Small Molecule CRISPR Enhancers

Small Molecules Enhance CRISPR Genome Editing

As the CRISPR-Cas9 system has become an increasingly popular tool for genome editing, the desire to improve efficiency of precise CRISPR-mediated gene editing has arisen. A number of studies have identified bioactive small molecules that enhance the overall efficiency and targeting precision of CRISPR/Cas-9-mediated genome editing. We offer many of these small molecules, in addition to making CRISPR-based tools available to the global research community.

The CRISPR-Cas9 system is an RNA-guided genome-editing tool that provides researchers a simple, easy, and quick way to modify the genomes of various organisms. Using this system, Cas9 is gudied to a target sequence where it cleaves the DNA to form a double-stranded DNA break (DSB). Cells repair the break through one of two approaches, non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Most commonly, cells will utilize NHEJ, which has been shown to have high efficiencies in Cas9-mediated genome modification. However, NHEJ is notably imprecise, typically resutling in insertions or deletions (INDELS) that result in unpredictable outcomes. Many investigators desire precision targeting to modify exact DNA sequences (including SNPs, knock-ins, conditional knock-outs, etc) in a controlled setting. In these cases HDR from a provided DNA template is the preferred mechanism of repair; however, this pathway is utilized by the cell less frequently than NHEJ. A number of studies have been performed to identify small molecules to modulate the NHEJ and HDR DNA repair pathways with the intention of improving the efficiency of Cas9-mediated gene editing.

Modulation of HDR within the context of CRISPR-genome editing has been investigated by many groups and led to the identification of small molecules that enhance CRISPR-mediated HDR efficiency in various cell types. These compounds have been shown to be cell type specific and context dependent, with authors demonstrating activity to varying degrees. The compound RS-1 (RAD51-stimulatory compound 1) is a known stimulator of the human homologous recombination (HR) protein RAD51, which specifically stimulates the DNA binding and recombination activity of RAD51. Song et al showed that RS-1 enhances Cas9-mediated knock-in efficiency in in vitro and in vivo rabbit embryos, likely by stimulating RAD51. RS-1 treatment was also shown to increase Cas9-stimulated HDR in human embryonic kidney HEK293A cells by Pinder et al. Pinder also investigated the potent β3-adrenergic receptor partial agonist, L755507. Though results from this group showed only a slight stimulation of HDR in HEK293A cells, Yu et al subsequently demonstrated that L755507 enhances CRISPR-mediated HDR in human iPS cells (iPSCs). An alternate approach to promote HDR is to decrease NHEJ. SCR7 was reported as an inhibitor of NHEJ by Srivastava et al and shown by Maruyama et al to increase the efficiency of HDR-mediated CRISPR-Cas9 genome editing in human and murine cultured cells. Following these papers, some inconsistencies were identified between the originally published structure of SCR7 and the actual structure used in CRISPR-related studies. It has been shown that the compound that actually yielded enhanced efficiency was SCR7 pyrazine, a product of spontaneous cyclization of SCR7. Subsequent studies using SCR7 pyrazine have shown that the effect of this compound on CRISPR-Cas 9 genome editing efficiency is cell type specific and context dependent. Learn more below about these and additional small molecules that have been studied for modulating CRISPR editing efficiency.

References

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5. Song J, Yang D, Xu J, Zhu T, Chen YE, Zhang J. 2016. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat Commun. 7(1): https://doi.org/10.1038/ncomms10548

6. Davis KM, Pattanayak V, Thompson DB, Zuris JA, Liu DR. 2015. Small molecule?triggered Cas9 protein with improved genome-editing specificity. Nat Chem Biol. 11(5):316-318. https://doi.org/10.1038/nchembio.1793

7. Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kühn R. 2015. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 33(5):543-548. https://doi.org/10.1038/nbt.3198

8. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. 2015. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. 33(5):538-542. https://doi.org/10.1038/nbt.3190

9. Yu C, Liu Y, Ma T, Liu K, Xu S, Zhang Y, Liu H, La Russa M, Xie M, Ding S, et al. 2015. Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells. Cell Stem Cell. 16(2):142-147. https://doi.org/10.1016/j.stem.2015.01.003