Abstract
Purpose :
Peripherin-2 is an integral membrane protein expressed in rod and cone photoreceptors and is involved in disk morphogenesis. It is specifically localized to the highly curved rim regions of the outer segment disks. The purpose of this investigation was to knock out peripherin-2 in X. laevis to aid in the study of the role and functions of this protein.
Methods :
Crispr/Cas9 methodology was used to knock out Xenopus peripherin-2 (xrds38). Multiple PAM sites in the DNA sequence were identified and synthetic guide RNA’s were designed and generated by in vitro transcription. sgRNA’s were coinjected with Cas9 mRNA into fertilized single cell X. laevis embryos. Injected embryos were raised at 18C on a 12:12 light cycle. At 2dpf (days post-fertilization), whole embryos were used to generate genomic DNA. The region surrounding the PAM site was amplified by PCR and DNA sequence analysis was performed to assess the efficiency of editing. At 14dpf, tadpoles were sacrificed and their eyes were enucleated for western blot and immunohistochemical analysis.
Results :
Xenopus laevis are pseudo-tetraploid and have two functional genes (and therefore four alleles) that encode xrds38. Several sgRNA’s to different PAM sites were investigated and a single sgRNA (sg3) was identified that efficiently edits both genes based on sequencing of genomic DNA. Western blots and antibody labeling of frozen sections confirmed that a subset of animals generated no longer expressed xrds38 in their retinas. Lack of xrds38 expression did not cause retinal degeneration.
Conclusions :
We were able to successfully knock out xrds38 expression in photoreceptors. The lack of retinal degeneration observed is likely because X. laevis also express a second peripherin-2 (xrds35/36) which is highly similar in sequence. Xrds35/36 is not however thought to be an orthologue of rom1. The ability to knock out xrds38 will allow us to obtain further insights into the role of peripherin-2 in disk morphogenesis and outer segment homeostasis. Future studies will include simultaneously knock out of both xrds38 and xrds35/36.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.