Tuesday, May 27, 2014

New web-based target and off-target locator for #CRISPR etc.: GT-Scan. gt-scan.braembl.org.au

... I haven't tried GT-Scan yet but it has features I like such as: (1) web-based, (2) user-defined flexibility for specifying the target.    GT-Scan: Identifying unique genomic targets.
 2014 May 23. pii: btu354. [Epub ahead of print]

Tuesday, May 13, 2014

#CRISPR plasmid mouse pronuclear injection service at Vanderbilt TMESCSR core facility.

A service request form is now online for mouse embryo CRISPR injection requests via the Vanderbilt TMESCSR (transgenic core facility).  Although from a technical standpoint the method is basically the same as standard pronuclear injections, we have modified our pronuclear injection form to reflect the different information that goes into a CRISPR gene targeting experiment.  

Thursday, May 1, 2014

#CRISPR genomic target structure clarified here in my blog post. Target = protospacer + PAM.

When discussing CRISPR "targets", it's important to use precise terms to describe the important features.    The genomic target of CRISPR-Cas9 is composed of two parts: (1) the protospacer and (2) the protospacer-adjacent-motif, or PAM.

Note that the PAM does not base-pair with the CRISPR/guide RNA at all.   It appears to interact with the Cas9 protein directly.  The CRISPR RNA only base pairs with the DNA base that are complementary to the protospacer.   

It's wise not to use "target" and "protospacer" interchangeably in CRISPR-related discourse; it causes headaches at the design stage.  Bottom line: screen your genomic sequences of interest for occurrences of the full target, but only the protospacer (not the PAM) becomes part of the CRISPR/guide RNA.