1. Generating Isogenic Cell Lines To Study The Influence
of The CRB1 Gene on Retinal Degradation
Sebastian Fonseca
B.S. Biomedical Engineering
Arizona State University
Mentored by Mei Jiang
PI Dr. Dennis Clegg

2. Retinal Dystrophies and CRB1
• Retinitis pigmentosa (RP) and leber
congenital amaurosis (LCA).
• Chronic and incurable diseases.
• Eventually lead to almost complete loss of
sight.
• Usually detected in early childhood stages.
http://www.isotineeyedrops.com/eye-care/symptoms-of-retinitis-pigmentosa/
http://www.medscape.com/viewarticle/774365
http://www.blindness.org/leber-congenital-amaurosis

3. Research Objectives
• Recreate a mutation at a specific site in the
CRB1 gene that has been associated with the
development of RP and LCA.
• Carry out the initial steps of the CRISPR/Cas9
genome editing technique creating nine
different clones with mutations in the CRB1
gene.
• Create isogenic cell lines that possess the
p.Cys948Tyr, p.Cys698Arg and p.Leu878Pro
mutations in order to observe if the
phenotype associated to retinal degradation
was in any way correlated.
Bujakowska, K., Audo, I., Mohand-Saïd, S., Lancelot, M.-E., Antonio, A.,
Germain, A., … Zeitz, C. (2012). CRB1 mutations in inherited retinal
dystrophies. Human Mutation, 33(2), 306–315.
http://doi.org/10.1002/humu.21653

4. Methodology: CRISPR/Cas 9 Complex
• Clustered regularly interspaced short palindromic repeats (CRISPR).
• Use of donor DNA in order to use the cell’s natural repair mechanisms to generate a mutation in the
genetic code of a group of cells.
• Use of an endonuclease (Cas9) and a small guide RNA in order to cut a cell’s DNA at the site of a
specific mutation for CRB1.

5. Methods and Experimental Techniques
1. Minipreparation
2. Vector digestion and dephosphorylation
3. Oligos annealing and phosphorylation
4. Ligation
5. Transformation and colony selection

6. Minipreparation (miniprep)
• Miniprep is a technique carried out in
order to obtained purified DNA from
cells.
• The procedure involves the use of
several buffers that help cell lysis,
alongside centrifuge spinning cycles
that allow unwanted material to be
discarded.

7. Vector Digestion and Dephosphorylation
• Vector digestion consists of creating a
double strand break in the DNA of the
vector.
• This break will be used to insert the
oligos sequence complementary to
the site of the mutation.
• Vectors have to be depohsphorylated
in order to avoid self-ligation.
• Self-ligation occurs when the two
ends of the vector attach to each
other and therefore do not allow for
the olligos to be inserted.

8. Oligos annealing and
phosphorylation
• The forward and reverse oligos
sequences are recombined to
form double stranded DNA.
• Phosphorylation functions as a
recognition process for the oligos
to attach to the ‘break’ site of the
vector.
Ligation
• In this step the oligos are attached
to the vector with the help of
specific enzymes called ligases.
• Many of the failed transformation
procedures are related to
complications in this step.

9. Transformation and Colony Selection
• The vectors with the oligos sequences inserted in them are transfected into
bacterial cells in order to increase the amount of DNA that can be later used.

10. Experimental Data

11. Vector Digestion

12. Annealing

13. Ligation Electrophoresis
Ligation products
Digested pgRNA-CKB vector
1kb ladder

14. Successful Ligation and Transformation
• Bacteria are growing on the ampicillin plate after using modified ligation protocol with oligos dilution.

15. Validation of Transformation with Gel Electrophoresis
• Bacteria were collected from the ampicillin plate
and purified plasmid DNA was obtained by using
the miniprep protocol.
• The plasmid DNA was mixed with restriction
enzymes (Esp3I), however because of the
absence of the original sequence in the vector,
the enzymes could not generate a double strand
break.

16. Sequencing

17. Future Work
• Deliver the vector containing the sgRNA alongisde a sample of donor DNA (containing the p.Cys948Tyr
mutation) into a CRISPR line of mammalian cells that already contain the Cas9 knock-in protein.
• Expand the group of cells containing the desired mutation.
• Perform validation and sequencing of these isogenic cell lines.

18. Questions

19. Acknowledgements
• Dr. Linda Petzold
• Stephanie Lupo
• ICB
• SABRE Program and
participants
• Family and friends
Clegg Lab:
• Professor Dennis Clegg
• Dr. Mei Jiang
• Katharine McLean
• Britney Pennington
• Leah Foltz
• Kelsy Siegel
• Qirui Hu
• Tracy Clevenger