Presentation regarding crop genome editing and its application

1. Amit Singh Rana
PhD Biochemistry
52633
Genome Editing for Crop
Improvement

2.  controlled change in the DNA
 DNA is deleted, modified, inserted or replaced
 Randomly inserts genetic
 Site specific locations
 CRISPR, 2009
 Easier
 Simpler
 Faster
 Cheaper
 Accurate
WHAT IS GENOME EDITING?

3. CONVENTIONAL BREEDING VS
GENETIC MODIFICATION VS
GENE EDITING

4. TOOLS
 Science as 2015 Breakthrough of the Year
 Meganucleases (MegaN)
 Zinc Finger Nucleases (ZFNs)
 Transcription Activator-Like Effector-based Nucleases (TALEN)
 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9)
system
 Create site-specific double-strand breaks (DSBs)
 NHEJ or HR, targeted mutations (edits)

5. DOUBLE STRAND BREAK
REPAIR
 Non-Homologous End Joining (NHEJ)
 Homology Directed Repair (HDR)
 NHEJ - directly join the DNA ends
 HDR- homologous sequence as a template
 Homologous to the flanking sequences
 Desired change at DSB
 Rate increases by at least three orders of magnitude

6. Genome editing with site-specific nucleases (SSNs). The double stranded breaks (DSBs) introduced by CRISPR/Cas9 complex can
be repaired by non-homologous end joining (NHEJ) and homologous recombination (HR).
(A)NHEJ repair can produce heterozygous mutations, biallelic mutations (two different mutations at each chromosome) and
homozygous mutations (two independent identical mutations) leading to gene insertion or gene deletion.
(B) (B) In the presence of donor DNA digested with the same endonuclease leaving behind similar overhangs, HR can be achieved
leading to gene modification and insertion.

7.  14 to 40 base pair unique or nearly-so in most genomes
 Very specific (>14bp)
 Best known are the LAGLIDADG family
 Costly and time (major disadvantage)
 Initial protein engineering stage for custom meganuclease
 technically challenging and hindered by patent disputes
MEGANUCLEASES (MegaN)

8. ZINC FINGER NUCLEASE
 Designed zinc finger domains
 Cys2-His2 zinc finger protein
 3–6 zinc finger domains (ZFPs)
 3 bp long target DNA sequence
 Non-specific nuclease domain FokI
 ZFNs pair bind and align in reverse
fashion
 Dimerization of FokI nuclease
domain
 First time reported in tobacco (Wright
et al. 2005) and Arabidopsis (Lloyd et
al. 2005)
 Tobacco, Maize, Arabidopsis,
Soybean, Canola and other plants

9. TAL EFFECTOR NUCLEASES
(TALENS)
 Secreted proteins bacterial genus Xanthomonas
 33–35 amino acid repeats
 12 and 13, repeat variable di-residues (RVDs)
 RVD recognizes one nucleotide
 Combination of repeat number and RVDs composition in the repeats
 Chimeric protein (DNA binding domain and FokI nuclease domain)
 Cut in desired DNA region
 Low off-target effects
 Rice, wheat, maize, tomato, potato,Arabidopsis, tobacco

10. CRISPR/CAS9
 Provide immunity to bacteria against bacteriophage
 Adaptation
Small fragment inserted into CRISPR locus.
Cas genes (CRISP associated), helicase and nuclease activity.
 Transcription
long pre-crRNA (poly-spacer precursor crRNA)
short crRNAs CRISPR RNA), 39–45 nucleotides containing one spacer sequence.
 Interference
Ternary Cas9-crRNAtracrRNA complex binds to Cas proteins.
crRNA complement with protospacer sequence (PAM)
tracrRNA required for Cas-mediated DNA interference
Cas-proteins cut foreign DNA sequences, DNA degradation.
 Cost-effective and easy-to-use technology.

11. Mechanism of CRISPR/Cas9 action: in the acquisition phase foreign DNA gets incorporated into the CRISPR loci of
bacterial genome. CRISPR loci is then transcribed into primary transcript and processed into crRNA with the help of
tracrRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with a crRNA and cleaves foreign
DNA near PAM region.

