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.