Abstract
Purpose :
Peripherin-2 is an integral membrane protein expressed in rod and cone photoreceptors and is involved in disk morphogenesis. It is localized to the highly curved rim regions of the outer segment disks. In X. laevis, rods have two distinct peripherin-2 homologs (xrds35/36 and xrds38). The purpose of this investigation was to knock out one of the peripherin2 homologs (xrds38) in X. laevis in order to study its functions.
Methods :
Crispr/Cas9 methodology was used to knock out X. laevis peripherin-2 (xrds38). Synthetic guide RNAs to multiple PAM sites in the gene sequence were coinjected with Cas9 mRNA into fertilized single cell Xenopus embryos. Chimeric F0 knockouts were raised to maturity and crossed to each other to generate full knockout lines in which all xrds38 alleles contain nonsense mutations. Confocal microscopy, Western blots and EM were performed on retinas of d14 chimeric F0 or full knock out F2 tadpoles.
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 knock out animals no longer expressed xrds38 in their retinas. Lack of xrds38 expression did not cause retinal degeneration and gross photoreceptor morphology was only subtly altered. The number or depth of incisures was reduced as suggested by the lack of labeling with anti-xrds35/36 antibody. TEM indicated that the characteristic hairpin structure of the disk rims was intact.
Conclusions :
We successfully knocked out xrds38 expression in X. laevis photoreceptors. Our analysis of these knockouts indicates that xrds35/36 alone is sufficient to drive disk synthesis and rim formation such that the retinas look grossly normal and did not undergo degeneration. However, xrds38 appears to play a role in outer segment incisure formation/organization. Future studies will include simultaneously knocking out both xrds38 and xrds35/36.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.