1. CRISPR/Cas9 Potential
Anna Dye, Beau Smith
Biol 443: Plant Development
and Genetic Engineering

2. Biology Background
Clustered Regularly Interspaced Short Palindromic Repeats:
Part of the Prokaryotic Adaptive Immune System (40% of Bacteria, 90% of Archaea)
Segments of DNA with repeating base
sequences. CAS9: Nuclease that complexes with
CRISPR sgRNA, allowing it to bind with
DNA of interest and cleave both strands
(two cleavage sites).

3. Motivation
There have not been many advances to tools used in genetic engineering.
Find a tool speed up and simplify genetic engineering while making it more
accessible.
Useful to prevent monoculture.
Efficient in both monocots and dicots (85% efficiency in rice).
Gene silencing over gene suppression.

4. RIN mutated tomatoes: Experimental Design
Gene of interest: RIN--Master regulator gene for fruit ripening in tomatoes,
mutations inhibit ripening of fruit.
Hypothesis:
CRISPR/Cas9/sgRNA complex will disrupt ripening in tomatoes by introducing
double stranded breaks.
Design:
Three different sgRNA: 1) middle of coding region (protein interaction with
MADS-box proteins) 2) middle of coding region (transcription activating), 3) RIN
start codon
Genomic DNA from leaf of regenerated plants. Guide 1 and 2 tested with
restriction enzymes, guide 3 tested with hetero-duplex mobility assay.

5. RIN mutated tomatoes: Construct and Transformation
Construct including sgRNA, NPTII, and Cas9 cloned into agrobacterium binary
vector with kanamycin resistance.
3’UTR::35S(P)::NPTII::hsp17.3(T)
PcUBI4-2(P)::Cas9::pea3A(T)
OsU3::gYSA
--Introduction into tomato explant by dipping in agrobacterium, and following
standard procedure.

6. RIN mutated tomatoes: Results
Analysis of two guide-1 mutants, one guide-2 mutant, and four guide-3 mutants
Bioassay
G3 plants eventually turned red
G2 and G1 plants showed ripening inhibition
Western blot
No RIN protein expressionin G1 or G2; G-3 had expression
PCR
Homozygous expression of mutation

8. RIN mutated tomatoes: Takeaway message
Knockout heritable mutations can be created with CRISPR: applicable to genetic
engineering
NHEJ is a useful way to create these knockout mutants
Proof of concept for more useful applications
No interruption of off-target endogenous genes
Ethylene application would induce ripening.
Useful for commercial application.

9. CRISPR Cas9-mediated Viral Interference in Plants
Experimental Design
Virus: Tomato Yellow Leaf Curl Virus (TYLCV), part of the Geminiviridae family:
Major threat to food security, worldwide.
Premis: Each genus of this family has conserved regions of DNA (Enter
CRISPR!). By having a plant express CRISPR Endonuclease against these
conserved regions, one can defend against multiple viruses.
Hypothesis: Plants overexpressing CRISPR endonuclease and primed sgRNA
specific to TYLCV Virus/ Geminiviridae conserved region will have lower levels of
infection of TYLCV/other Geminiviruses.

10. CRISPR CAS9 Viral Interference Methods/Construct

11. CISPR Viral Interference in Plants: Major Results
CRISPR Interfered with Replication, Decreased TYLCV Accumulation of both
natural infection and laboratory infection, and reduced symptoms of viral
infection
CRISPR was capable of targeting multiple different Geminiviruses with a
single sgRNA template targeting a conserved region of the families genome
CRISPR was capable of multiplexing (targeting multiple regions of viral
genome) further reducing symptoms

13. Conclusions/Future Directions
Proof of concept
Advances basic scientific research
Multiplexing solves issues of editing polyploid plants
Potentially faster introduction of edited crops into market
Accessibility for niche interests
More efficient and less expensive than other gene editing technology
Can target chromatin

14. Complications or Concerns for these Methods
Regulation concerns
Off-gene targets: May target other genes with partial homology to sgRNA
InDel: Mutations that may result in gene disruption via frameshifting and/or by
creating premature stop codons.
Transgene escape is still a possibility: Gene-editing is heritable; however cas9
mediated immunity requires exposure to sgRNAs via a viral vector.

15. Future Directions Cont’d
Knock-in Genes for Plants: Shown to work in Zebrafish
Bioremediation: Use CRISPR to Eliminate transgenes in wild-type plants
Exploration of orthologs/synthesis of orthologs via targeted mutagenesis:
Creating more efficient/specific/utilizable Cas9
Targeting of RNA not just DNA

16. References
Regalado, A. DuPont Predicts CRISPR Plants on Dinner Plates in Five Years [internet]. MIT Technology Review; 2015 Oct [cited 2015 Dec 3].
Available from http://www.technologyreview.com/news/542311/dupont-predicts-crispr-plants-on-dinner-plates-in-five-years/
Ito, Y., Nisizawa-Yokoi, A., Endo, M., Mikami, M. CRISPR/Cas9 mediated mutagenesis of the RIN locus that regulates tomato fruit ripening.
Biochemical and Biophysical Research Communication [internet] 2015 [cited Dec 2015]; 467, 76-82. Available from
http://ac.els-cdn.com/S0006291X15306343/1-s2.0-S0006291X15306343-main.pdf?_tid=d7c482c0-9913-11e5-8f68-00000aacb362&acdnat=144907
4800_2de4ad6bf4eb5fc6ca4a5f003d46ad2a
Belhaj, K, Chaparro-Garcia, A.,Kamoun, S., Nekrasov, V. Plant genome editing made easy: targeted mutagenesis in model and crop plants using
the CRISPR/Cas system. Plant Methods [internet] 2013 [cited Dec 2015] 9:39. Available from http://www.plantmethods.com/content/9/1/39
Ali, Z., Abulfaraj, A., Idris, A., Ali, S., Tashkandi, M., Mahfouz, M. M. CRISPR/Cas9-mediated viral interference in plants. Genome Biology [internet]
Nov 2015 [cited Dec 2015] 16:238. Available from http://www.genomebiology.com/2015/16/1/238
Ma, X., et al. A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Molecular
Plant [internet] Aug 2015 [cited Dec 2015] Volume8, Issue 8, p 1274-1284. Available from
http://www.cell.com/molecular-plant/abstract/S1674-2052(15)00204-X
Kimura, Y., Hisano, Y.,Kawahara, A., Higashijima, S. Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by
CRISPR/Cas9-mediated genome engineering. Scientific Reports [internet] Jul 2014 [cited Dec 2015] 4:6545. Available from
http://www.nature.com/articles/srep06545
Grissa, I., Vergnaud, G., & Pourcel, C. (2007). The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and
repeats.BMC bioinformatics, 8(1), 172. Available from http://www.biomedcentral.com/1471-2105/8/172
Kermicle, J. L., Evans, M. M. S. 2010. The Zea Mays Sexual Compatibility Gene ga2: Naturally Occurring Alleles, Their distribution, and Role in
Reproductive Isolation.