1. CRISPR-Cas9 Mediated Gene Edit of Holocentric Binding
Protein 2 in Caenorhabditis elegans
Jackson Higginbottom, H. LaMascus, and L. Moore.
Department of Biology, College of Natural and Health
Sciences, Oklahoma Christian University.
2. Background
• Cancer Associated Gene (CAGE)-1
• Testes-specific expression in normal tissues
• Over-expressed in cancer
• Holocentric Binding Protein (hcp)-1
• Ortholog to CAGE-1
• Holocentric Binding Protein (hcp)-2
• Paralog to hcp-1
J Cell Biol., 147(3), 471–480.
3. Caenorhabditis elegans
• ~1mm long roundworm
• Live in soil and feed on bacteria
• Advantageous model organism
• Simple anatomy
• Invariant cell lineage
• Short life cycle
• Large brood size
• Amenable to genetic analysis
Genetics, 77(1), 71–94
4. Objectives
• Generate a strain of C. elegans containing a deletion of hcp-
2
• Characterize the phenotypes of hcp-2 deleted strains
• Test how hcp-2 gene dosage affects C. elegans
development
5. CRISPR-Cas9 System
Cas9 is an endonuclease
• Base pairing between 5’-sgRNA and target DNA
• Presence of PAM site
Homologous Recombination
• Nucleotide sequences are exchanged between two
similar or identical molecules of DNA
Genetics, 200(4), 1035–1049.
6. Methods
Figure 1. Plasmid Construction
Hygromycin resistanceHeat-shock Cre
Dominant roller LoxP recognition site
8. Results
Figure 4.Figure 3.
upstream
downstream
gDNA
23.1 kbp -
9.41 kbp -
6.56 kbp -
4.32 kbp -
2.3 kbp -
2.0 kbp -
1.5 kbp -
1.0 kbp -
0.564 kbp -
0.300 kbp -
Successful design of a
CRISPR/Cas9 system
for C. elegans induced
a deletion of hcp-2.
9. Conclusions and Future Directions
• Generate a strain of C. elegans containing a deletion of hcp-
2
• Design a functional CRISPR/Cas9 system for C. elegans
• Confirm successful homologous recombination by the
presence of two dominant markers and gel electrophoresis.
• Characterize the phenotypes of hcp-2 deleted strains
• Test how hcp-2 gene dosage affects C. elegans
development
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10. Citations
1. Brenner, S. (1974). The Genetics of Caenorhabditis elegans. Genetics,
77(1), 71–94.
2. Dickinson, D. J., Pani, A. M., Heppert, J. K., Higgins, C. D., & Goldstein,
B. (2015). Streamlined Genome Engineering with a Self-Excising Drug
Selection Cassette. Genetics, 200(4), 1035–1049.
3. Moore, L. L., Morrison, M., & Roth, M. B. (1999). Hcp-1, a Protein
Involved in Chromosome Segregation, Is Localized to the Centromere
of Mitotic Chromosomes in Caenorhabditis elegans. The Journal of Cell
Biology, 147(3), 471–480.
Design a functional CRISPR/Cas9 system for C. elegans
Confirm successful homologous recombination by the presence of two dominant markers, gel electrophoresis, and PCR
Talk about what happens once SEC is removed
The red arrow indicates…
A brief recap of what we have accomplished so far…
The next step for this project is to characterize.. And test hcp-2 gene dosage.