# Surveyor Assay or T7EI Assay

## Target Site

The CRISPR target site is depicted in the form N20NGG, and the location of the cutsite (3-4 nucleotides upstream of the PAM site) is indicated by the arrow. After cleavage in the cell, mutations and indels will be centered around the cutsite.

## Heteroduplex formation

First, flanking primers will be used to amplify the genomic region surrounding the CRISPR target site. It is essential to have a clean PCR product with only one specific band on a gel. It is also a good idea for the left and right primers to be spaced differently with respect to the cutsite, resulting in asymmetric cut bands that are easy to discern on a gel. Next, the purified PCR product is denatured and slowly re-annealed to promote the formation of heteroduplexes. DNA strands should randomly re-shuffle and anneal to each other, creating bulges and helix distortions where the sequences differ.

## Heteroduplex Cleavage

Both Surveyor and the T7EI enzyme recognize and cleave DNA at mismatched bases due to imperfect complementarity between the two DNA strands (heteroduplex). This process allows for bulk mutation detection that would not be detectable by standard Sanger sequencing of the PCR product.

## Gel

The products of the Surveyor or T7EI assay are resolved on an agarose or PAGE gel. Here, the two cut bands are shown as two clearly resolved products due to an asymmetric cutsite with respect to the PCR primer locations.

## Quantification

The resolved products from the gel analysis are visualized and quantitated using software like ImageJ. The negative control lane indicates no cleavage, meaning that the DNA strands are perfectly matched. The positive control indicates full-cleavage, which would be detected if every DNA helix was a heteroduplex. More typical experimental results are shown in lanes 3 and 4. The ratios of the uncut to cut bands can be used to approximate the bulk percentage of mutations in the starting population. The equation is:

$$\%\ Modification = 1-\sqrt{(1-fraction\ cleaved)} * 100$$

where:

$$fraction\ cleaved = \frac{cut\ bands}{(uncut\ band + cut\ bands)}$$

Alternatively, if ‘a’ and ‘b’ are equal to the integrated area of the cleaved bands, and ‘c’ is equal to the integrated area of the parental band the equation is:

$$\%\ Modification = 1-\sqrt{1-\frac{(a+b)}{(a+b+c)}} * 100$$

The equation to paste into Excel would be:

=100*(1-(SQRT(1-((A1+A2)/(A1+A2+A3)))))

where:

Cut band 1 = A1
Cut band 2 = A2
Parental band = A3

And, A1, A2, and A3 refer to cells in Excel.

# Restriction Digest

## Target Site

The CRISPR target site is depicted in the form N20NGG, and the location of the cutsite (3-4 nucleotides upstream of the PAM site) is indicated by the arrow. The sequence corresponding to a convenient restriction site is indicated in red. After cleavage in the cell, mutations and indels will be centered around the cutsite. This will cause disruptions in the restriction sequence that would not be recognized or cut by the enzyme.

## Digestion

The PCR products are simply digested with the enzyme. (Unlike the mismatch assays, this is performed without prior heteroduplex formation.) Unmodified PCR products retain the restriction site and are completely digested, while PCR products with indels and mutations that disrupt the restriction site, due to genome modification, remain uncut.

## Gel

The products of the Surveyor or T7EI assay are resolved on an agarose or PAGE gel. Here, the two cut bands are shown as two clearly resolved products due to an asymmetric cutsite with respect to the PCR primer locations.

## Quantification

The resolved products from the gel analysis are visualized and quantitated using software like ImageJ. The negative control lane indicates no cleavage, meaning that the restriction site sequence has been changed. The positive control indicates full-cleavage, and the presence of an intact restriction site. More typical experimental results are shown in lanes 3 and 4. The ratios of the uncut to cut bands can be used to approximate the bulk percentage of mutations in the starting population. The equation for quantification by restriction analysis is:

$$\%\ Modification = \frac{cut\ band}{(uncut\ bands + cut\ bands)} * 100$$

Alternatively, if ‘a’ and ‘b’ are equal to the integrated area of the cleaved bands, and ‘c’ is equal to the integrated area of the parental band the equation is:

$$\%\ Modification = \frac{(a+b)}{(a+b+c)} * 100$$

The equation to paste into Excel would be:

=100*((A1+A2)/(A1+A2+A3))

where:

Cut band 1 = A1
Cut band 2 = A2
Parental band = A3

And, A1, A2, and A3 refer to cells in Excel.