Surveyor Nuclease Assay.

Surveyor nuclease assays were performed on amplicons across the C site using primers P1 (5′-cgctgcgtgacaagagaata-3′) and P2 (5′-cacacacacttctggggaca-3′) and HiFi Platinum PCR Supermix (Thermoscientific), and genomic DNA was purified as described (25). The amplification conditions were 94 °C for 5 min followed by 34 cycles of 95 °C for 45 s, 59 °C for 45 s, and 68 °C for 1 min and a final extension at 68 °C for 7 min. A fraction of the amplicon was quantified using gel electrophoresis and densitometry analysis, and 200–300 ng amplicon was used for the surveyor nuclease assay according to the manufacturer’s instructions (IDT) and as described (17). Briefly, the first step of this assay is to melt and anneal to allow for heteroduplex formation. Specifically, 200–300 ng amplicons were heated at 95 °C for 10 min followed by cooling to 85 °C at 2 °C/s and subsequently, 25 °C (−0.3 °C/s), with a 1-min hold at every 10°. After heteroduplex formation, 1 μL surveyor nuclease and 1 μL enhancer were added to each reaction, and the samples were incubated at 42 °C for 1 h. Reactions were resolved on a 2% (wt/vol) agarose gel and stained with ethidium bromide, and images were captured using an FC2 Flourchem Gel Imaging System (Alpha Innotech).

The relative staining intensity of the 385-bp (intact) and 251-bp (cleaved) fragments for each sample was quantified using ImageJ. To account for the smaller size of the 251-bp fragment, the staining value was multiplied by 1.53 (adjusted cleaved fragment). To calculate percentage of cleaved product, the normalized cleaved band intensity was divided by the sum of the normalized cleaved fragment intensity and the uncleaved fragment intensity. The unmodified alleles are present in excess to the modified alleles in the original sample, such that after amplification of the C site and initial denaturing of the amplicons, each strand of the modified amplicon may form a heteroduplex with a complementary strand from the unmodified amplicon (17). Thus, to account for the phenomenon that each modified allele can contribute to two heteroduplex products, we used the previously described formula to convert the fraction of cleaved products to percentage of C-site mut: 100 × (1 − (1 − cleaved)^0.5) (17). The cleaved products caused by heteroduplex formation from short-indel mutations are likely to comigrate with the 0.25-kb cleaved band in the gel separation. However, the cleaved products from long-indel mutations could potentially migrate away from the cleaved band and hence, may not be represented in this assay. Thus, the surveyor assay may underrepresent indel mutation frequencies.