"Transgene-free CRISPR/Cas9 genome-editing methods in plants" by Matthew R. Willmann, Ph.D. Director, Plant Transformation Facility College of Agriculture and Life Sciences, School of Integrative Plant Science, Cornell University.

1. Transgene-free CRISPR/Cas9
genome-editing methods in plants
Matthew R. Willmann, Ph.D.
Director, Plant Transformation Facility
College of Agriculture and Life Sciences, School of Integrative Plant Science
Cornell University

2. What do we do at the CALS Plant Transformation
Facility?
• We make transgenic and genome-edited plants of time-consuming or
hard-to-transform species with a particular interest in New York
State crops

3. How do we help our users?
• Plant transformation and
genome editing
• Tissue culture projects
• User access to equipment
• Gene guns
• Consulting
• General consulting
• Transformation of a new crop or
genotype
• Method development
Rice Maize

4. What are our upcoming plans?
• Increasing services related to maize
• In negotiations with Pioneer to license the BBM/WUS high efficiency
transformation technology for monocots
• New crops
• Cucurbits (starting with watermelon)
• Brassicas (starting with canola)
• Industrial hemp

5. Research interests
• Developing and improving plant transformation methods
• Uncovering the genotypic basis for the genotype dependency of plant
transformation and regeneration
• Developing and improving genome-editing methods
• Transgene-free methods
• Rice (with Adam Bogdanove)
• Apple (with Awais Khan)
• Testing ways to increase the efficiency of CRISPR/Cas9-mediated insertions
and allelic swaps (with Adam Bogdanove)

6. What is genome editing?
• The ability to make specific genetic changes within a genome
• Specific location
• Specific type of alteration (insertion, deletion, point mutation, etc.)
• Specific final sequence
• Common in bacteria, yeast, and mammalian research for decades
• Takes advantage of errors in normal DNA repair processes

7. Broken DNA happens
• Double-strand DNA breaks play a
key role in crossing over during
gamete formation (meiosis)
• Double-strand DNA breaks occur
in somatic cells in response to
irradiation, UV-light, and certain
chemicals
• Usually repaired without errors
• But, sometimes . . .
Modified from http://ib.bioninja.com.au/

8. Mutations induced by DNA repair in somatic
cells
Modified from Sandler and Joung 2014

9. What if we could direct where double-strand
breaks happen?
• Requirements:
• Ability to recognize a specific DNA sequence
• Ability to cleave DNA (nuclease)
• The result would be a site-directed or sequence-specific nuclease
• Types of site-directed nucleases
• Protein recognition of DNA
• RNA recognition of DNA

10. RNA recognition of DNA
• Clustered regularly interspaced short palindromic repeat
(CRISPR)/Cas9 system (2013)
• A prokaryotic adaptive immune response against viruses
• Transformed into a genome editing tool
Bortesi and Fischer 2014

11. Bortesi and Fischer 2014
Different types of possible edits

12. How is genome editing performed?
• To utilize CRISPR/Cas9 gene-editing in plants, you need to find a way
to get the following into a plant cell and then regenerate a whole
plant from that cell
• Cas9 protein
• Guide RNA(s)
• Donor template DNA

13. How is genome editing performed?
• Transgenic methods
• Physically insert genes for Cas9 and
guide RNA(s) into the genome
• Regenerate a organism from the
transgenic cell
• Eliminate the transgenes after editing
has occurred
• Non-transgenic methods
• Insert Cas9 protein and guide RNA(s)
into the cells
• Regenerate a whole organism from
the edited cell
• The proteins and RNAs are naturally
degraded over time
Genome
Cas9 and gRNA genes
Cas9 protein and in vitro-transcribed gRNAs
Bortesi and Fischer 2014

14. Using transgenic plants to perform
CRISPR/Cas9 gene-editing in plants
• Transgenic plants
• Transformation of a construct
encoding Cas9 and guide RNA(s)
Modified from sigmaaldrich.com
Selection
marker gRNA Cas9
LB RB

15. Using transgenic plants to perform
CRISPR/Cas9 gene-editing in plants
• Transgenic plants
• Transformation of a construct
encoding Cas9 and guide RNA(s),
and containing donor template
Modified from sigmaaldrich.com and Schiml and Puchta 2016
homology homologysequence of
interest
Selection
marker gRNA
Cas9
LB RB

16. How to eliminate the transgene?
• Inbred lines
• Segregate the mutation from the transgene by selfing or backcrossing to the
original line
• Non-inbred lines or clonally propagated plants (Ex. Apple or grape)
• Very hard to segregate the transgene away without losing the character of the
line
• Molecularly removing the transgene
• Cre/loxP
• FLP/FRT
• piggybac transposon

17. Cre/loxP system
https://www.jax.org/news-and-insights/jax-blog/2011/october/cre-lox-breeding-for-dummies-part-ii

18. piggybac transposon
http://www.bioscience.co.uk/products/piggybac-transposon-vector-construction

19. Using non-transgenic methods to perform
CRISPR/Cas9 gene-editing
• Non-transgenic methods
• Particle bombardment
• Protoplasts
• Viral replicons

20. Using non-transgenic methods to perform
CRISPR/Cas9 gene-editing
• Particle bombardment
• Cas9 protein, guide RNA(s), and
donor template followed by
regeneration of whole plants
• Can also use Cas9 in RNA form,
but is less efficient
Modified from Zhang et al. 2016
Protein
RNA/protein coating

21. Using non-transgenic methods to perform
CRISPR/Cas9 gene-editing
• Protoplasts
• Transient expression of Cas9 and
guide RNA(s), and donor template
in protoplasts followed by
regeneration of whole plants
• Transfection of protoplasts with
Cas9 protein and guide RNA(s)
followed by regeneration of whole
plants