12. Various genome-editing tools.
(A)Zinc-finger nucleases (ZFNs) act as dimer. Each monomer consists of a DNA binding domain and a nuclease domain. Each DNA
binding domain consists of an array of 3–6 zinc finger repeats which recognizes 9–18 nucleotides. Nuclease domain consists of type II
restriction endonuclease Fok1.
(B)Transcription activator-like nucleases (TALENs): these are dimeric enzymes similar to ZFNs. Each subunit consists of DNA
binding domain (highly conserved 33–34 amino acid sequence specific for each nucleotide) and Fok1 nuclease domain.
(C)CRISPR/Cas9: Cas9 endonuclease is guided by sgRNA (single guide RNA: crRNA and tracrRNA) for target specific cleavage. 20
nucleotide recognition site is present upstream of protospacer adjacent motif (PAM).

13. CRISPR/CAS SYSTEM
 Cas protein in the ribonucleoprotein system can vary.
 Makarova et al. (2011) classified CRISPR/Cas systems into three types:
 type I, type II, and type III
 presence of signature Cas3, Cas9 and Cas10 proteins respectively.
 Further modified into two class-five type classification systems
 Class 1 CRISPRs have multiple subunit effector complexes
 Class 2 CRISPRs concentrates most of their functions with single protein
effectors

14. CLASSIFICATION OF CRISPR/CAS9
SYSTEM.

15. CAS9 VARIANTS WITH THEIR
ORIGIN AND SPECIFICATIONS.

16. Mega v ZNF v TALEN v Cas9

17. CRISPR-CPF1
 CRISPR-Cpf1, (Prevoltella and Francisella), advanced tool
 Uses single Cpf1 protein for crRNA processing, target site recognition, and DNA
cleavage.
 Differs substantially in many aspects.
 Ribonuclease that processes precursor crRNA
 Recognizes thymine rich (like 5’-TTTN-3’) PAM sites.
 Cpf1 generates 4 bp overhangs.
 Sticky ends provide more efficient genomic insertions.

19. TIMELINE
OF
CRISPR

20. LIST OF GENES TARGETED BY
GENOME EDIITNG TOOLS

21. CRISPR SPECIFICATIONS IN PLANTS
 Optimal promoters for expression.
 Suitable vector system
 Efficient target sites and transformation method.
 Specific expression vectors
 sgRNA regulated promoters AtU6, TaU6 etc
 Cas9 promoters like ubiquitin promoters.
 Choice varies
 sgRNA and Cas9 can be co-expressed in a single plasmid ex. pFGC-pcoCas9,
pRGEB32, pHSE401.
 https://www.addgene.org/ crispr/plant/

22. WEB BASED TOOLS FOR GUIDE
RNA SYNTHESIS ALONG WITH OFF
TARGET PREDICTION

23. PLASMIDS
USED
IN
EDITING

24.  Minimum or No off-target effects.
 COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and
Deletions).
 potential off-targets 3% (soyabean).
 No detectable off-targets found in A. thaliana, wheat, rice and sweet orange.
 Target specific oligonucleotides (20 nt)
 sgRNA + Cas9 sequence (a binary vector)
 Individually
 Transformed using a suitable method.
 Delivery systems vary based on plant species, research purpose, and
requirements.
 Restriction enzyme digestion suppressed PCR (RE-PCR) method.
 Whole genome sequencing.
SELECTION OF TARGET SITE

25. Simplified flow chart representing CRISPR/Cas9 mediated plant genome editing. After the selection of the target site, sgRNAs are
designed using various bioinformatic softwares and packed into specific vectors along with codon optimized Cas9. After delivery
into plant cells, putative transformants can be screened by multiple assays and used for further analysis.

27. Applications of
genome editing for
crop improvement

28. ABIOTIC STRESS RESISTANCE

29. BIOTIC STRESS RESISTANCE

30. IMPROVED NUTRITIONAL QUALITY

31. DNA FREE MODIFICATIONS OF
PLANT GENOME
Extra DNA frequently integrate into the plant genome.
 Disruption, plant mosaicism and off target disruptions.
 Lessens the efficiency of gene editing and gene insertion.
 Uses Cas9 ribonucleoproteins (RNPs).
 Cas9 RNPs are in vitro pre-integrated Cas9 nucleases and gRNA, delivered
into plant
 Equally efficient to plasmid based expression.
 RNPs are delivered in isolated plant protoplasts.
 Tobacco, Arabidopsis, lettuce, rice, Petunia, and wheat.

32. Proposed workflow for DNA free genome editing. Cas9 is expressed purified from E. coli. In vitro transcription of single guide RNA
(sgRNA) and transcribed in vitro and RNP complex formation. RNPs and DNA precipitation onto 0.6 µm gold particles followed by
Particle bombardment in targeted cells. Plants regeneration without any selective agent from bombarded cells and screened for mutations
via PCR/restriction enzyme assay and deep sequencing.

