So many genes, so little time: targeting systems for gene destruction Thomas Jacobs Center for Applied Genetic Technologies Institute for Plant Breeding, Genetics & Genomics http://www.vib.be/en/news/PublishingImages
Double-stranded RNA induces gene-silencing • Viruses Dicer Dicer dsRNA • Transgenes • Inverted-repeats siRNA siRNA 5’CAP Poly A AGO 5’CAP Poly A Degradation
Inducing RNAi with hairpin vectors Inverted repeat DNA Transcription White light Blue light mRNA AGO
Trans-acting siRNA (tasiRNA) pathway MIR gene miRNA AGO1 mRNA Recognition sequence RDR6 Terminator Promoter Sequence of gene to be silenced 22 nt
Proof-of-concept • Identified 6 miRNA recognition sequences – Case study with miR1514 • P450 Endogenous Target – Highly expressed all tissue types – Nulls are embryo lethal in arabidopsis 1514:P450 target construct recognition sequence
P450 mRNA reduced in 1514:P450 events 180% 160% Relative Expression 140% 120% 100% 80% 60% 40% 20% 0% 1514:P450 Empty target vector
Validating six tasiRNAs miRNA Recognition Sequences Fused to GFP Target Empty Wild vector 1509 type 1510 1510a.2 1514 3514 5770
qRT-PCR and protein abundance 160% 140% qRT-PCR Relative to Empty Vector Control 120% Fluorescence 100% 80% 60% 40% 20% 0% Empty Wild 1509 1510 1510a.2 1514 3514 5770 vector type n = 10, P < 0.0001, LSD α = 0.05
Silencing P450 in stable events • Roots are nice, but really need transgenic plants 160% P450 expression relative to 140% 120% non-transformed 100% 80% 60% 40% 20% 0% • 12 expressing lines in duplicate • Non-transformed 38% expression of non-transformed • Lethal gene effect?
CRISPR/Cas as genome editing tool Science Pennisi 2013 • NHEJ results in indels
CRISPR mutagenesis in soybean Possible targets (GN 20 GG) Cas9 + 5’ Target Wild type
CRISPR mutagenesis in soybean GFP 5’ Target % reads ∆ CACCTTCACCTACGGCGTGCAGTGCTTCAGCCGC CACCTTCACCTACGG--TGCAGTGCTTCAGCCGC 47.7% -2 CACCTTCACCTACG---TGCAGTGCTTCAGCCGC 47.2% -3 CACCTTCACCTACGGC----AGTGCTTCAGCCGC 48.0% -4 CACCTTCACCTACG-CGTGCAGTGCTTCAGCCGC 45.6% -1 CACCTTCACCTACG---TGCAGTGCTTCAGCCGC 57.9% -3 CACCTTCACCTACGG--TGCAGTGCTTCAGCCGC 29.3% -2 CACCTTCACCTACG--------TGCTTCAGCCGC 49.4% -8 CACCTTCACCTACGG------GTGCTTCAGCCGC 48.4% -6 CACCTTCACCTACG-CGTGCAGTGCTTCAGCCGC -1 68.4% CACCTTCACCTACGGCGTGCAGTGCTTCAGCCGC wt 16.1% • Showing two most common sequences per event
Targeting orthologous genes • Individually? • Both at same time? An example with soybean DDM1 41 unique targets Chr11 … … Chr1 Shared: 21 targets 49 unique targets
