Precision Genome Engineering in plant based model crops and their applications

1. WELCOME
TO
CREDIT SEMINAR II
PBG 692
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Darshana Patra 18123K01

2. PRECISION BREEDING IN PLANTS
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3. Outline
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4. Objectives of Plant Breeding
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Prime objective is to increase crop yield and improve quality of crop produce
https:// www.agronomy.org/science-news/understanding-genetic-basis-drought-tolerant-crops/Biotech info center
Until recently, our ability to generate allelic diversity in plants was limited to
introduction of variants from domesticated and wild species by breeding via
uncontrolled recombination or the use of chemical and physical mutagens—
processes that are lengthy and costly or lack specificity.

5. Farm To Fork Strategy
• for a fair, healthy and environmentally friendly food system
• new innovative genomic techniques accelerate the development of
bio based products
• may play a role in increasing sustainability along the food supply
chain
• provided they are safe for consumers and the environment while
bringing benefits to society as a whole.
• accelerate the process of reducing dependency of pesticides
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6. Molecular Marker Assisted Selection Breeding
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MAS refers to the use of DNA markers that are tightly-linked to target loci
Assumption: DNA markers can reliably predict phenotype

7. MAS Breeding
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Pros :
• Similar to traditional breeding , not regulated
• Accelerating breeding process
• Easier for stacking multiple traits within the same
cultivar
Cons:
• Must know genomic and genetic background
• Very costly
• False markers

8. Don’t underestimate the power of genetic variability
Classical Breeding by selecting stem, lateral bud, terminal bud, flower cluster, stem & flower, leaf
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9. Traditional Mutagenesis
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Further crossing
to remove
undesirable
mutations and to
obtain optimal
varieties

10. Mutagenesis
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Undirected
DSB induction
by X rays

11. Enhancing genetic variability with mutagens
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12. Pros and Cons of Mutation Breeding
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Pros:
• Induction of desirable mutant which is absent in natural plant materials
• Not regulated ecologically, environmentally friendly
• Straightforward phenotypic selection, technically easy
Cons:
• Generally random and unpredictable
• Good mutations come with bad mutations
• Need large mutant pool to identify good one
• Costly and slow

13. Enhancing genetic variability with
mutagens
• Used in the last 70 years for breeding.
• More than 3000 cultivars worldwide
• Many further unwanted (off-site) mutations
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14. High Precision : no or less non-targeted mutations
Traditional Mutagenesis Vs Targeted Mutagenesis
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15. Going “Bio” : Enzymes can do it better
Site-specific induction of DSBs : Natural inheritance is
governed by molecular scissors
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16. Plant GE Toolbox: Edit Delete Move
• Gene editing provides a faster and precise way to create new variation,
dominated by the creation of short insertion and deletion mutations leading
to loss of gene function, due to the dependence of editing outcomes on DNA
repair pathway choices intrinsic to higher eukaryotes.
• Other types of edits such as point mutations and precise and pre-designed
targeted sequence insertions have rarely been implemented , despite
providing means to modulate the expression of target genes or to engineer
the function and stability of their protein products.
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17. Designer Plants
• Custom editing by regulation of repair pathway choices or by
taking advantage of alternative types of DNA repair
• The advent of novel gene editing tools are independent of
DNA double-strand break repair, and methods completely
independent of host DNA repair processes
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18. Precision Breeding
• A plant breeding approach in which a phenotypic trait of
interest is selected by means of identifying a functional
marker that is directly derived from the genomic region of a
trait-controlling gene
• Selections are based on the polymorphic genic regions linked
with a trait of interest
• Availability of genomic resources are of utmost importance
for making FM
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19. The next step : Breeding at the speed of light
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20. CRISPR Loci induce acquired
immunity in bacteria against the virus
infection or plasmid transfer
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21. 3 Options : Precise edits with CRISPR
• Make use of host cell HDR
– Involve a DSB.
– Capable of very large insertions
• Use enzymatic base editing
– No DSB repair. Single base edits typically
• Use prime editing
– No DSB repair. Capable of all edits <80 bases
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22. Gene editing via CRISPR/Cas
• CRISPR/Cas is more efficient and cheaper
• Directed , off-site mutations can be avoided
• Any gene can be targeted
• A row of improved traits introduced in different crops already
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Steps
• Binding with NGG
PAM (spcas9)
• R loop = ds to ss DNA
• sgRNA binding with
homologous
sequence
• Cas9 makes a cut