22. Using non-transgenic methods to perform
CRISPR/Cas9 gene-editing
• Viral delivery
• Different ways of introducing the
virus
• Direct infection (TRV)
• Agrobacterium (ssDNA)
• Bombardment
• Protoplasts
• Method can allow you to avoid
tissue culture steps if you are able
to infect whole plants, the
germline cells are infected, and
you harvest seeds from the
infected plants
Modified from Zaidi and Mansoor 2017

23. Currently available virus systems for use with
CRISPR/Cas9 in plants
Zaidi and Mansoor 2017
Note: RNA viruses cannot be used to supply donor templates

24. Comparing transgenic and non-transgenic
methods
Transgenic methods
• Time required for eliminating the
transgene and proving its absence
• Higher off-target rates
• Lower success of homologous-
recombination-mediated editing
because only one copy of the
donor template per cell
• Many issues with regulation and
public perception
Non-transgenic methods
• Time required for generating and
examining more regenerated plants
• Lower off-target rates
• Higher success of homologous-
recombination-mediated editing
because potentially many copies of
the donor template per cell
• Likely fewer issues with regulations
and public perception

25. Using particle bombardment for transgene-
free genome editing of rice
• Established optimal
bombardment conditions of
immature rice embryos using
plasmid encoding 35S::GUS

26. Testing particle bombardment for transgene-
free genome editing of rice
• Established optimal
bombardment conditions in rice
using plasmid encoding
35S::GUS

27. Testing particle bombardment for transgene-
free genome editing of rice
• Tested the ability of Cas9 protein
and in vitro transcribed gRNAs to
direct cleavage when introduced
by particle bombardment
• ART1 gene
• ~1:4 molar ratio of Cas9:gRNAs
5’ target
site
ART1
3’ target
site
1609 bp
WT band
149 bp
△ band
F3 and R2
primer pair
F3
R2
R2
F3
223 bp
△ band
F2 and R1
primer pair
F2
R1
R1
F2
TA-rich
1460 bp deletion

28. Upcoming steps
• Sequence PCR products to confirm deletions
• Regenerate plants from embryos bombarded with Cas9 protein and in
vitro-transcribed gRNAs and identify edits
• Use the same bombardment system for homologous recombination-
induced allelic swaps at the same locus
• Compare editing efficiencies between transgenic and non-transgenic
genome-editing methods
• Test protoplast and viral replicon non-transgenic methods and
compare to bombardment

29. Summary
• There are at least three methods for non-transgenically genome-
editing plants
• Non-transgenic genome-editing methods are more advantageous for
homologous-recombination-based edits, induce fewer off target
mutations, and can avoid regulatory issues
• Non-transgenic genome-editing methods require the screening of
more regenerated plants
• When developing or testing new genome-editing methods it is
recommended that each of the steps involved is tested individually

30. New journal: The CRISPR Journal
• Published by Mary Ann Liebert,
Inc., Publishers
• Manuscripts are already being
accepted
• First issue scheduled for
February 2018
• Editors include CRISPR pioneers
Jennifer Doudna, Emmanuelle
Charpentier, George Church
• Editors from the plant sciences
include Caixia Gao, Jian-Kang
Zhu, Matthew Willmann
• More information found at
www.theCRISPRjournal.com

31. Acknowledgements
• PTF Staff
• Zach Lindskoog
• Elsa de Becker
• Jordon Zonner
• Shaumik Ashraf
• Alvina Gul Kazi
• PTF Faculty Advisory Board
• Adam Bogdanove, Chair
• Maureen Hanson
• Susan McCouch
• Joss Rose
• Mike Scanlon
• Joyce Van Eck
• Collaborators
• Adam Bogdanove (Cornell)
• Susan McCouch (Cornell)
• Awais Khan (Cornell)
• Mark Sorrells (Cornell)
• Joyce Van Eck (Boyce Thompson Institute)
• Luz Stella Barrera (Corpoica)
• Funding sources
• Cornell, CALS
• NSF PGRP grant to Adam Bogdanove, Susan
McCouch, Erin Doyle, Jan Leach, Boris
Szurek, and Dan Voytas
• Apple Research and Development Program
(ARDP) grant to Awais Khan and Matthew
Willmann

32. Contact information for the PTF
Matthew R. Willmann, Ph.D.
mrw6@cornell.edu
B18 Weill Hall (Office), B22 Weill Hall (Lab)
Office: 607-254-1466; Cell: 508-243-2495
http://sips.cals.cornell.edu/research/plant-
transformation-facility

33. Using non-transgenic methods to perform
CRISPR/Cas9 gene-editing
• Using single-stranded DNA
donors?
• Modified or non-modified
Renaud et al. 2016Bortesi and Fischer 2014

34. Current PTF research projects
• Developing and improving plant transformation methods
• Rice (with Adam Bogdanove and Susan McCouch)
• Azucena
• Carolina Gold
• O. glaberrima cv. CG14
• Llanura 11 (with Luz Stella Barrera of Corpoica)
• Wheat (with Mark Sorrells)
• Spring wheat Glenn
• Winter wheat Medina

35. Current PTF research projects
• Uncovering the genotypic basis for the genotype dependency of plant
transformation and regeneration (with Joyce Van Eck and Susan
McCouch)
Modified from Indoliya et al. 2016

36. Current PTF research projects
• Developing and improving CRISPR/Cas9 genome-editing methods
• Developing transgene-free methods
• Rice (with Adam Bogdanove)
• Apple (with Awais Khan)
• Testing ways to increase the efficiency of CRISPR/Cas9-mediated insertions
and allelic swaps (with Adam Bogdanove)