Bioinformatic analysis of SARS-CoV-2 genomes and gRNA and RPA primer design

gRNA design

Extensive Cas12a-gRNA design instructions can be found here (steps 1-8): https://www.nature.com/articles/s41596-020-0367-8?proof=t

Primer design

RPA requires two pre-amplification primers (forward and reverse).

The primer lengths should be ~25–35 nt, and the total amplicon size should be 80–140 bp. The region that is targeted by the Cas12a should be in the amplicon, not part of the primers. Primers are typically designed with melting temperatures between 54 and 67 °C. We typically design 4-6 forward and reverse primers, following instructions from RPA kit manufacturers to find an optimal pair that is specific and sensitive. The CRISPR detection reaction is very specific, thus it is not necessary to evaluate primer sets using gel electrophoresis to look for specific amplicons. 

We recommend the following Nature protocols paper for reference (https://www.nature.com/articles/s41596-019-0210-2?proof=t). It contains an extensive protocol on Cas/RPA reactions for Cas13a. A key difference between Cas12a (the enzyme used in miSHERLOCK) and Cas13a is that Cas12a targets dsDNA; Cas13a target RNA. Thus, for miSHERLOCK, the T7 promoter upstream of the target region is not required.

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