| Popis: |
CRISPR has become widespread as a method for generating targeted mutations in C. elegans. Screening for introduced point mutations in C. elegans is aided by the use of Co-CRISPR strategy, which takes advantage of co‑editing an unrelated locus (such as dpy-10) to visibly identify the worms in which at least one efficient Cas9 mediated homologous recombination has occurred (Arribere et al. 2014; Kim et al. 2014; Paix et al. 2015). In most current methods being used, detection of the edit is performed using restriction digestion of a PCR product and sequencing 100-150 potential candidates. To make the process of screening for point mutations more efficient and a one step process (a simple PCR), we have adapted the tetra-primer amplification refractory mutation system (ARMS)-PCR method for use with CRISPR (Sullenberger and Maine 2018). The ARMS-PCR method utilizes 4 PCR primers (Figure 1A), two outer primers at different distances from the point mutation, and two primers in opposing directions which terminate on the point mutation, with a wildtype primer in one direction containing the wildtype nucleotide at its 3’ end, and a mutant primer in the reverse direction containing the mutant nucleotide at its 3’ end (Figure 1A). While this single 3’ mismatch in a primer does not prevent amplification of the incorrect template, an additional mismatch is added to both primers 1-3 nucleotides from the 3’ end to further destabilize the internal primers. This additional mismatch decreases the amplification of the incorrect primer to a greater extent than the correct primer, allowing each primer to only amplify from its respective template. Since the primer binding is unstable, however, the detection rate of false positives and false negatives (due to improper primer destabilization) is relatively high. Significant optimization of PCR conditions, including concentrations of PCR components such as Mg2+, is often required to obtain a high level of specificity for amplification of the allele specific product (Medrano and de Oliveira 2014). This degree of PCR condition optimization in the absence of an existing allele is not possible and thus the ARMS-PCR is not useful for screening CRISPR-Cas9 based genome edited mutations. Here we present CEPAD-PCR, an allele-specific PCR detection method based on generating silent mutations around the site of the desired genetic lesion during the CRISPR-Cas9 genome editing process. |
