the delivered payload's level of expression and corresponding basal editing efficiencies. Notably, the Repsox impact remained intact, irrespective of these factors.

The major obstacle to genome editing is the delivery of CRISPR/Cas9 components. VLP delivery modality and treatment with small compounds like Repsox can decrease off-target mutations as compared to other approaches like nucleic acid delivery by transfection and integrating viral vectors because of the short half-life of RNPs.

The efficacy of loss-of-function editing might be influenced by TGFβ type 1 receptor (TGFβRI)-dependent signaling. By employing complementary methods, the researchers also demonstrated that Repsox promotes editing by blocking upstream signaling mediated by the TGFβ receptor.

Repsox inhibits adenosine triphosphate (ATP) interaction with TGFβR-1/ activin receptor-like kinase receptor 5 (ALK5), thereby causing hindrance to TGFβ signaling and phosphorylation of the SMAD2/3 pathway. Thus, the genetic deletion of this upstream component renders the cell immune to Repsox activity.

TGFRI inhibition by Repsox therapy possibly provokes a shift in DSB repair from the classical non-homologous end joining (C-NHEJ) route to the more fault-prone substitute-NHEJ pathway, resulting in improved loss-of-function editing. Of note, the Repsox-mediated editing augmentation resulted in a human immunodeficiency virus (HIV)-resistant pool of primary human CD4+ cells and has applications in research and clinical areas.


The study findings demonstrated that Respox, a TGFβ signaling inhibitor, is a potential editing efficiency booster for CRISPR/Cas9 following several rounds of screening. In a panel of regularly used primary cells and cell lines for biomedical research, the bio-active small molecule Repsox consistently improved CRISPR/Cas9 loss-of-function editing.

The current study also found that using Repsox to treat cells from various tissue origins increases loss-of-function editing, irrespective of Cas9 mode or guide ribonucleic acid (gRNA) expression. Repsox-mediated editing amplification in primary human CD4+ T-cells also resulted in the production of highly efficient HIV-1 resistant cells.

Taken together, the present work shows the capacity of small compounds reversibly targeting cellular pathways to enhance genome editing for research purposes and is likely to aid gene therapy efforts.

Journal reference:
Shanet Susan Alex

Written by

Shanet Susan Alex

Shanet Susan Alex, a medical writer, based in Kerala, India, is a Doctor of Pharmacy graduate from Kerala University of Health Sciences. Her academic background is in clinical pharmacy and research, and she is passionate about medical writing. Shanet has published papers in the International Journal of Medical Science and Current Research (IJMSCR), the International Journal of Pharmacy (IJP), and the International Journal of Medical Science and Applied Research (IJMSAR). Apart from work, she enjoys listening to music and watching movies.


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