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