33. CASE STUDY

34. POWDERY MILDEW
 Fungal disease.
 Erysiphales, with Podosphaera xanthii (a.k.a. Sphaerotheca fuliginea), most
commonly reported cause.
 Easy to identify.
 Display white powdery spots on the leaves and stems.
 Layer of mildew made up of many spores forms across
the top of the leaves.
 Chemical methods, bio organic methods,
genetic resistance.
 Slow down the growth of plant, in severe cases reduces
fruit yield and quality.

35. BACKGROUND
 MILDEW RESISTANT LOCUS O (Mlo), encodes a membrane-associated protein
with seven transmembrane domains.
 Confer susceptibility to Oidium neolycopersici.
 Homozygous loss-of-function mutations (mlo) result powdery mildew resistance.
 16 Mlo genes, SlMlo1 to SlMlo16, SlMlo1 major contributor to powdery mildew
susceptibility.
 Natural non-transgenic loss-of-function slmlo1 mutants, but introgression mutations
is lengthy and laborious process.
 Generated transgene-free genetically edited slmlo1 tomato (Tomelo) variety using
the CRISPR/Cas9 system.
 Deleted SlMlo1 locus using the double sgRNA strategy.

36. METHODS
 Plasmid pAGM4723::Cas9_sgRNA1_sgRNA2
 Plant transformation
 GCR7589, a derivative of tomato cultivar Moneymaker, was
transformed
T0 transgenic plants were selected with kanamycin medium medium
and then transferred
into soil.
 Plant DNA extraction.
 Detection of Cas9-induced deletions and T-DNA in plant genomic DNA using
PCR
 Whole genome Illumina sequencing.

37.  10 primary transformants (T0) carrying T-DNA expressing Cas9 and sgRNAs,
analysed for altered electrophoretic mobility, indication SlMlo1 modifications.
 8 out of 10 tested T0 transformants showed a mobility shift indicating the
presence of mutations.
 3 transformants (2, 8 and 10), showed band shift consistent with the expected
deletion of 48 bp.
 All three transformants that showed the expected band shift proved to carry
homozygous (transformants 2 and 8) or biallelic (transformant 10) mutations
at the SlMlo1 locus.
 To generate non-transgenic slmlo1 tomato lines, T-DNA segregated by selfing T0
transformant and cultivating next generation (T1) plants.
 5 slmlo1 T-DNA-free individuals identified.
RESULTS

38. Disease resistance assays revealed all the slmlo1 mutant plants fully resistant.
All tested wild-type plants were susceptible.
slmlo1 mutant plants morphologically similar and produced harvested fruit weight
similar to the wild type.

39. Generating non-transgenic slmlo1
tomato lines resistant to powdery
mildew.
(a) The SlMlo1 locus
was targeted by two sgRNAs
(b) T0 tomato transformants were
tested for the presence of deletions
using PCR
(c) Selected T0 transformants
genotyped using the PCR band
shift assay alongside wild
type (WT)
(d) SlMlo1 sequencing reads from
selected T0 transformants
(e) Leaves of tomato plants
inoculated with Oidium
neolycopersici (5 weeks post
inoculation)
(f) PCR genotyping of the T1
generation for the presence
T-DNA and the slmlo1 mutation.
The agarose gels presented in panels
(b and c) were cropped.

40. Illumina sequencing data
(a) Quantification of Illumina
sequencing reads matching the
T-DNA or vector backbone in
wild type and slmlo1 T1
progeny lines;
(b) Coverage of the T-DNA by
Illumina reads
(c) Coverage of the SlMlo1 locus
by Illumina reads.

41. CONCLUSION
Named the powdery mildew resistant slmlo1 tomato variety Tomelo.
 9.5 months from the DNA transformation step.
 slmlo1 mutation could be readily introduced into elite or locally adapted varieties in
less than a year with relatively little effort or investment.

42. FUTURE PROSPECTS
 Recent advances have revolutionized genome editing but certain issues and challenges
like the SSN/DNA delivery methods, off-target effects needs to be addressed for better
efficiency and output.
 Some aspects of the editing system are still unclear, including the catalytic activity of
Cas9, target sites identification and the importance of PAM sites.
 Approaches like Digenome-seq and GUIDE-seq have been developed to detect off
target activities in human cells and need to be adapted to plants too for better efficiency
and specificity of Cas9.
 Till date, the genome editing systems are mainly used to destroy genes in plants by
inducing DSBs following NHEJ repair that introduces Indels at the target site.

43. THANK YOU!