Repurposed from a prokaryotic adaptive immune response, the CRISPR/Cas9 system has become a powerful RNA-guided genome-editing tool for targeting and manipulating virtually any genomic sequence, since its inception in 2012.  Nowadays CRISPR is being used in a plethora of applications including the elucidation of gene function involved in disease, the correction of disease-causing mutations, and genome-wide screens that identify drug-target or disease-resistance genes, etc. amongst other novel and constantly expanding applications beyond gene-editing[11].

Not only is this technique simple, easy, and quick, but it is also versatile, allowing researchers to modify the genome of various organisms with many (ingenious) variations of the technique available.

Along with the popularity of this technique, the need for improved efficiency and precision has arisen. Once a double-strand DNA break (DSB) is established via RNA-guided (type II) Cas9, the cell employs one of two major (competitive) repair mechanisms to repair the break: non-homologous end joining (NHEJ) or homology-directed repair (HDR), with the NHEJ pathway being the predominate one in mammalian cells, operating in all phases of the cell cycle. In the absence of a repair template, error-prone NHEJ ligates the DNA ends, introducing non-specific (random) indels or substitutions at the DNA site, convenient for generating gene knockouts[1].

For precise targeted sequence changes, the cell must favor HDR over NHEJ, which is initiated with a (double-stranded or single-stranded) donor template containing a sequence of interest, flanked by homology arms. Obtaining a high efficiency of the HDR pathway however is quite challenging in most eukaryotic cells as only a small proportion (< 10%) of cells undergo this pathway, which is temporally restricted to late S and G2 phases of the cell cycle[2, 3].   

One simple and effective strategy for enhancing CRISPR precision involves the use of small bioactive molecules identified by various research groups to modulate specific targeting processes. Studies have shown that these small molecules either directly enhance the HDR pathway or indirectly increase the HDR pathway through inhibition of the NHEJ pathway[3].  A third group of molecules enhance HDR efficiency through cell cycle synchronization and timed delivery of pre-assembled Cas9 ribonucleoprotein (RNP) complexes, so that CRISPR gene editing is performed exclusively during the S and G2 phase[4]. Yet another strategy involving small molecules involves chemically reducing Cas9’s activity once it has had sufficient opportunity to modify the target locus, decreasing the amount of off-target DNA cleavage activity[5]. 

Such chemical approaches offer a relatively simple, cheap, and safe way to rapidly inhibit or activate specific targeting processes, often in a reversible manner with the ability to fine-tune through treatment dosage and/or length of exposure adjustments. As these “precision enhancing” molecules are cell type specific and context dependent, it is important to choose the optimal one for your application.  To assist in your choice, refer to the carefully curated list of molecules offered by J&K Scientific, organized by the small molecule’s mechanism of action in the targeting process and successful CRISPR applications via cell type.

HDR Enhancers
RS-1

HDR Inhibitors (that result in enhanced NHEJ)
Azidothymidine

NHEJ Inhibitors
SCR7 pyrazine, KU5778, NU7026, M3814, VE-822

Small molecules that increase HDR via cell cycle synchronization
Aphidicolin, Thymidine, Nocodazole, ABT-751, XL413

Small molecules with undetermined pathways
L755507, Brefeldin A, Resveratrol, VPA

Small molecule (post-translational) control of Cas9 to improve off-target cleavage
(Z)-4-Hydroxytamoxifen

References:
[1] Jiang, F., & Doudna, J. A. (2017). CRISPR-Cas9 Structures and Mechanisms. Annual review of biophysics, 46, 505–529. https://doi.org/10.1146/annurev-biophys-062215-010822
[2] Pinder, J., Salsman, J., & Dellaire, G. (2015). Nuclear domain 'knock-in' screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing. Nucleic acids research, 43(19), 9379–9392. https://doi.org/10.1093/nar/gkv993
[3] Bischoff, N., Wimberger, S., Maresca, M., & Brakebusch, C. (2020). Improving Precise CRISPR Genome Editing by Small Molecules: Is there a Magic Potion? Cells, 9(5), 1318. https://doi.org/10.3390/cells9051318
[4] Lin, S., Staahl, B. T., Alla, R. K., & Doudna, J. A. (2014). Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife, 3, e04766. https://doi.org/10.7554/eLife.04766
[5] Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A., & Liu, D. R. (2015). Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nature chemical biology, 11(5), 316–318. https://doi.org/10.1038/nchembio.1793