DDM1 example Chromosome 11 Line AAGAGGAGGTACAGTGTGAGGAGGTACC Line AAGACCCATTT.GATCAGCAAGAATCCC AAGAGGAGGTA---TGTGAGGAGGTACC AAGACCCATTT.GATCAGCAAGAATCCC 48.4% 68.4% 1 5 AAGAGGA-------TGTGAGGAGGTACC AAGACCCATTT.--TCAGCAAGAATCCC 44.5% 13.2% AAGAGGAGGTA---TGTGAGGAGGTACC AAGACCCATTT.GATCAGCAAGAATCCC 50.3% 92.6% 2 6 AAGAGGAGGTA----GTGAGGAGGTACC AAGACCCATTTTGATCAGCAAGAATCCC 47.2% 2.8% … Chr11 … Chr1 Chromosome 1 Line AAGACCCATTT.GATCAGCAAGAATCCCATT AAGACCCATTT.GATCAGCAAGAATCCCATT 63.7% Line AAGCTACTTGAAGCTAGGATAAAGGAAGAG 5 AAGACCCATTTTGATCAGCAAGAATCCCATT 25.5% AAGCTACTT-----------AAAGGAAGAG 46.0% 3 AAGACC-----.-----GCAAGAATCCCATT 34.2% AAGCTACTTGAAG------TAAAGGAAGAG 6 43.2% AAGACCCATTT.G----GCAAGAATCCCATT 33.6% AAGCTACTTGAAG------TAAAGGAAGAG 86.0% 4 AAGCTACTTGAAGCTAGGATAAAGGAAGAG 8.3%
Each target works, at different efficiencies Indel Frequency 100% 10% 20% 30% 40% 50% 60% 70% 80% 90% 0% GFP 5’ GFP 3’ 07g14530 01g 38150 11g07220 1 gRNA 01g 38150 11g07220 1 gRNA 04g36150 06g18790 miR1509 miR1514
Whole plant modifications ATTCTTGCTGATCAAA.TGGGTCTTGGGAA ATTCTTGCTGATCAAA.TGGGTCTTGGGAA 80.31% ATTCTTGCTGA--AAA.TGGGTCTTGGGAA 10.13% ATTCTTGCTGA---AA.TGGGTCTTGGGAA 4.75% ATTCTTGCTGATCAAAATGGGTCTTGGGAA 1.13% ATTCTTGC-----AAA.TGGGTCTTGGGAA 0.32% ATTCTTGCTGATCAA-.TGGGTCTTGGGAA 0.21% ATTCTTGCT---CAAA.TGGGTCTTGGGAA 0.14% ATTCTTGCTGAT-AAA.TGGGTCTTGGGAA 0.13% 25% • 3/24 lines have 20% Indel frequency complete Cas9 15% Time1 • Agrobacterium Time2 10% transformation may 5% be necessary 0% 1 11 1 11 1 11 DDM target 10 21 24 Lines
Cot-node transformation • CRISPR target to phytoene synthase – Sent vectors to Bob Stupar’s lab Photo: Jean-Michel Michno
CRISPR take-home • CRISPRs are working in soybean • Vectors available from • More information available at http://parrottlab.uga.edu/parrottlab/Vectors/index.htm
Acknowledgements PI: Wayne Parrott Labmates: Undergrads: Maria Ortega David Dyck Lisa Kanizay Aaron Umansky Technicians: Andrew Disharoon Donna Tucker Pete LaFayette Noah Lawler Jade Newsome Dena Mehalakes Collaborators : Lauren Lail Lila Vodkin – small CJ Tsai – Poplar RNA datasets Xiaohong Zhou John Finer – GFP line Bob Stupar Bob Schmitz Jean-Michel Michno
Off-targeting results of 5 gRNAs Off-target indel frequency 0% 20% 40% 60% 80% 100% Off gRNA Off target sequence target TTGTGAATGTTTATTGTCGTCGG 1g Off-target TCTAAGATGTTTATTGTGGTGGG 4g Cas9 Control GTGTGAATGTTTAAGGTGGTTGG 8g 13g GTTCTAATGTTTATTGTGGTTGG 13g2 TTGTGAATGTTTATTGTTGTCGG 07g14530 13g3 ATGTTAATGTTTATTGTGTTAGG 15g ATGTTAATGTTTATTGTGTTAGG 17g TTGTGAATGTTTGTTGTCGTCGG 18g TCATGAATGTTTATTGTCGTTGG 18g2 TCTAAGATGTTTATTGTGGTGGG DDM1 chr1 Chr11 TTTAATTGAAGCTAGGATGAAGG DDM1 chr11 Chr1 GGAAGAGGAGGTGCAGTATGAGG 20g GGAATGGTGCGGTCTCTGAATGG Met1 CTAGAAGATAGGAAAGGAAATGG 5g miR1514 18g GAATGGGAGAGGAAAGGAAATGG
Recommend
More recommend
