CRISPR小分子增强剂


 小分子增强CRISPR基因组编辑

随着CRISPR-Cas9系统成为越来越流行的基因组编辑工具,提高CRISPR介导的精准基因编辑效率的呼声不断高涨。大量研究发现了增强CRISPR/Cas-9介导的基因组编辑的整体效率和靶向精准的生物活性小分子。我们除了为全球研究界提供CRISPR工具之外,也提供许多这种小分子

CRISPR-Cas9系统是一种RNA引导的基因组编辑工具,为研究人员提供一种简单、容易和快速的方法修饰各种生物的基因组。使用这个系统,Cas9被引导到靶序列切割DNA形成DNA双链断裂(DSB)。细胞通过两种方法中的一种修复断裂,非同源末端连接(NHEJ)或同源重组修复(HDR)。细胞最常利用的是NHEJ,它在Cas9介导的基因组修饰中具有高效率。然而,NHEJ的精确度很差,经常导致插入或缺失(INDELS)而引发难以预测的后果。许多研究人员期望在可控条件下精准靶向修饰准确的DNA序列(包括SNP、基因敲入、条件性基因敲除等)。在这些情况下,来自提供的DNA模板的HDR是优选的修复机制;然而,细胞利用该途径的频率低于NHEJ。为了提高Cas9介导的基因编辑的效率,大量研究已经进行筛选调控NHEJ和HDR修复途径的小分子。

许多团队研究了CRISPR基因组编辑背景下的HDR调控,因而发现各种细胞类型中增强CRISPR介导的HDR效率的小分子。这些化合物是细胞类型特异性和上下文依赖(context dependent)的,各作者描述的活性程度不同。RS-1(RAD51-stimulatory compound 1,RAD51蛋白激活剂)是已知的人同源重组(HR)蛋白RAD51的刺激剂,可特异刺激RAD51的DNA结合和重组活性。Song等人表明了RS-1可能通过刺激RAD51增强兔胚胎体外和体内Cas9介导的基因敲入效率。Pinder等表明RS-1处理也加强了人胚胎肾细胞HEK293A中Cas9激活的HDR。  Pinder还研究了有效的β3-肾上腺素能受体部分激动剂,L755507。虽然该团队的结果显示HEK293A细胞内只有少量HDR激活,但Yu等随后证明L755507加强了人iPS细胞(iPSCs)中CRISPR介导的HDR。促进HDR的另一种方法是抑制NHEJ。Srivastava等人报道了SCR7可作为NHEJ的抑制剂,Maruyama等表明它增强人和鼠培养细胞中HDR介导的CRISPR-Cas9基因组编辑。这些论文之后,发现最初发表的SCR7结构和CRISPR相关研究使用的实际结构之间有些不一致。真正产生增强效率的化合物是SCR7吡嗪,一种SCR7自发环化的产物。之后使用SCR7吡嗪的研究显示这种化合物对CRISPR-Cas9基因组编辑效率的影响是细胞类型特异性和上下文依赖的。下面详细了解这些及其他用于CRISPR编辑效率调控研究的小分子。


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 参考文献

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