CRISPR Cas9 Explained

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.

[eCitizen]

This is like listening to a foreign language spoken entirely in English.

[emilyse]

When you know nothing and must learn everything in a week...

[chalknduster]

This revolutionary technique deserved the 2020 Nobel prize. Women in science deserved it.

[invertebrateargus]

This makes things so much easier. Thank you

[tru-fab]

i didn't realize it was this easy. Thanks

[Viraj_]

Awesome very helpful to understand this technique! Thank you very much ORIGENE team!

[ashtongrist]

But how do I learn what those words are?

[billstuart]

I think I watched a similar video describing the Turbo Encabulator.

[OriGeneMarketing]

A more in-depth webinar given by 3 CRISPR experts are available at
CRISPR Webinar by Drs. Barrangon, Zhang and Church

[OriGeneMarketing]

OriGene also recorded an in-depth webinar at
Webinar: Genome Editing with CRISPR/Cas9

[edyoucate]

Wow nice video, thanks for helping me understand this topic

[rayfox]

I just want a kit so I can turn all my plants florescent with luciferase

[fireteamomega]

How do you acquire pam for cleavage specifications if there's no donor required ?

[davidpullen]

Scary stuff here!!... just know it's a technology to "edit" the genes of Humans in the it's "smooth sailing" for genome editing...

[Julia]

can any of this help someone who has had strokes and sever brain damage?

[philg]

I saw an ad similar to this in an old parallel Kubrick sci flick in a tangential universe

[vitorcasarin]

Hi there ! If you modified the DNA of an adult, do you believe his/her body would eventually adapt to those changes? (Such as growing taller, etc) Or does it only works with embryos? Thanks !

[nazra]

So, how long are we away from actually using this in humans to cure diseases? Centuries? Decades?

[ferryjap]

Is there any CRISPR product for therapeutic ? How can I order it.?

[karthicr]

Hi, this vedio is really easy to understand the complex system. I would like to make a gene knock out in Lamda phage. I am little confused, how to use this sytem for lamda phage gene knockout?