基因組編輯工具-CRISPR/Cas9
繼ZFN（Zinc Finger Nucleases）技術后，Merck在2013年推出新一代基因組編輯工具--CRISPR/Cas9，讓研究人員以更快、更經濟的方式實現基因組特定位點的編輯。

基因組編輯工具-CRISPR/Cas9

繼ZFN（Zinc Finger Nucleases）技術后，Merck在2013年推出新一代基因組編輯工具--CRISPR/Cas9，讓研究人員以更快、更經濟的方式實現基因組特定位點的編輯。憑借過去10年在基因組編輯領域的豐富經驗積累以及專業的生物信息學平臺，Merck已經成功設計出覆蓋人類，小鼠和大鼠三個物種的所有基因的CRISPR/Cas9載體，并可以提供在線定制服務，以及完整的CRISPR實驗workflow解決方案。此外，默克與Sanger Institute合作開發了人、小鼠全基因組CRISPR 文庫，以幫助科學家實現基因功能的快速篩選、規模化的模型建立以及藥物作用篩選等。

• 高效：優化的載體設計，大限度提高轉染效率，簡化篩選工作
• 特異：特殊的gRNA設計和雙切口酶系統，大限度提高特異性
• 全面：產品齊全，可提供質粒、RNA、慢病毒載體、RNP等形式，涵蓋人、大鼠、小鼠、植物等多個物種，更有Sanger Arrayed和Broad Pools全基因組文庫以及重要通路的亞文庫
• 掌控：強大的慢病毒全基因組文庫可輕松進行高通量篩選，全面掌控人或小鼠的基因組

 

CRISPR/Cas9 基因編輯工具

• Sanger Arrayed Lentiviral CRISPR   Libraries • Lentiviral CRISPR Pools Libraries • CRISPR/Cas9單載體表達系統 • CRISPR Cas9-D10A雙切口酶系統 • SygRNA® Cas9 RNP系統 • CRISPR/Cas9基因激活表達載體 • CRISPR/Cas9在植物中的應用 • Cas9蛋白 • CRISPR對照 (DNA and Virus)

CRISPR-based Gene Activation

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.

 

Product no.	Description
DCAS9P300	Sigma CRISPR dCas9-p300 Activator Expression Plasmid

 

 

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.

 

References

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).