Table 1.

Major characteristics of general methods for rare allele enrichment and detection

Method	Nuclease	Guide/blocker	Sensitivity	Specificity	Enrichment efficiency	Multiplex	Target motif requirement	Tight temperature control	Operations
COLD-PCR ( 6,11)	-	-	0.01% with NGS	Medium	500-fold	Yes	No	Yes	PCR
BDA ( 5 )	-	DNA blockers	0.01% with qPCR	Medium	10000-fold	Yes	No	No	PCR
HOLMESv2 ( 26 )	Cas12	∼120 nt sgRNA	0.1% with fluorescence detection	High	-	No	PAM	No	LAMP + Nuclease reaction
SHERLOCKv2 ( 28 )	Cas12, Cas13 and Csm6	∼120 nt sgRNA	0.6% with fluorescence detection	High	-	Yes	PFS/PAM	No	RPA + Nuclease reaction
PAND ( 41 )	PfAgo	16 nt gDNA	0.1% with fluorescence detection	High	-	Yes	No	No	PCR/tHDA + Nuclease reaction
Cut-PCR ( 27 )	Cas9	∼120 nt sgRNA	0.01% with NGS	High	18-fold	No	PAM	No	Nuclease reaction + PCR
NAVIGATER ( 36 )	TtAgo	16 nt gDNA	0.01% with XNA-PCR	High	60-fold (0.5% VAF)	Yes	No	No	Nuclease reaction + dPCR
A-Star	PfAgo	16 nt gDNA	0.01% with TaqMan qRT-PCR	High	5500-fold	Yes	No	No	Nuclease reaction in PCR