Sanger Sequencing for Screening and Confirmation of Mutations Generated from CRISPR Genome-editing

A simple introduction of CRISPR

Clustered Regularly Interspaced Short Palindromic Repeat or CRISPR as a part of bacterial defense system against bacteriophage is used as a dominating gene-editing tool to make changes in targeted DNA sequences. The most popular CRISPR-Cas9 system is developed to use guided RNA that targets sequence of interest, which is bound by the Cas9 protein, the DNA nuclease enzyme, to cleave the sequence next to the PAM (protospacer adjacent motif) site. The double strand break (DSB) created is further repaired and a mutation (point mutation or indels-insertions or deletions) is created by non-homologous (non-homology end joining, NHEJ) and homologous repair (homology-directed repair, HDR), respectively. A thorough review on CRISPR and applications provides good information on the technology.

Screening of mutations generated by CRISPR

Since the CRISPR is does not always generate a mutation after introduction into a specific cell line, it is necessary to identify the mutations and get a pure mutant clone. Generally, it will need the following two steps: (1) screen a population of cells and check for gene-editing efficiency and (2) isolation and confirmation of individual clones that contain the desired mutations.

There are multiple ways to screen the cell population for specific mutations, including PCR, PCR followed by restriction enzyme digestion, T7E1, Sanger sequencing, and NGS. Although PCR is often the first step in some of these methods, PCR by itself is very limited. It works well when insertion or deletion can be clearly detected by difference in size (>50bp) when the mutation is created by HDR. But this method failed to detect mutations created by NHEJ), which could be an indel of only a few nucleotides. Therefore, other methods add a different spin to make detection possible without considering the size difference between the wildtype and the mutant.

PCR followed by restriction enzyme digestion uses restriction enzyme site design at the cleavage site, either by adding or removing a unique restriction site. Therefore, the restriction enzyme digestion of the PCR products is able to distinguish the mutants from the wildtype sequences. But the design is necessary and specific for each sequence, not generic for all the mutant screenings.

T7E1 assay uses endonuclease 1 from T7 phage (T7E1) to cleave at the heteroduplex mismatched sequences. Although the method can usually detect insertions or deletions (indels) with three nucleotides, it fails to detect a single nucleotide indels. The method also failed to tell if the mutation is homogenous when both strands contain the same mutations. In addition, this method is also tedious and time consuming.

Traditional Sanger sequencing analysis is not able to tell sequences with mixed population. However, multiple software packages developed to deconvolute the mix sequence information (see below) have been successfully used and is cost-effective compared to NGS if only a small screening project is needed. NGS, a high throughput sequencing tool better for large projects and sample sizes, is powerful for identification of target as well as off-target effect of CRISPR while Sanger sequencing can’t tell any off-target effect.

Sanger sequencing for screening and mutation verification

Sanger sequencing is a powerful tool for screening of guide RNAs and estimating editing efficiencies. Sanger sequencing can accurately identify the mutations quantitatively with the help of the software mentioned above. For sanger sequencing, the PCR products are first made to cover the targeted cleavage site using the DNA templates from the heterogenous population of the transfected cells. The PCR products containing both the wildtype and mutations are then sequenced by Sanger sequencing and the data are analyzed by the software. In addition to mutation screening, Sanger sequencing is also used for verification of isolated pure clones selected from a pool of mixed clones.

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