CRISPR and protoplasts: Hand-inhand towards high-throughput gene silencing in cassava

Transcription

1 CRISPR and protoplasts: Hand-inhand 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

2 Importance of cassava 1. Food security crop in Southern Africa 2. Source of starch for industrial purposes A B A. Intercropping of cassava and maize. B. Distribution of global consumption of cassava

3 Geographical distribution of cassava-infecting geminiviruses Cassava Geminiviruses

4 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

5 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

6 Method

7 Preliminary bioinformatic analysis of target gene 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

8 Recombinant Cas9 plasmid construction Cas9 grna

9 Genomic grna target identification

10 grna design

11 Plant Growth A B Growth of cv.60444, T200 and TME3 cassava cultivars. A. Nodal cultures. B. One month-old plants.

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

13 Protoplast Quantification Flow cytometric quantification of released cassava protoplasts. A02. cv A03. T200 A04. TME3

14 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.

15 Protoplast Transformation Assay A B Fluorescent cv protoplasts viewed using fluorescence microscopy at 100X. A. egfp filter image. B. Bright field and egfp filter composite image.

16 Amplification of target region transcript bp UT V C VC UT V C VC UT V C VC cv60444 T200 TME3

17 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

18 References

19 ACKNOWLEDGEMENTS Prof Rey Plant Biotech Research team National Research Foundation

20 END