This document summarizes a study that used CRISPR-Cas9 to generate a strain of the nematode Caenorhabditis elegans containing a deletion of the hcp-2 gene. The objectives were to generate a C. elegans strain with an hcp-2 deletion, characterize the phenotypes of hcp-2 deleted strains, and test how hcp-2 gene dosage affects development. A CRISPR-Cas9 system was successfully designed for C. elegans and induced a deletion of the hcp-2 gene, as confirmed by gel electrophoresis. Future work will characterize phenotypes of the hcp-2 deleted strain and test how hcp-2 gene dosage impacts development.

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

7. Figure 2. Cas9-triggered Homologous Recombination and SEC Removal
Methods (Cont.)

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
✔
✔
✔

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.

11. Questions?