Quick learning of CRISPR/Cas9
What is CRISPR? What is Cas9? How does CRISPR system work? What is needed to perform genome engineering in your lab? This short video introduces the basics of this novel technology, as well as genome editing/knockout products offered by OriGene. For more details, please visit http://www.origene.com/CRISPR-CAS9/
Targeted genome modification has been hampered by the lack of an efficient tool to create a double-stranded break at a designed location. The CRISPR/CAS system is an RNA-guided nuclease system for the targeted introduction of double stranded DNA cleavage.
Originally discovered in bacteria as an acquired defense against foreign pathogens, CRISPR/CAS technology has been demonstrated to work successfully in all the common model organisms, making it one of the hottest technology breakthroughs in 2013.
Targeted genomic cleavage requires two key elements: a homing device and an endonuclease. In the CRISPR/CAS platform, the homing device is guide RNA, or gRNA and a CAS nuclease.
The gRNA contains a twenty-nucleotide target sequence immediately upstream of a Protospacer Adjacent Motif or PAM, linked to a tracrRNA scaffold. This is sufficient to direct the CAS9 nuclease to the complementary site in the genome and create a double-stranded break.
Compared to previous genome editing methods, such as ZNF and TALEN, CRIPSR/CAS is cheaper, quicker, and more accessible for researchers.
Due to its amazing simplicity, CRIPSR-based genome editing can be achieved with a simple transfection.
OriGene offers an all-in-one vector, pCas-Guide, engineered with all the essential elements for targeted cleavage.
• The codon-optimized Cas9 expression
• A cloning site for a twenty nucleotide target sequence
• And a gRNA scaffold downstream of the cloning site to be transcribed into a complete gRNA by a U6 promoter.
With a target sequence cloned into this vector, cells transfected with these constructs will express Cas9 nuclease and the guide RNA, which will then lead to a sequence-specific double-stranded break in the genome.
Other vector variations are also available to provide added convenience, such as GFP for transfection monitoring or Lenti-backbone for viral delivery. For researchers who perform mRNA microinjection or mRNA transfection, a T7 driven expression vector is available.
With the CRISPR/CAS system, genome editing is now smooth sailing. Two types of editing are commonly used. Homologous recombination utilizes a repair template. The desired changes are flanked by left and right homologous arm sequences. Upon double crossover, the desired change is integrated into the genome.
Applications include gene knockout, gene-tagging, and site-specific mutagenesis.
Non-homologous end joining, where the ends of the break are joined together, introduces random mutations, insertions, and deletions.
This can also be used to create variant libraries.
Applications for CRISPR/Cas9 are endless. You can generate an in-del variant library at a targeted locus.
You can knock out a target gene and simultaneously knock-in a functional cassette, such as a mammalian selection marker or a fluorescent marker.
And you can introduce pre-designed mutations.
One common application is the promoter study. It requires the insertion of a reporter gene, such as luciferase and/or a GFP, at the first exon of a target gene. This results in knocking out the target gene and knocking in the reporter, therefore enabling the researcher to monitor the promoter activity.
For this application, OriGene offers a complete solution. Simply search with a gene symbol, and you can find a fully functional kit containing
• Two gRNA vectors,
• One donor vector,
• And one scramble control vector
Another common application is the safe-harbor insertion of transgenes. Compared to other transgene insertion methods, this method provides total control, for insertion site, orientation, as well as for copy number.
A ready-to-use kit for this application is coming soon from OriGene as well.
A ready-to-use kit for this application is now available from Origene.
Origene brings you a complete CRISPR/Cas solution that is simple and easy to use. To learn more please visit the Origene website.