Direct delivery of CRISPR-Cas9 complex in plant cells
Woo, J. W. et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat. Biotechnol. 10–13 (2015). doi:10.1038/nbt.3389
Photo Credit: Je Wook Woo
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RNA-guided endonucleases (RGENs)
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Cas9 protein-gRNA ribonucleoproteins(RNPs)
purfied Cas9 protein + *2~10 gRNAs
protoplast of Arabidopsis, rice, tobacco and lettuce
PEG transformation
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Mutation frequency test: T7 endonuclease I (T7E1) assay; targeted deep sequencing
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in arabidoposis: 2 DSBs- targeted deletion of intervening sequence
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mutation detected after 24h after transfection!
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off target: almost no in lettuce
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mutation maintained afrer regeneration; transmittable to progeny
Advantage of preassembled RGEN RNP delivery:
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no foreign DNA plasmid (bypassing regulation?)
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fast mutation after transfection
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degraded rapidly by proteases in cells (reduce mosaicism and off-target)
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no need to optimize codon and promoter (widely applicable)
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enables in vitro prescreening to guide the choice of highly active gRNAs and genotyping of mutant clones via restriction fragment length polymorphism (RFLP) analysis.
Direct delivery of meganuclease and TALENs in plant cells
Luo, S. et al. Non-transgenic Plant Genome Editing Using Purified Sequence-Specific Nucleases. Mol. Plant 8, 1425–1427 (2015).
meganuclease delivery:
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smallest size of RGENs- easy delivery?
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I-SceI meganuclease alone: no mutagenesis detected
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I-SceI +Trex2 DNA: 7.7% mutagenesis
TALENs delivery:
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TALEN monomers: ALS2T1L/1R in tobacco
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TALEN protein: 1.4% mutagenesis
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TALEN plasmids: 18.6% mutagenesis