Table 2 Advantages and disadvantages of different methods for CRISPR/Cas9-mediated mutant screening. (Adapted from Bao et al. 2019)
Method | Advantages | Disadvantages | Reference |
|---|---|---|---|
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 mutants | Limited 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 indel | Requires 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 mutants | Cannot 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 mutants | Time 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 mutants | Requires 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 mutants | Requires designing specific primers and is time consuming and/or labor intensive | (Hua et al. 2017) |
PCR- and labeling-based assay | Simple, effective, and sensitive | Not able to reveal the exact nucleotide change in the mutant | (Biswas et al. 2019) |
Whole-genome sequencing | Identifies on-target and off-target mutations | Costly and time consuming | (Tang et al. 2018) |