Table 5 Practical guidance for the implementation of CRISPR technology in filamentous fungi based on data obtained for T. thermophilus in this study
From: Practical guidance for the implementation of the CRISPR genome editing tool in filamentous fungi
Plasmid-based approach | RNP-based approach | |
|---|---|---|
Preparation of nuclease | Cloning of the nuclease into a plasmid prior transformation is mandatory. When constitutively expressed, risk of off-targets should be considered. When present on AMA-plasmid, the risk should be lower but still present | Cloning of the nuclease into a plasmid allowing heterologous expression, e.g. in E. coli, is a prerequisite. Once established and purified, the nuclease can be aliquoted and stored prior to use. As the protein does not become expressed in the targeted fungus, the risk of off-targets should be very small |
Preparation of guide RNA | Plasmid-based, thus more stable during handling and storage | Involves in vitro transcription, hence potentially sensitive to handling errors |
Transformation procedure | Easy | Easy but requires preassembly of RNPs |
Transformation rate | Very high also with four targets | Very high for single and double targets Low for three and four targets |
Single-targeting efficiency of FnCpf1, AsCpf1, SpCas9 | Locus-dependent | Locus-dependent |
Multiplex-targeting efficiency of FnCpf1 | High (34 % ± 6 % in this study) | Low (13 % ± 2 % in this study) |
MTP-based down-scaling for FnCpf1 | Possible with no loss in efficiency with respect to single and double targeting | Possible with no loss in efficiency with respect to single targeting* |