Abstract
Purpose :
The CRISPR-Cas9 knockin mouse (NOD.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J) was originally reported by Platt et al (2014) and is now commercially available from Jax Laboratories, Bar Harbor, Maine. The mouse, which has a CAG-3xFLAG-SpCas9-P2A-EGFP cassette inserted at the Rosa26 locus, theoretically displays ubiquitous Cas9 and EGFP expression in nucleated cells. Previous studies showed Cas9 expression and successful gene knockdown across bone marrow, brain, and lung tissues using AAV and lentiviral-delivered sgRNA (Platt et al., 2014). Our aim was to characterise retinal expression of SpCas9 to determine the suitability of this mouse as a model for optimising retinal gene knockdown.
Methods :
Cas9EGFP homozygotes were killed by cervical dislocation. For Western blot, retinae were dissected and underwent dissociation and magnetic-activated cell sorting (MACS) into CD73+ve (rod photoreceptors) and CD73-ve (remaining retinal cells) populations. Western blot was carried out for FLAG, SpCas9 and α -tubulin. For immunohistochemistry (IHC), eyes were fixed in 4% PFA for 30 minutes, embedded in OCT and frozen. Cryostat sections were stained for SpCas9, FLAG, PKC-a, and calbindin, with secondary Alexa-Fluor goat antibodies, and imaged on a Zeiss LSM710 confocal microscope. In vivo imaging took place using the Heidelberg SLO and OCT.
Results :
Western blot showed clear whole retina SpCas9 expression, but weak expression in the CD73+ve extracted cell population (rods). IHC showed Cas9 and EGFP expression in the retinal ganglion cell and inner nuclear layer, but not in the outer nuclear layer or photoreceptor outer segments. Cas9 and EGFP expression did not co-localise with PKC-α or with calbindin, indicating limited horizontal and bipolar cell expression, but showed an expression pattern characteristic of Müller glia.
Conclusions :
The NOD.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J mouse would be a suitable model for optimising Cas9 knockdown in retinal ganglion cells or Müller cells, but may be a poor model for photoreceptor targets. The apparent lack of both Cas9 and EGFP in this model may reflect variable expressivity from the ROSA26 locus in retinal cell populations.
Reference:
Platt et al. Cell 2014
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.