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Table 2 Advantages and disadvantages of different methods for CRISPR/Cas9-mediated mutant screening. (Adapted from Bao et al. 2019)

From: How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis

MethodAdvantagesDisadvantagesReference
CAPS
A RE site within the DNA target site is destroyed by a genome-editing mutation
Simple, fast, economical, and can detect homozygous and heterozygous mutantsLimited to the original target sequences(Shan et al. 2014)
indCAPS
A RE site is created using mismatch primers next to a DNA target site
More flexibility for different types of indelRequires designing specific primers to distinguish known indel alleles(Hodgens et al. 2017)
T7E1 cleavage assay
T7 endonuclease 1 digests mismatched heteroduplexes formed between wild-type strands and mutated strands
Simple, fast, economical, and can detect heterozygous mutantsCannot detect homozygous mutants(Vouillot et al. 2015)
PAGE
Homoduplex DNA migrates faster than heteroduplex DNA in native PAGE
Simple, fast, economical, and can detect homozygous and heterozygous mutantsTime consuming and low throughput(Zhu et al. 2014)
HRM
Homozygous DNA has a unique melting temperature (Tm), while mutated heterozygous DNA has a lower Tm
Fast and efficient for detecting SNPs and indels in mutantsRequires specific instrumentation and sensitivity is affected by amplicon size(Thomas et al. 2014)
ACT-PCR
A critical annealing temperature in PCR suppresses the mismatched annealing of the primer to the template, inhibiting the production of amplicons
Simple, fast, economical, and can detect homozygous mutantsRequires designing specific primers and is time consuming and/or labor intensive(Hua et al. 2017)
PCR- and labeling-based assaySimple, effective, and sensitiveNot able to reveal the exact nucleotide change in the mutant(Biswas et al. 2019)
Whole-genome sequencingIdentifies on-target and off-target mutationsCostly and time consuming(Tang et al. 2018)