Table 2.

Screening of sgRNA activity

MethodDescription
Surveyor or T7E1 mismatch cleavage assayUses enzyme to cleave mismatched heteroduplex DNA after denaturation and reannealing; cleaved fragments represent mutated DNA on agarose gel, allowing estimation of efficiency
Tracking of indels by decomposition (https://tide.nki.nl/)Online program that determines the ratio of mutated alleles in a background of wild-type alleles; chromatogram from sequencing of the region around the sgRNA site is uploaded, and the algorithm calculates the ratio of mutated alleles
Deep sequencingThe most sensitive method: it provides not only efficiency of sgRNA edits but also, detailed sequence–based information on the kinds of mutations that each gRNA can generate