23. Features of CRISPR/Cas9 GE
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• High precision, high efficiency with unprecedented ability to generate targeted
and specific mutations
• Procedures are identical to genetic modification
• Final products are similar to traditional breeding
• Deactivating one or multiple genes, up/down regulating genes, early pathogen
detection
• Precise modifications using base editors, prime editing and HDR
• Targeted insertion of transgene

24. Creating Precise Mutation ?
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Turn off Turn on

25. Genomic double- strand break (DSB) generation is followed by different cellular repair pathways. Error-prone non-homologous
end joining (NHEJ) and microhomology-mediated end joining (MMEJ)pathways create the majority of mutations throughout the
cell cycle. Homology directed repair (HDR), active in S/G2 phases of the cell cycle, repairs DSB without error.
CELLULAR REPAIR PATHWAY
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26. High efficiency : Time
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27. Time ? Precision ?
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28. 31-08-2021 Darshana Patra Chen et al 2019 28
Staggered cuts by SpCas9
– HNH cleaves target strand at - 3 position
– RuvC can make a cut at either -3, -4, -5, or even further

29. Hypothetical model
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Staggered cuts by SpCas9
• HNH cleaves target strand at - 3
position
• RuvC can make a cut at either
-3, -4,-5, or even further
Generation of 1 bp insertion during
CRISPR/Cas9-induced DSB repair

30. Predicting precise edited products
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Prediction of CRISPR/Cas9-induced
mutations

31. Scientific risk assesment of gene
edited plants
• DSB repair is a natural process, mutations occur spontaneously all of the time
• Plants with CRISPR/Cas-induced or spontaneous mutations cannot be discriminated and
are nature-identical
• Classical mutagenized plants are exempted from regulation due to a ‘long safety record ‘
• Edited Plants are at least as safe as mutagenized crops
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32. Out of total cellular repair events..
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33. Precision editing by HDR
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• Coincident error prone NHEJ repair confounds HDR strategies
• Yields a majority of random mutants and a minority of accurate HDR
corrections
• NHEJ occurs in G1/S while HDR occurs in G2/M
• A Cas9-geminin fusion imparts G2/M expression on Cas9
• Biased towards HDR events

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Published : 8 July 2021
• Here, fast, efficient and reproducible targeted nucleotide substitutions in sugarcane
reported, enabling precise co-editing of multiple alleles via template-mediated and
homology-directed repair (HDR) of DNA double strand breaks induced by the
programmable nuclease CRISPR/Cas9
• Selected gene variants into elite cultivars without crossing and associated linkage
drag.

35. Precise editing by HDR mediated allele
replacement (gene targeting)
• In the GT approach, a DRT is constructed by flanking the desired
sequence modifications on each side by regions of homology to the
target locus, often referred to as homology arms.
• When the DRT is delivered to a target cell and a DSB is
simultaneously induced in the genome, the DRT can be used for
HDR that proceeds via synthesis-dependent strand annealing (HDR
repair in plants see Knoll et al. 2014), resulting in incorporation of
the edits in the genome.
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36. Low HDR frequency in somatic plant cells
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37. Multi allelic precision ns conferred
herbicide tolerance
• Frequency of HDR in somatic cells is negligible, making routine allele replacement at scale in
plants impractical.
• In fact, GT is usually successful in only 0.1–1% of recovered plants.
• Although some of the improvements described below have increased these efficiencies,
surpassing the 10% mark can be considered exceptional.
• In stark contrast, NHEJ repair of targeted DSBs can generate indels with close to 100%
efficiency in some species (Pan et al. 2016; Ueta et al.2017; Lee et al. 2019b; Malzahn et al.
2019).
• This difference is partly due to the restriction of HDR to the late S and G2/M phases of cell
cycle, as opposed to NHEJ which operates in both dividing and non- dividing cells (Mao et al.
2008; Charbonnel et al. 2011).
• Simply put, the main hurdle to effective GT is the need to create conditions under which HDR
is favored over NHEJ.
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38. Strategies to improve HDR mediated genome editing
Manipulation
of DNA repair
pathway
Manipulation
of donor
template
Adding new
functions to
Cas enzymes
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Combinatorial approach

