USP26 gene variants have been reported to be associated with male infertility, such as non-obstructive azoospermia (NOA), oligozoospermia, and asthenoteratozoospermia, and may be associated with altered sperm sex chromosome segregation patterns. Among the reported variants, USP26 cluster variants (c.370_371insACA/c.494 T > C/c.1423C > T) represent a frequently reported variant pattern in certain populations. Rapid and convenient detection of USP26 cluster variants may serve as a powerful tool for male infertility-associated cohorts. Here, we develop a CRISPR/Cas12a-based assay to rapid detection USP26 variants in human samples. Candidate CRISPR RNAs (crRNAs) and RPA primers were screened and optimized, followed by incorporation of restriction enzymes to reduce wild-type-derived amplification interference. Using controlled artificial DNA mixtures, the assay detected mutant-template fractions down to 5% under the tested conditions. The complete workflow could be performed within 60 min using basic laboratory equipment. The results of the CRISPR/Cas12a system are consistent with Sanger sequencing in 91 male infertility research samples. Overall, our study establishes a rapid molecular detection method for detecting USP26 cluster variants and provides an alternative approach for genetic studies of male infertility-associated cohorts.
