Table 1.
Comparison of different CRISPR-Cas9-based multiplexed gene-editing systems
| CRISPR systems | Efficiency | gRNA processing | Cas9 pre-transformation | Required additional procedures | Total time spenta |
|---|---|---|---|---|---|
| Plasmid-required systems | |||||
| HI-CRISPR14 | 3 targets with 100% | Endogenous | No | Gene synthesis | 10–13 days |
| CRISPRm7 | 1–3 targets with 81–100% | Self-cleavable ribozymes | No | Helper plasmid | 8–10 days |
| Csy4-based CRISPR8 | 4 targets with 96% | Csy4 cleavage | Yes | Helper plasmid | 11–13 days |
| CasEMBLR5,26 | 1–5 targets with 50–100% | Individual cassette | Yes | Helper plasmid | 11–13 days |
| CRISPR by Mans and Rossum et al.15 | 6 targets with 65% | Individual cassette | Yes | 3 plasmids for 6 gRNAs | 8–9 days |
| GTR-CRISPR (this work) | 8 targets with 87% | tRNA processing | No | No | 6–7 days |
| Cloning-free systems | |||||
| CRISPR by Generoso et al.16 | 2 targets with <15% | Individual cassette | No | No | 2 days |
| CAM17,27 | 3 targets with 64% | Individual cassette | Yes | No | 5 days |
| Lightning GTR-CRISPR (this work) | 4 targets with 96% 6 targets with 60% |
tRNA processing | No | No | 3 days |
CRISPR Clustered Regularly Interspaced Short Palindromic Repeats, gRNA guide RNA
aTotal time was calculated including Cas9 pre-transformation, gene synthesis, plasmid construction, and yeast transformation (details as shown in Supplementary Table 2)