TABLE 4.
List of selected Cas9 and Cas12a homology-directed gene targeting mechanisms and their efficiencies in crops and animals.
| Nuclease | HDR Enhancing System | Target Gene | Target Organism/plant | HDR efficiency percentage | References |
| FnCas12a and LbCas12a | Repairing DNA template flanked by 1000 bp homology DNA fragments/arms | Chlorophyllide-a oxygenase gene (CAO1) | Rice | 3-8 | Begemann et al., 2017 |
| LbCas12a | Ribonucleo Protein (RNP) Complex | slc45a2(albino) and tyr (tyrosinase) | Zebrafish and Xenopus | 5-7 | Moreno-Mateos et al., 2017 |
| Synthesis dependent repair with donor repair template coupled with left homologous arm is sufficient for HDR mechanism | Acetolactate synthase gene (ALS) | Rice | 0.014 | Li et al., 2018a | |
| 0.021 | |||||
| Ribozyme based strategy to synthesize crRNA’s and DNA repair template | Acetolactate synthase gene (ALS) | Rice | 4.6 | Li et al., 2019a | |
| 1.7 | |||||
| Utilization of homologous recombination enhancers | Acetolactate synthase gene (ALS) | Arabidopsis | 1.47 | Wolter and Puchta, 2019 | |
| Utilization of viral multi replicon system (de novo engineered geminiviral replicon system) to increase the availability of donor template | Salt-tolerant (SlHKT1;2) | Tomato | 4.5 - 9.8 | van Vu et al., 2020 | |
| CRISPR-SpCas9 | SpCas9, sgRNA and single-stranded DNA oligo’s (72 base pair) into plant cells | Phytoene desaturase-OsPDS | Rice | 6.8 | Shan et al., 2013 |
| CRISPR-Cas9 | gRNAs targeting Lig4 gene were transformed with Cas9- Lig4 knockout resulted in enhancement of HDR | Acetolactate synthase gene (ALS) | Rice | 0.15-1.0 | Endo M. et al., 2016 |
| Geminivirus based vectors to release abundant HDR template | Actin-1 (ACT1) | Rice | 6.8 - 19.4 | Wang et al., 2017a | |
| Glutathione S-transferase (GST) | 7.7 | ||||
| CRISPR-SpCas9 | DNA donor template contains constitutively expressing PAT gene with 1 kb homologous arm surrounding the target gene | Liguleless1-LIG1 | Maize | 0.2 - 4.61 | Svitashev et al., 2015 |
| Male fertility genes - Ms26 | 0.13 - 3.11 | ||||
| Male fertility genes - Ms45 | 0.47 - 1.87 | ||||
| Acetolactate synthase gene (ALS1 and ALS2) | 1.35 - 2.23 | ||||
| CRISPR-Cas9-VirD2 | Cas9-VirD2 chimeric protein helps in DSB and bringing close proximity of phosphorothioate mediated template DNA through VirD2 relaxase | Acetolactate synthase gene (ALS) | Rice | 4.1 - 20.8 | Ali et al., 2020 |
| Histone Deactylase (HDT) | 0.2 - 8.7 |