Post on 17-Feb-2021
transcript
A novel system for co-expression of multiple gRNAs
using RNA polymerase II promoters for multiplex
gene editing with CRISPR/Cas9
Udhaya Kannan
University of Saskatchewan &
AAFC Saskatoon
08 September 2017
2nd International Symposium on Innovations in Plant and Food Sciences
Cell press
CRISPR-Cas9 for crop improvement
Choice of system for desired manipulation
Knockout, edit, activation, repression
Design of guide RNA
Construct assembly
Delivery of construct - development of stable transgenics
Detection of mutation events
Detect effect of mutations
Utilization for plant breeding
CRISPR-Cas9 for crop improvement
Choice of system for desired manipulation
Knockout, edit, activation, repression
Design of guide RNA
Construct assembly
Delivery of construct - development of stable transgenics
Detection of mutation events
Detect effect of mutations
Utilization for plant breeding
CRISPR - Cas9
Repressor
Domain
Doudna and Charpentier, Science 2014
CRISPRi – Casilio system
Cheng et al 2016, Cell Research 26: 254–257
Conventional Casilio
R R
R
R
GOI
CRISPRi using EAR
• Ethylene response element binding factor –
Associated amphiphilic Repression (EAR)
• Consensus sequence patterns – LxLxL / DLNxxP
• EAR – Histone deacetylase mediated chromatin
modification
• TPL is a corepressor via EAR
Kagale and Rozwadowski, 2011 Epigenetics 141-146
Multiple gRNA-tRNA assembly
Xie et al, 2015 PNAS 112, 3570-3575
Cas9 Pol III promoter tRNA gRNA tRNA gRNA
Scaffold Scaffold Terminator Pol II promoter NLS FLAG
NLS
Terminator
Polycistronic tRNA - gRNA (PTG) processing gRNA expression
Cas9 Pol III promoter
gRNA gRNA
Scaffold Scaffold Terminator NLS
Terminator
Terminator Pol II promoter
Arrayed gRNA cassette expression
Pol III promoter NLS
FLAG
• Pol III - Requirement of first nucleotide in gRNA
– G(U6) and A(U3)
– Difficulty - gRNAs design at AT rich regions
• Pol II better studied than Pol III in non-model plants
• Pol II – allows spatial and temporal control of expression in vivo
RNA Pol II vs Pol III promoters for PTG
Construct assembly Gateway and Golden gate cloning
Gateway Cloning
Golden gate cloning
BsaI BsaI
BsaI
Thermo Fisher Scientific Inc.
New England Biolabs Inc.
BsaI
http://2012.igem.org/wiki/images/9/90/S%C3%A9lection_165.png
Validation of PTG in Nicotiana benthamiana
Transformation into Agrobacterium tumefaciens
Construct assembly
Infiltration in N. benthamiana leaf tissues
Guide RNA design
Detection of deletion events
https://www.google.ca/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwiMjbzuhMvUAhXhzIMKHStZA4AQjRwIBw&url=https://www.researchgate.net/publication/51901937_The_Use_of_Agroinfiltration_for_Transient_Expression_of_Plant_Resistance_and_Fungal_Effector_Proteins_in_Nicotiana_benthamiana_Leaves&psig=AFQjCNEJ9NG7f6ExvSiJyfsJyMZH7VyDdQ&ust=1498000481736072
Guide RNA Design
*
*
*
*
* *
* *
Partial NbBAK1 gene
Partial NbBak1 gene
* Not to scale
54 bp 46 bp
1328 bp
gRNA6 gRNA3 gRNA5 gRNA2
Lowder et al 2015, Plant Physiology 169, 971-985
• PTG in N. benthamiana
- deletions of 54 bp, 46 bp and 1328 bp
- confirmed by sequencing
• PTG will be validated in wheat – wheat protoplast – TaPDS
• CRISPRi - validated in N. benthamiana - NbPDS
- validated in wheat protoplast system – TaPDS
• Generation of stable wheat transgenics
Results and work in progress
Summary
• The novel PTG system using Pol II promoters has
been validated in N. benthamiana
• Base constructs are available that can be used for
any desired manipulation for genes
• CRISPR Cas9 system can be efficiently used to
create desired gene manipulation and aid crop
improvement
Acknowledgments
• Dr. Kevin Rozwadowski
• Dr. May Hijazi
• Merek Wigness
• Brent Mooney
• Phytotron & Greenhouse Crew
• Dr. Curtis Pozniak
• Dr. Sateesh Kagale
• Dr. Andrew Sharpe
Thank you