crispr and protoplasts: hand-in-hand towards high ... · • current methods for evaluating gene...
TRANSCRIPT
CRISPR and protoplasts: Hand-in-hand towards high-throughput
gene silencing in cassava
Dr Patience Chatukuta Post-doctoral Fellow
Plant Biotechnology Research Program, Wits University
ACGT Forum, Wits Professional Development Hub 18 May 2018
Importance of cassava
1. Food security crop in Southern Africa 2. Source of starch for industrial purposes
A. Intercropping of cassava and maize. B. Distribution of global consumption of cassava
A B
Cassava Geminiviruses
• Geographical distribution of cassava-infecting geminiviruses
Gene silencing in functional genomics
• Different approaches are currently being used to broadly assign functions to unknown genes
• Reverse genetics approaches have been used to disrupt genes and create loss-of-function mutants
• The CRISPR/Cas9 system is one of the more recently developed tools that can be harnessed to silence targeted genes with high specificity, easy manipulation, high efficiency and high throughput
• This has been proven in Arabidopsis, wheat, rice, among others
Gene silencing in cassava
• Cassava is recalcitrant to transformation; existing methods are time-consuming • Current methods for evaluating gene function in cassava are lengthy, space
inefficient, require frequent assessment of plants by skilled personnel • Several recent reports detail new approaches such as virus-induced gene
silencing, RNA interference via viral antisense RNAs and siRNAs, and genome editing using CRISPR/Cas9
• Agrobacterium-mediated delivery of CRISPR/Cas9 has been used to silence the phytoene desaturase (MePDS) gene, study cassava brown streak disease by silencing the translation initiation factor 4E (eIF4E), to silence the viral AC2 and AC3 genes involved in gene activation and replication enhancement respectively
• These involved generation of whole transformed plants, a process which takes at least 8 months
• The use of protoplasts provides a rapid in vivo route of assaying the effects of gene silencing
Method
Manes.12G069400: •Belongs to the RING/U-box superfamily • Is co-expressed with a ubiquitin-protein complex adapter and various binding proteins • Involved in the biotic and abiotic stress response of cassava • Involved in the ubiquitination process which (a) modulates protein function (b) is reprogrammed by geminiviruses to achieve full host infection • Involved in selective, non-covalent interaction with zinc ions and other proteins/protein complexes •Up-regulated in SACMV-infected TME3, according to transcriptome data (unpublished data) •One of 105 genes annotated as candidates for association with CMD resistance
Preliminary bioinformatic analysis of target gene
Recombinant Cas9 plasmid construction Cas9
gRNA
Genomic gRNA target identification
gRNA design
Plant Growth
Growth of cv.60444, T200 and TME3 cassava cultivars. A. Nodal cultures. B. One month-old plants.
A B
Protoplast Isolation
Cassava protoplasts under bright field microscopy at 40X. A. cv.60444 B. T200 C. TME3
A B C
Protoplast Quantification
Flow cytometric quantification of released cassava protoplasts. A02. cv.60444 A03. T200 A04. TME3
Protoplast Viability Assay
Fluorescein diacetate staining of 48h-old cassava TME3 protoplasts. Protoplasts viewed using fluorescence microscopy at 40X. A. Green filter image. B.. Bright field and green filter image.
Protoplast Transformation Assay
Fluorescent cv.60444 protoplasts viewed using fluorescence microscopy at 100X. A. eGFP filter image. B. Bright field and eGFP filter composite image.
A B
Amplification of target region transcript
bp
400 200
cv60444 T200 TME3
UT V C VC UT V C VC UT V C VC
To do list
1. Detect and quantify efficiency of targeted mutagenesis in transformed protoplasts
2. Conduct transient expression assays of transformed protoplasts 3. Conduct deep sequencing of edited genomic target region 4. Silence more genes implicated in cassava-SACMV interactions to
assist in creation of functional networks
ACKNOWLEDGEMENTS
• Prof Rey • Plant Biotech Research team • National Research Foundation
END