A mixing heteroduplex mobility assay (mHMA) to genotype homozygous mutants with small indels generated by CRISPR-Cas9 nucleases

Abstract

The development of gene editing technologies, especially the CRISPR-Cas9 system, has been pivotal for understanding the functional role of proteins. Rapid and efficient genotyping methods are necessary to screen for generated mutations and streamline the isolation of homozygotes. CRISPR-Cas9 system targeting a single site in the gene typically results in small indels. Many genotyping methods utilize the heteroduplex that is formed when wild-type and mutant amplicons with small indels anneal during PCR creating a bubble due to mismatched strands. These methods include T7 endonuclease/Cel-I assay, high resolution melting (HRM) analysis, and heteroduplex mobility assay (HMA). Our protocol explains a simple, two step method of a mixing HMA (mHMA) to identify homozygous mutants, a modification of the previously published HMA. We have utilized the mHMA for screening and genotyping numerous CRISPR generated models. The mHMA method to differentiate homozygous wild type from homozygous mutant animals eliminates - •DNA sequencing, even with small indels that can be difficult to discern on a gel.•additional enzymatic reaction steps, such as with the T7EI/Cel-I assay.•specialized equipment and analysis tools, such as with HRM analysis.

Keywords: Gene editing; Genotyping; HMA; Homozygosity; Mixing heteroduplex mobility assay (mHMA) for screening homozygous mutants; PCR; Simple CRISPR-Cas9; Zebrafish.

Graphical abstract

Fig. 1

CRISPR homozygous null mutation detection by mixing heteroduplex mobility assay (mHMA) in zebrafish. Image of ethidium bromide stained polyacrylamide gel (6%) showing separation of homoduplex and heteroduplex PCR amplicons. Amplicons were obtained from three HMA-identified homozygous F1 zebrafish generated from a G1 heterozygous cross. Small and large brackets indicate homoduplex and heteroduplex bands respectively. The initial F1 HMA PCR showing a single band (1–3) is loaded first with mixed WT/F1 PCR (1+, 2+, 3+) loaded in the next lane to the right for direct comparison. Underlined # + = inferred homozygous mutant; Not underlined # + = inferred homozygous wild type; L = 100 bp ladder; G1 = G1 heterozygous parent; C = wild-type control.