![]() For instance, iHDA requires 200-bp probe for mutation detection where the probe needs to be synthesized through a two-step PCR or cloning 13. Though these techniques can detect a wide range of indels, they does involve multiple steps. Improved versions of the HMA called improved heteroduplex analysis (iHDA) 13 and Probe-Induced HMA (PRIMA) 14 use DNA probes to detect 1-bp pair indels after gene editing. Heteroduplex Mobility Assay (HMA) can also distinguish between the heteroduplexes and homoduplexes DNA in native PAGE but indels smaller than 3 bp in size can be missed 11, 12. This assay, however, still cannot provide information about the presence or absence of frameshift mutations. Unlike T7E1/Surveyor assay, it can detect homozygous biallelic mutants. Another commonly used strategy for genotyping is CRISPR/Cas9-derived RNA-guided engineered endonucleases in Restriction fragment length polymorphism analysis (RGEN-RFLP) analysis 10. In addition, this technique does not provide information about types of mutations as well as the number of indels, which hampers researchers to screen out indels of nucleotides of multiples of three. A weak point of the technique is that it cannot differentiate homozygous biallelic mutants from wild-type nor heterozygous biallelic mutants from heterozygous monoallelic mutants. They can detect heteroduplexes formed by the hybridization of wild-type and mutated DNA strands or two differently mutated DNA strands 9. The T7E1/Surveyor assays 7, 8 are broadly used for detecting modified genes. In these cases, initial screening is important to choose “best possible” candidates before sequencing. However, in many cases, genotyping with genomic DNA is required in cases where reliable antibodies are not available or the samples are not cell lines, e.g. Western blot analyses can be chosen for screening if reliable antibodies are available and the samples are cell lines. ![]() One of the major challenges to perform screening of knockout (KO) clones that possess CRISPR/Cas9-induced frameshift mutations is the need for simple and cost-effective strategies that do not require special equipment and are available in standard laboratories and institutes. These indels are of interest to researchers to acquire frameshift null mutants for gene functional studies 6. These DSBs are predominantly repaired by error-prone non-homologous end-joining (NHEJ) pathway often producing mutations of small nucleotide substitutions or insertion/deletion (indel) at the targeted sequence 5. The Cas9 nuclease combined with a short single-guide RNA (sgRNA) allows researchers to create site-specific double-strand breaks (DSBs) 2– 4. Thus, DST-PCR is a simple and fast method to screen KO candidates generated by the CRISPR/Cas9 system before the final selection of clones with sequencing.Ī prokaryotic RNA-mediated adaptive immune system known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been developed into a revolutionary genome-editing technology 1. In addition, this technique also allowed the identification of heterozygous and homozygous biallelic functional KO candidates. This method allowed us to detect 1-bp to 2-bp insertion and 1-bp to 4-bp deletion in one or both alleles of mutant cell clones. We present actual cases of screening of CRISPR/Cas9-engineered knockout (KO) cells for six genes, where we screen indels to obtain potential KO cell clones utilizing our approach. Generated amplicons are directly subjected to TBE-High-Resolution PAGE, which contains a high concentration of bis-acrylamide, for mutant clones detection with 1-bp resolution. Here, we developed a simple genotyping method called DST-PCR (Double-strand break Site-Targeted PCR) using “face-to-face” primers where the 3’ ends of forward and reverse primers face each other at the position between 3-bp and 4-bp upstream of the PAM sequence, which is generally the Cas9-mediated double-strand break site. Technically simple and costly reasonable genotyping methods are awaited to efficiently screen the frameshift null mutant candidates. The introduction of small insertion/deletion (indel) mutations in the coding region of genes by the site-specific nucleases such as Cas9 allows researchers to obtain frameshift null mutants.
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