CRI RISPR a R and prot otop oplasts: Ha Hand-in in- hand - - PowerPoint PPT Presentation

CRI RISPR a R and prot

• top
• plasts: Ha

Hand-in in- hand towards h high-thr hroughput hput gen ene s silenci cing in c n cassa assava

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 Ge Geminivi viruses es

• Geographical

distribution of cassava-infecting geminiviruses

Gene silencing in functi tional 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

Me 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

Prelimi minary bioi

• infor
• rmatic analysis of t

target gene

Recombinant C Cas9 plasmid constru ruction

Cas9 gRNA

Ge Genom

• mic gRNA

NA ta targ rget identification

gRNA NA design

Pl Plant Growth th

Growth of cv.60444, T200 and TME3 cassava cultivars.

• A. Nodal cultures. B. One month-old plants.

A B

Protoplast t Isolati tion

Cassava protoplasts under bright field microscopy at 40X. A. cv.60444 B. T200 C. TME3 A B C

Protoplast Quantifi fication

Flow cytometric quantification of released cassava protoplasts. A02. cv.60444 A03. T200 A04. TME3

Protoplast t Viability A 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.

Prot

• top
• plast Transfor
• rmation
• n 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

References

patience.chatukuta@wits.ac.za patience.chatukuta@gmail.com

ACKNOWLEDGEMENTS

• Prof Rey
• Plant Biotech Research team
• National Research Foundation

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