In the CRISPR-Cas9 system, different guide RNA (gRNA) can be designed based on the target gene. The DNA cutting efficiency of Cas9 can vary depending on the gRNA and target gene. It is therefore important to be able to accurately verify the activity of the CRISPR-Cas9 system in a given assay, in order to maximize the cleavage and gene knock-in/knock-out efficiency of CRISPR-Cas9. Synbio Technologies provides SSA activity assays, in vitro cleavage activity assays, and endogenous activity assays to detect gRNA activity.
SSA Activity Assays
Single-strand annealing（SSA）assays can verify whether the target gRNA plasmid can mediate Cas9 to cut naked DNA, It is a common method to assess the activity of CRISPR-Cas9. The plasmid-based SSA assay contains two inactive luciferase-encoding fragments, which contain a stop codon and a segment of gRNA in the middle of the target sequence. If CRISPR-Cas9 can recognize and cleave the target site, the resultant double-stranded break can be repaired by the SSA mechanism, rescuing the now-active luciferase gene. Thus the SSA assay can be used to predict the cleavage activity of CRISPR-Cas9 by fluorescence detection.
Verification of in vitro Cleavage Assay
The target DNA sequence is digested with Cas9 in vitro to obtain two DNA fragments. Agarose gel electrophoresis allows analysis of gRNA-mediated DNA cleavage by comparing the yield of the smaller, digested fragments to the remaining undigested target DNA. Verification of in vitro cleavage is another way to determine the activity and efficiency of CRISPR-Cas9.
Endogenous Activity Assay
Surveyor method is called mismatch endonuclease assay. It uses T7E1 endonuclease. The suitable primers are first designed on both sides of the target, followed by the PCR amplification of the DNA containing potentially mismatched mutation sites. T7E1 endonuclease can recognize and cleave mismatched DNA heteroduplexes. The digested product is finally analyzed by agarose gel electrophoresis to estimate the ratio of the cleavage and un-cleaved bands, reflecting the activity of CRISPR-Cas9.