The latest tool in genome editing – CRISPR/Cas9 – allows for specific genome disruption and replacement in a flexible and simple system resulting in high specificity and low cell toxicity. The CRISPR/Cas9 genome editing system requires the co-expression of a Cas9 protein with a guide RNA. With the protospacer-adjacent motif (PAM – the sequence NGG) present at the 3′ end, Cas9 will unwind the DNA duplex and cleave both strands upon recognition of a target sequence by the guide RNA. The functional cassette in the rescue donor vector can then be inserted into the unwound DNA. The repaired genome will now express your desired sequence with or without tags.
OriGene provides all the vectors and service for the Cas9-genome editing. http://www.origene.com/CAS9/
pCas-Guide vector (SKU# GE100002) encodes Cas9 enzyme and the guide RNA. You can use OriGene free gRNA design tool to design your guide RNA. Have them synthesized by any commercial company such as IDT . Anneal them together to make a double strand DNA and ligate the dsDNA to pCas-Guide vector. OriGene also offers an annealing buffer (SKU# GE100007) for you to make the dsDNA.
pCas-Scramble (SKU#GE100003) contains a scrambled sequence in pCas-Guide vector. It is a good negative control that you can use for your work.
The gene specific rescue donor vector can be made through gene synthesis (www.blueheronbio.com) or from your own donor constructs. OriGene also provides donor vector construction service. To order your donor vector service, please go to http://www.blueheronbio.com/Services/Genome-Editing.aspx
You can also select the following premade functional cassettes:
Cas9 Further Reading
- Genome editing of human pluripotent stem cells to generate human cellular disease models. Dis Model Mech. 2013 Jun 10
- Biotechnology: Rewriting a genome. Nature 2013 Mar 7;495(7439):50-1
- RNA-Guided Human Genome Engineering via Cas9. Science 2013 Feb 15;339(6121):823-6
- Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 2013 Feb 15;339(6121):819-23