基因激活表达载体 Sigma-Aldrich 代谢组学

继ZFN（Zinc Finger Nucleases）技术后，Merck在2013年推出新一代基因组编辑工具--CRISPR/Cas9，让研究人员以更快、更经济的方式实现基因组特定位点的编辑。凭借过去10年在基因组编辑领域的丰富经验积累以及专业的生物信息学平台，Merck已经成功设计出覆盖人类，小鼠和大鼠三个物种的所有基因的CRISPR/Cas9载体，并可以提供在线定制服务，以及完整的CRISPR实验workflow解决方案。此外，默克与Sanger Institute合作开发了人、小鼠全基因组CRISPR 文库，以帮助科学家实现基因功能的快速筛选、规模化的模型建立以及药物作用筛选等。

• GX：优化的载体设计，限度提高转染效率，简化筛选工作
• 特异：特殊的gRNA设计和双切口酶系统，限度提高特异性
• 全面：产品齐全，可提供质粒、RNA、慢病毒载体、RNP等形式，涵盖人、大鼠、小鼠、植物等多个物种，更有Sanger Arrayed和Broad Pools全基因组文库以及重要通路的亚文库
• 掌控：强大的慢病毒全基因组文库可轻松进行高通量筛选，全面掌控人或小鼠的基因组

CRISPR/Cas9 基因编辑工具

• Sanger Arrayed Lentiviral CRISPR   Libraries • Lentiviral CRISPR Pools Libraries • CRISPR/Cas9单载体表达系统 • CRISPR Cas9-D10A双切口酶系统 • SygRNA® Cas9 RNP系统 • CRISPR/Cas9基因激活表达载体 • CRISPR/Cas9在植物中的应用 • Cas9蛋白 • CRISPR对照 (DNA and Virus)

CRISPR for Epigenetic Editing – dCas9

Epigenome editing is a tool in which the DNA or histone is modified at specific sites in the genome using engineered molecules. This strategy requires precise targeting which is accomplished through the use of nuclease-deficient Cas9 (dCas9). However, unlike genome editing, epigenome editing does not affect genome DNA sequence.

p300-dCas9 Induced Targeted Histone Acetylation

Histone acetylation, carried out by histone acetyltransferases (HATs), plays a fundamental role in regulating chromatin dynamics and transcriptional regulation. The importance of histone acetylation in cancer has been clinically validated with several inhibitors of HDACs as anti-tumor agents. p300/CBP is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells. The p300 HAT domain (1284-1673) is catalytically active and can be fused to nucleases for targeted epigenome editing.

Sigma has developed an approach for efficient targeted histone acetylation using CRISPR. We demonstrated that gRNA can successfully direct dCas9 fused to p300 HAT catalytic domain to increase levels of histone acetylation and endogenous gene expression. This strategy for investigating functions of histone acetylation at specific genomic loci has enormous potential for research and therapeutic applications.

dCas9-p300 CRISPR Gene Activator

The dCas9-p300 CRISPR Gene Activator system is based on a fusion of dCas9 to the catalytic histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300. This approach has been independently validated by the Gersbach lab (Duke University) to activate genes at both proximal and distal locations relative the transcriptional start site (TSS). The dCas9-p300 histone acetylation approach represents a distinct mechanism of action relative to dCas9-VP64 or other similar gene activation motifs. While activation domains, such as VP64, help recruit transcription complexes to the promoter region, they are at the mercy of the epigenetic state of the gene and dependent on the availability of additional transcriptional proteins. Conversely, the p300 histone acetyltransferase protein opens a transcriptional highway by releasing the DNA from its heterochromatin state and allowing for continued and robust gene expression by the endogenous cellular machinery.

Above is a map for the plasmid which expresses the dCas9p300 fusion protein. This plasmid also co-expresses GFP from the same transcript as dCas9p300 to easily monitor delivery and expression in your target cell type. The dCas9p300 plasmid can be co-transfected with custom-made U6-gRNA plasmids to target activation near transcription start sites or other regulatory locations. An Oct4 control plasmid is available (CRISPR17-1EA) which can be used to help establish the dCas9p300 approach in your laboratory. This Oct4 control has been shown to work in HEK293 cells (Figure below). We recommend testing the Oct4 control in HEK293 cells alongside any new cell type you wish to try.

OCT4 (POU5F1) is one of the most difficult targets to be activated

Octamer-binding TF 4 (Oct4, POU5F1) is the key transcription factor (TF) that maintains pluripotency of stem cells. It also plays a critical role in reinstating cellular pluripotency. The expression of OCT4 is stringently silenced in differentiated cells. Activation of silenced OCT4 gene has become a hallmark event during epigenetic reprogramming into induced pluripotent stem cells (iPSCs).

p300-dCas9 activated endogenous OCT4

p300 HAT domain (1284-1673) was fused to dCas9 (D10A, H840A) to generate p300-dCas9. Nine gRNAs were designed to target OCT4. Six of the nine gRNAs tested, along with p300‑dCas9, resulted in 2-fold or higher OCT4 transcript levels compared to control HEK293 cells.

1. Hilton, Isaac B., et al. "Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers."Nature Biotechnology (2015).
2. Ji, Qingzhou et al. “Engineered zing-finger transcription factors activate OCT4 (POU5F1), SOX2, KLF4, c-MYC (MYC) and miR302/367”.Nucleic Acids Research (2014).