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CRISPR Cas9 System Activity Detection.pdf


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CRISPR Cas9 System Activity Detection
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 and are 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.

Construction of plasmid-based SSA

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