Carrington et al (2015) describe another approach to functionally assessing guide RNAs for use with Cas9 that is quick, relatively easy and inexpensive.
After designing and assembling guide RNAs to be used to direct Cas9 to specific targets, investigators would like to be able to make an empirical assessment of those guide RNAs prior to fully investing time and effort in attempting to create genetically modified lines with the desired mutation. There are lots of approaches – sequencing lots of clones or small libraries, high resolution melt analysis (HRMA) and/or Surveyor/T7 endonuclease assays. All of these approaches have their advantages and limitations.
Carrington et al. work with zebrafish which are highly polymorphic leading, according to them, to false positive results when using HRMA and Surveyor/T7 endonuclease assays.
The authors were looking for an assay that was relatively easy and practical for users generating small numbers of mutants, and at the same time sensitive and quantitative.
What they describe is a fluorescent PCR-based method they call CRISPR-Somatic Tissue Activity Test or CRISPR STAT. The concept is simple – they use high resolution capillary electrophoresis to separate PCR products, some of which contain indels as a result of Cas9 cleave and end-joining repair. This collection of size variants can be resolved with single nucleotide resolution using capillary electrophoresis.
The authors show that these data provide a quantitative measure of a guide RNAs effectiveness.
More interesting perhaps is the finding that the correlation between the CRISPR-STAT results and the germline mutagenesis rates was very good. Of the 28 guide RNAs tested only 3 resulted in positive CRISPR-STAT results but did not yield germline mutations.
So if one has ready access to capillary electrophoresis, CRISPR-STAT may be a good way to assess one’s guide RNAs
Blake Carrington, Gaurav K. Varshney, Shawn M. Burgess, and Raman Sood (2015) CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity. Nucl. Acids Res. first published online August 7, 2015 doi:10.1093/nar/gkv802