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Transgene-free CRISPR/Cas9
genome-editing methods in plants
Matthew R. Willmann, Ph.D.
Director, Plant Transformation Faci...
What do we do at the CALS Plant Transformation
Facility?
• We make transgenic and genome-edited plants of time-consuming o...
How do we help our users?
• Plant transformation and
genome editing
• Tissue culture projects
• User access to equipment
•...
What are our upcoming plans?
• Increasing services related to maize
• In negotiations with Pioneer to license the BBM/WUS ...
Research interests
• Developing and improving plant transformation methods
• Uncovering the genotypic basis for the genoty...
What is genome editing?
• The ability to make specific genetic changes within a genome
• Specific location
• Specific type...
Broken DNA happens
• Double-strand DNA breaks play a
key role in crossing over during
gamete formation (meiosis)
• Double-...
Mutations induced by DNA repair in somatic
cells
Modified from Sandler and Joung 2014
What if we could direct where double-strand
breaks happen?
• Requirements:
• Ability to recognize a specific DNA sequence
...
RNA recognition of DNA
• Clustered regularly interspaced short palindromic repeat
(CRISPR)/Cas9 system (2013)
• A prokaryo...
Bortesi and Fischer 2014
Different types of possible edits
How is genome editing performed?
• To utilize CRISPR/Cas9 gene-editing in plants, you need to find a way
to get the follow...
How is genome editing performed?
• Transgenic methods
• Physically insert genes for Cas9 and
guide RNA(s) into the genome
...
Using transgenic plants to perform
CRISPR/Cas9 gene-editing in plants
• Transgenic plants
• Transformation of a construct
...
Using transgenic plants to perform
CRISPR/Cas9 gene-editing in plants
• Transgenic plants
• Transformation of a construct
...
How to eliminate the transgene?
• Inbred lines
• Segregate the mutation from the transgene by selfing or backcrossing to t...
Cre/loxP system
https://www.jax.org/news-and-insights/jax-blog/2011/october/cre-lox-breeding-for-dummies-part-ii
piggybac transposon
http://www.bioscience.co.uk/products/piggybac-transposon-vector-construction
Using non-transgenic methods to perform
CRISPR/Cas9 gene-editing
• Non-transgenic methods
• Particle bombardment
• Protopl...
Using non-transgenic methods to perform
CRISPR/Cas9 gene-editing
• Particle bombardment
• Cas9 protein, guide RNA(s), and
...
Using non-transgenic methods to perform
CRISPR/Cas9 gene-editing
• Protoplasts
• Transient expression of Cas9 and
guide RN...
Using non-transgenic methods to perform
CRISPR/Cas9 gene-editing
• Viral delivery
• Different ways of introducing the
viru...
Currently available virus systems for use with
CRISPR/Cas9 in plants
Zaidi and Mansoor 2017
Note: RNA viruses cannot be us...
Comparing transgenic and non-transgenic
methods
Transgenic methods
• Time required for eliminating the
transgene and provi...
Using particle bombardment for transgene-
free genome editing of rice
• Established optimal
bombardment conditions of
imma...
Testing particle bombardment for transgene-
free genome editing of rice
• Established optimal
bombardment conditions in ri...
Testing particle bombardment for transgene-
free genome editing of rice
• Tested the ability of Cas9 protein
and in vitro ...
Upcoming steps
• Sequence PCR products to confirm deletions
• Regenerate plants from embryos bombarded with Cas9 protein a...
Summary
• There are at least three methods for non-transgenically genome-
editing plants
• Non-transgenic genome-editing m...
New journal: The CRISPR Journal
• Published by Mary Ann Liebert,
Inc., Publishers
• Manuscripts are already being
accepted...
Acknowledgements
• PTF Staff
• Zach Lindskoog
• Elsa de Becker
• Jordon Zonner
• Shaumik Ashraf
• Alvina Gul Kazi
• PTF Fa...
Contact information for the PTF
Matthew R. Willmann, Ph.D.
mrw6@cornell.edu
B18 Weill Hall (Office), B22 Weill Hall (Lab)
...
Using non-transgenic methods to perform
CRISPR/Cas9 gene-editing
• Using single-stranded DNA
donors?
• Modified or non-mod...
Current PTF research projects
• Developing and improving plant transformation methods
• Rice (with Adam Bogdanove and Susa...
Current PTF research projects
• Uncovering the genotypic basis for the genotype dependency of plant
transformation and reg...
Current PTF research projects
• Developing and improving CRISPR/Cas9 genome-editing methods
• Developing transgene-free me...
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Transgene-free CRISPR/Cas9 genome-editing methods in plants

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"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.

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Transgene-free CRISPR/Cas9 genome-editing methods in plants

  1. 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. 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. 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. 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. 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. 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. 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. 8. Mutations induced by DNA repair in somatic cells Modified from Sandler and Joung 2014
  9. 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. 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. 11. Bortesi and Fischer 2014 Different types of possible edits
  12. 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. 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. 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. 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. 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. 17. Cre/loxP system https://www.jax.org/news-and-insights/jax-blog/2011/october/cre-lox-breeding-for-dummies-part-ii
  18. 18. piggybac transposon http://www.bioscience.co.uk/products/piggybac-transposon-vector-construction
  19. 19. Using non-transgenic methods to perform CRISPR/Cas9 gene-editing • Non-transgenic methods • Particle bombardment • Protoplasts • Viral replicons
  20. 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. 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. 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. 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. 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. 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. 26. Testing particle bombardment for transgene- free genome editing of rice • Established optimal bombardment conditions in rice using plasmid encoding 35S::GUS
  27. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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)

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