Optimized protocol to generate genome-wide inactivated Cas9-expressing murine T cells

Laprie-Sentenac, Marguerite; Cretet-Rodeschini, Clara; Menger, Laurie

doi:10.1016/j.xpro.2022.101922

Cited by 1 publication

(1 citation statement)

References 14 publications

(21 reference statements)

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“…A previous study in Rosa26-Cas9 knock-in mouse showed that constitutive SpCas9 expression did not result in any detectable toxicity or morphological abnormalities in multiple organs (including the brain, kidney, liver, spleen, and heart tissue) of this transgenic strain [ 24 ]. Multiple studies have provided evidence of the safety of Cas9 mice by showcasing the utilization of diverse tissues and cells from these murine models [ 34 , 35 , 36 ]. However, long-term SpCas9 expression and its associated toxicity have not been characterized in ocular tissues in these mice, which can be bred for germline transmission of the Cas9 allele.…”

Section: Discussionmentioning

confidence: 99%

Long-Term Evaluation of Retinal Morphology and Function in Rosa26-Cas9 Knock-In Mice

Mohan

Dubey

Jung

et al. 2023

IJMS

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The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.

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