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Table 5 Practical guidance for the implementation of CRISPR technology in filamentous fungi based on data obtained for T. thermophilus in this study

From: Correction to: Practical guidance for the implementation of the CRISPR genome editing tool in filamentous fungi

 Plasmid-based approachRNP-based approach
Preparation of nucleaseCloning 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 presentCloning 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 RNAPlasmid-based, thus more stable during handling and storageInvolves in vitro transcription, hence potentially sensitive to handling errors
Transformation procedureEasyEasy but requires preassembly of RNPs
Transformation rateVery high also with four targetsVery high for single and double targets
Low for three and four targets
Single-targeting efficiency of FnCpf1, AsCpf1, SpCas9Locus-dependentLocus-dependent
Multiplex-targeting efficiency of FnCpf1High (34 ± 6% in this study)Low (13 ± 2% in this study)
MTP-based down-scaling for FnCpf1Possible with no loss in efficiency with respect to single and double targetingPossible with no loss in efficiency with respect to single targetinga
  1. aDouble targeting was not tested