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. Sigma offers 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.

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References

Chen Yu, Yanxia Liu, Tianhua Ma, Kai Liu, Shaohua Xu, Yu Zhang, Honglei Liu, Marie La Russa, Min Xie, Sheng Ding, Lei S Qi
Cell Stem Cell 2015-02-05
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...Read More
Van Trung Chu, Timm Weber, Benedikt Wefers, Wolfgang Wurst, Sandrine Sander, Klaus Rajewsky, Ralf Kühn
Nature Biotechnology 2015-05-01
The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we ...Read More
Yuanwu Ma, Wei Chen, Xu Zhang, Lei Yu, Wei Dong, Shuo Pan, Shan Gao, Xingxu Huang, Lianfeng Zhang
RNA Biology 2016-07-02
Precise modifications such as site mutation, codon replacement, insertion or precise targeted deletion are needed for studies of accurate gene function. The CRISPR/Cas9 system has been proved as a powerful tool to generate gene knockout and knockin animals. But the homologous recombination (HR)-directed precise genetic modificat...Read More
Diane Yang, Marissa A Scavuzzo, Jolanta Chmielowiec, Robert Sharp, Aleksandar Bajic, Malgorzata Borowiak
Scientific reports 2016-01-01
Efficient gene editing is essential to fully utilize human pluripotent stem cells (hPSCs) in regenerative medicine. Custom endonuclease-based gene targeting involves two mechanisms of DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR is the preferred mechanism for common applications such knoc...Read More
Jun Song, Dongshan Yang, Jie Xu, Tianqing Zhu, Y Eugene Chen, Jifeng Zhang
Nature Communications 2016-01-01
Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nuc...Read More
Jordan Pinder, Jayme Salsman, Graham Dellaire
Nucleic Acids Research 2015-10-30
CRISPR is a genome-editing platform that makes use of the bacterially-derived endonuclease Cas9 to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs. We developed a nuclear domain knock-in screen, whereby the insertion of a gene encoding the green fluorescent protein variant Clo...Read More
Kevin M Davis, Vikram Pattanayak, David B Thompson, John A Zuris, David R Liu
Nature Chemical Biology 2015-05-01
Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen-responsive intein at sp...Read More
Takeshi Maruyama, Stephanie K Dougan, Matthias C Truttmann, Angelina M Bilate, Jessica R Ingram, Hidde L Ploegh
Nature Biotechnology 2015-05-01
Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA r...Read More
Steven Lin, Brett T Staahl, Ravi K Alla, Jennifer A Doudna
eLife 2014-01-01
The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by...Read More