CRISPR Disruption of YFP in Zebrafish Lens

Anna Colando1, Andrew Foerder1, Colton Kray1, Jasmine Shirey1, Owen Lawrence1, John I. Clark1,2

1Department of Biological Structure, 2Department of Ophthalmology, University of Washington, Seattle, WA.

Research Hypothesis

CRISPR targeting of transgenic Yellow Fluorescent Protein (YFP) lens expressing

zebrafish will result in a noticeable reduction of YFP over the 3-8 day post fertilization period.

CRISPR-Cas9 is a Bacterial Immune System(Clustered Regularly Interspaced Short Palindromic Repeat)

• CRISPR functions to inactivate infecting viruses.

• Bacterial Cas9 requires two distinct RNA transcripts to induce Double Stranded Breaks (DSB) in infecting viral RNAs:

o CRISPR RNA (crRNA) o Trans-acting CRISPR RNA

(tracrRNA)

• Over the past few years, investigators have modified the CRISPR system to allow easy manipulation of research genomes.

CRISPR-Cas9 Generates Double Stranded Breaks

• Bacterial CRISPR-Cas systems require multiple proteins, but we used type II which requires a single endonuclease, Cas9.

• crRNA and tracrRNA are reconfigured as a single guide RNA (gRNA).

• Cas9 enzyme executes a DSB using gRNA for specificity.

CRISPR-Cas9 DSB Repaired by NHEJ

• DSBs can be repaired through nonhomologous end joining (NHEJ).

• NHEJ repair generates InDels

• InDels can alter the reading frame and induce a truncation, thus inactivating the targeted protein.

CRISPR-Cas9 Tissue-Specific Gene Disruption

Procedure

1. Choose gRNA nucleotides to target YFP gene.

2. Annealing of gRNA oligonucleotides.

3. Construction of the tissue-specific CRISPR vector.

4. Injection of the CRISPR construct in zebrafish embryos.

CRISPR-Cas9 Targeting of YFPFound published gRNA targeting GFP

SCIENCE VOL 339 15 FEBRUARY 2013

Annealing of Gene Specific Oligonucleotides

Construction of a Tissue-Specific CRISPR Vector

1. Recombination reaction performed with:1. Destination vector (pDestTol2CG2-U6:gRNA)2. 5’ entry vector containing alphaA crystallin promoter3. Middle entry vector (Cas9/p3E-polyA)

2. Positive ampicillin selection

3. Extraction of plasmid DNA to confirm recombination reaction success with various digestions

4. BseRI digestion of resulting CRISPR vector

5. Ligation of YFP-specific seed sequence into digested destination vector

6. Positive ampicillin selection

7. Extraction of plasmid DNA to conduct sequencing

Construction of a Tissue-Specific CRISPR Vector

1. Recombination reaction performed with:1. Destination vector (pDestTol2CG2-U6:gRNA)2. 5’ entry vector containing alphaA crystallin promoter3. Middle entry vector (Cas9/p3E-polyA)

2. Positive ampicillin selection

3. Extraction of plasmid DNA to confirm recombination reaction success with various digestions

4. BseRI digestion of resulting CRISPR vector

5. Ligation of YFP-specific seed sequence into digested destination vector

6. Positive ampicillin selection

7. Extraction of plasmid DNA to conduct sequencing

Process Overviewalpha A Crystallin Promoter: promotes protein expression exclusively in the lens of the zebrafish.

Polymerase III promoter: promotes expression of the gRNA in all zebrafish tissues.

Recombination Reaction

Goal: Assemble and prepare destination vector for gRNA sequence insertion

• Destination vector (pDestTol2CG2-U6:gRNA)• 5’ entry vector containing alphaA crystallin promoter• Middle entry vector (Cas9/p3E-polyA)

BseR1 Sites in the Newly Made VC (V:pDestTol2CG2-

U6:gRNA C:Cas9)

BseRI DigestionGoal: Perform digestion with single-cleaving BseRI enzyme to open up the vector and ready it for gRNA sequence insertion.

Digestion Results

6000

3000

1000

1a 2a 3a 3b Uncut

YFP-Specific Seed Sequence Ligation

Goal: Clone the YFP-specific gRNA sequence into the destination vector.

CRISPR Vector Destination Vector

Kpn1/PflM1 Double Digest Results

VCVCC

Sequencing of CRISPR Vector

Goal: Confirm success of correct gRNA sequence insertion.

• Inject zebrafish embryos with the CRISPR construct.

•Phenotypic analysis to evaluate success.

•Generation of stable lines.

Next Steps in CRISPR-Facilitated YFP Reduction

Acknowledgements• John I. Clark Lab

• George M. Church Lab

• Owen Lawrence

• UW Biological Structure Department

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Charpentier, Emmanuel, and Jennifer Doudna. "Rewriting a Genome." NATURE.com. Macmillin Publishers Limited, 7 Mar.

2013. Web. 30 July 2015.

Mali, Prashant, Kevin Esvalt, and George Church. "Cas9 as a Versatile Tool for Engineering Biology." Naturemethods.com.

Nature Publishing Group, 27 Sept. 2013. Web. 30 July 2015.

"Protocol for Tissue-specific Gene Disruption in Zebrafish." Web. 30 July 2015.

<file:///C:/Users/imager/Downloads/LZon_CRISPR Vector Methods.pdf>.

"Zebrafish and Retinoic Acid." Zebrafish and Retinoic Acid. Hampden-Sydney College. Web. 30 July 2015.