Genetic screening of key genes regulating hair cell (HC) differentiation is of particular importance in gene therapy toward HC restoration in deaf patients in clinic.
There is a high degree of similarity regarding the developmental process and functions of auditory system between mice and human beings, especially in sound receptor cells, HCs. While transcriptomic profiles of HCs at multiple postnatal ages are currently available, it remains poorly understood in terms of which genes are essential in regulating HC differentiation and maturation because generating mutant Founder 0 (F0) mice, germ-line transmission and subsequent inter-breeding are time-consuming and laborious.
In a study published in Development, Dr. LIU Zhiyong’s group from the Institute of Neuroscience (ION), Center for Excellence in Brain Science and Intelligence Technology (CEBSIT) of the Chinese Academy of Sciences (CAS), established a rapid genetic screening method.
Previous work of Dr. LIU’s group has established a protocol which is able to inactivate three non-lethal genes simultaneously in F0 mice ready for immediate phenotype analysis. However, there are 25% of mammalian genes whose homozygous mutants will lead to embryonic or neonatal lethality, thus the strategy is not suitable for those lethal or essential genes. The typical solution is to use conditional knock out strategy, but it is very time consuming.
Is there an alternative approach to rapidly screen those essential genes? This study provides a novel genetic strategy named ‘mosaic CRISPR-stop’ to generate F0 mice with homozygous but mosaic mutations. By injecting base editor components and gene specific sgRNAs into one of the two blastomeres, offspring cells with or without gene editing are distributed randomly throughout various organs.
To assess efficiency of mosaic CRISPR-stop, the researchers chose Atoh1 for the first proof-of-concept study. Atoh1 mosaic mutant mice have much less HCs. The phenotype of shortened cochlear in Sox10 mutants is also reproduced by mosaic CRISPR-stop. Rbm24 is another essential gene expressed in cochlear HCs.
With mosaic CRISPR-stop, the researchers, at the first time, found that Rbm24 is of importance for the survival of cochlear outer hair cells (OHCs), which is further validated by independent Rbm24 conditional knockout model.
This study highlights that mosaic CRISPR-stop is a reliable method and will pave the way for genetic screening of developmentally essential genes in the mouse inner ear and other organs.
Reference: Guangqin Wang, Chao Li, Shunji He, Zhiyong Liu, “Mosaic CRISPR-stop enables rapid phenotyping of nonsense mutations in essential genes”, Development 2021 148: dev196899 doi: 10.1242/dev.196899 Published 9 March 2021
Provided by Chinese Academy of Sciences