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40. Anti Nutritional Factor : Gliadin
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Ingestion of antigenic
compound i.e. foreign
body Gliadin develop auto
immune response, destroy
the vili (absorb nutrients
from food)structure of
intestine

41. Target epitope as antigenic : MALDI-TOF analysis of the gliadin extracts from T545 , T553 and the BW208 wild type.
PBJ : Susana Sanchez-Leon et. al {18 September 2017}
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42. Wild species that are heat
and salt stress resistant
can be transformed into
novel crops
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43. Genetic Engineering
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Pros :
• Fast way to verify gene function
• Precisely modify crop productivity and quality
Cons :
• Necessary to know gene function
• Very costly, complicated procedures
• Heavily regulated

44. Complimentary to Traditional
Breeding : Solution to Linkage Drag
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Linkage Drag - Nightmare for Traditional Plant Breeders
Gene editing can remove linkage drag to create better plants
Never has progenies with good
flavor and better postharvest
quality,diseaseresistant tomato
Breaking genetic linkages by CRISPR/Cas

45. Crop Breeding : towards precision
Genome editing: improving a trait by precisely modifying the target genes or regulatory elements
or rearranging chromosomes in elite varieties.
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46. What makes it Different?
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47. CRISPR Base editing without DNA cleavage
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• Recruit catalytic domains of DNA deaminases
• Use D10A nickase to nick non-edited strand
to stimulate repair
• No DSB
• Nick repair is very accurate and fast
• Can target most disease associated SNPs

48. Prime editing - CRISPR without DNA breaks
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49. Prime Editing - CRISPR without DSB
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• Does not rely on host cell DSB repair to make an edit
• A Cas9 nickase is used to locate and prime the editing
• The intended edit is added to the guide RNA template
• A fused reverse transcriptase incorporates the edit
• Host cell repair incorporates the edit

50. DNA repair during Prime editing
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51. Broad applications of Prime editing
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52. Technologies for allele replacement in plants
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54. 31-08-2021 Darshana Patra Puchta et al 2021 54

55. Application of GE Techniques for Crop
Improvement
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56. Most efficient allele replacement methods for
major plant model and crop species
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The choice of BE for each application in order to avoid potentialbystander mutationsand other editing byproducts
should be carefullyconsideredbased on :
• the availabilityof PAM sequence properlypositioned in the target sequence
• size of the editing window
• specificity
Citationsand application of selection refer to the highestachieved editing efficiencyfor the given approach and species.
*Direct selection for the gene editing event, DRT donor repair template

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60. Challenges
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• Availability of Genomic Sequence
• Efficient plant transformation pipe line
• DNA-free genetic transformation method for
perennial plant species

61. Battles
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• Consumer acceptance
• Ecological concern
• Health concern
• Ethical concern

62. Take home message
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• Genome editing requires similar procedures used for Genetic
Engineering (GMOs), yet creates precise mutation in plant genomes
containing non-foreign DNA
• Resulting products are indistinguishable from products of natural
variability or mutagenesis, yet genome edited plants are regulated
case-by-case
• Limitation of genome editing application are plant transformation
pipeline and genome availability

63. References
• Chen, Kunling, et al. "CRISPR/Cas genome editing and precision plant breeding in
agriculture." Annual review of plant biology 70 (2019): 667-697.
• García-Molina, María Dolores, et al. "Gluten free wheat: are we there?." Nutrients 11.3
(2019): 487.
• Oz, Mehmet Tufan, et al. "CRISPR/Cas9-Mediated Multi-Allelic Gene Targeting in Sugarcane
Confers Herbicide Tolerance." Frontiers in Genome Editing (2021): 15.
• Sedeek, Khalid EM, Ahmed Mahas, and Magdy Mahfouz. "Plant genome engineering for
targeted improvement of crop traits." Frontiers in plant science 10 (2019): 114
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