Background

CRISPR/Cas9 nuclease is a ribonucleoprotein that is capable of cleaving a DNA double strand at a specific 22 nucleotide sequence. The major advantage of the CRISPR/Cas9 system compared to other nucleases such as zinc-finger nucleases and Transcription Activator-Like Effector Nucleases (TALENs) is that the sequence specificity is conferred by the RNA and does not require separate proteins for each target sequence. This reduces the cost dramatically, and a single construct can target as many as 32 targets. Due to this low cost and efficiency, the CRISPR/Cas9 system is now widely used in many plant species (Baltes and Voytas, 2015; Belhaj et al., 2015).

When a double-stranded DNA break induced by CRISPR/Cas9 is erroneously repaired by the NHEJ pathway, the repaired sequence most frequently results in small indels, among which one bp indels are the most common (Ma et al., 2015; Pan et al., 2016; Ren et al., 2016). One bp indel is too small for a polyacrylamide gel electrophoresis-based method to detect, therefore alternative methods capable of detecting one-bp indels are needed (Zhu et al., 2014).

Currently, the most commonly used method for small indel detection takes advantage of enzymes such as T7 endonuclease (T7E1) and CEL nuclease that cleave mismatches (Yeung et al., 2005; Vouillot et al., 2015). In these methods, the region including the target sequence is amplified by PCR, followed by the melting-annealing cycle to generate heteroduplexes and digestion by the mismatch detecting enzymes. While these methods are very effective in detecting large indels, they are not very effective in detecting a one-bp deletion. Even with T7E1 nuclease, which is better suited for the detection of small indels compared to the CEL nuclease, the efficiency decreases as the indel size decreases (Gohlke et al., 1994).

An alternative method, high-resolution melting (HRM), offers multiple advantages. HRM detects a small shift in the melting temperature (e.g., caused by heteroduplexing) using a dsDNA-binding dye. Firstly, it does not require additional pipetting steps after the PCR amplification step of the target region. Secondly, the method reliably detects single bp indels at a low (5%) concentration. In this protocol, we describe the procedure to analyze the HRM curves of PCR fragments containing CRISPR-generated small indels (Denbow et al., 2017). Four steps required for the detection of CRISPR-induced indels in Arabidopsis include: i) genomic DNA extraction, ii) optimizing the PCR condition, iii) PCR step for analyzing either T1 plants or putative homozygous plants carrying a single bp deletion, and iv) data analysis.