Table 1.

Comparison of rare allele enrichment methods

	dCas9-based method (12)	Cas9-DASH (10) and Cut-PCR (11)	NAVIGATER	NaME-PrO (36)	COLD-PCR (40)	Blocker (PNA or DNA)-PCR (37,41)
Enzyme	deactivated CRISPR Cas9 (high cost)	CRISPR Cas proteins (low cost)	TtAgo (low cost)	DSN (high cost)	N/A*	N/A
Guide/blocker	∼120 nt sgRNA (high cost)	∼120 nt sgRNA (high cost)	15/16 nt DNA (low cost)	20–25 nt DNA (low cost)	N/A	PNA (high cost) DNA (low cost)
PAM site requirement	Yes	Yes	No	No	No	No
Multi-turnover enzyme	No	No	Yes	Yes	N/A	N/A
Target	DNA	DNA	DNA & RNA	DNA	DNA	DNA
Incubation time	∼7 h	2 h	<1 h	22 min	∼2 h	∼2 h
Tight temperature control	No (37°C)	No (37°C)	No (66–86°C)	Yes** (98°C, 67°C)	Yes (thermal cycling)	Yes (thermal cycling)
Fraction detection limit in clinical samples	0.1% with allele-specific qPCR	0.01% with targeted deep sequencing	0.01% with XNA-PCR	0.01% with HRM/Sanger sequencing	0.1%-0.5% with MALDI-TOF	0.1%-1%
Enrichment level	Medium	High	High	High	High	High
Specificity	Medium	Medium-high	High	Medium	Medium	Medium
Multiplexing capability	Yes	Yes	Yes	Yes	Yes	Yes
Requirement for pre-amplification	No	No	No	No	No	No
Enrichment of cfDNA mutant fragments	No limitation	No limitation	No limitation	No limitation	No limitation	No limitation
Enrichment of mutant genomic DNA	No limitation	No limitation	Low efficiency	No limitation	No limitation	No limitation

*N/A – not applicable.

**After incubation at 98°C for 2 min, additional operations are required such as incubation at 67°C and then opening the tube to add DSN.