CRISPR/Cas9 PotentialAnna Dye, Beau SmithBiol 443: Plant Development and Genetic Engineering

Biology BackgroundClustered 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 withCRISPR sgRNA, allowing it to bind withDNA of interest and cleave both strands(two cleavage sites).

MotivationThere 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.

RIN mutated tomatoes: Experimental DesignGene 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 codonGenomic DNA from leaf of regenerated plants. Guide 1 and 2 tested with restriction enzymes, guide 3 tested with hetero-duplex mobility assay.

RIN mutated tomatoes: Construct and TransformationConstruct 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.

RIN mutated tomatoes: ResultsAnalysis of two guide-1 mutants, one guide-2 mutant, and four guide-3 mutantsBioassay

G3 plants eventually turned redG2 and G1 plants showed ripening inhibition

Western blotNo RIN protein expressionin G1 or G2; G-3 had expression

PCRHomozygous expression of mutation

RIN mutated tomatoes: Takeaway messageKnockout 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.

CRISPR Cas9-mediated Viral Interference in PlantsExperimental DesignVirus: 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.

CRISPR CAS9 Viral Interference Methods/Construct

CISPR Viral Interference in Plants: Major ResultsCRISPR 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

Conclusions/Future DirectionsProof 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

Complications or Concerns for these MethodsRegulation 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.

Future Directions Cont’dKnock-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

ReferencesRegalado, 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/

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