The
unc-52 gene in Caenorhabditis elegans encodes for the protein UNC-52 and is a homolog of the mammalian gene Heparan Sulfate Proteoglycan 2 (HSPG2), which encodes for the protein perlecan (Rogalski et al., 1993; Mullen et al., 1999). HSPG2 is implicated in the human diseases Schwartz-Jampel Syndrome type 1 and Dyssegmental Dysplasia, Silverman-Handmaker type (Arikawa-Hirasawa et al., 2001; Stum et al., 2006). The mutated
unc-52 gene expresses the phenotype for uncoordinated movement (Unc), which involves progressive paralysis and retarded sarcomere construction (Martinez et al., 2018). The UNC-52 protein is localized in the striated muscle dense bodies and the basement membrane, where it plays an important role in developmental processes such as cell adhesion, cell migration, and signal transduction (Kihira et al., 2012). Within the
unc-52 gene, an RGD (Arg- Gly- Asp) sequence is located at amino acid locations 746, 747, and 748 in exon 7. Exon 7, containing RGD748, is included in all three major isoforms, short (S), medium (M), and long (L) (Mullen et al., 1999). The RGD sequence is part of the Laminin IV type A domain and primarily functions as a cell attachment and adhesion site for integrins (Rogalski et al., 1993; Mullen et al., 1999). In this study, two separate mutations were performed on this
unc-52 RGD sequence using CRISPR-Cas9 technology. The
unc-52(
kq748) mutation replaced the aspartic acid (D) located at the 748 amino acid position with a glutamic acid (E) (Takahashi et al., 2007). The
unc-52(
kq745) mutation removed the RGD sequence by deleting amino acids 746, 747, and 748. Previous studies have shown that
unc-52 gene mutations cause the disorganized distribution of
pat-3 integrin, which is a receptor for extracellular matrix proteins (Rogalski et al., 1995). In order to study the cellular phenotypes of the
unc-52(
kq748) mutation, a double mutant was created:
pat-3::GFP;
unc-52 (
kq748). Staining showed that localization of the
pat-3::GFP reporter in the double RGD mutant appeared normal, with dense bodies and M-lines alternating along the muscle filaments (Figures 1A and 1B). In order to visualize the actin filaments in the body wall muscles of N2 wild type and
unc-52(
kq748) mutants, staining was performed using 0.4 U/mL rhodamine-conjugated phalloidin, Thermo Fisher Scientific, Waltham, MA (Figures 1C and 1D). This
unc-52(
kq748) staining showed no obvious abnormalities outside of interruptions due to fixation and slicing procedures (Figure 1D). Additional assays were performed to investigate any other physical or behavioral phenotypes. The thrashing assay, in which worms were placed in drops of M9 buffer and the number of thrashes were counted for 15 seconds, showed a 27% average decrease in thrashes per second for the
unc-52(
kq748) mutants compared to the N2 worms, and a 33% decrease in
unc-52(
kq745) mutants (Figure 1E). A one-way ANOVA statistical test was performed to check the statistical significance of these results. The p-value from the ANOVA test was 3.1×10-9, which is less than the 0.05 needed to be statistically significant. Post hoc testing was then done to see which groups differed. Both the
unc-52(
kq748) and
unc-52(
kq745) groups differed significantly from the N2 wild-type group, as shown by p-values of 7.1×10-6 and 4.63×10-8 respectively. These results show that there was a statistically significant decrease in motility for both
unc-52(
kq748) and
unc-52(
kq745) mutants.