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X-linked diseases typically exhibit more severe phenotypes in males than females. In contrast, protocadherin 19 (PCDH19) mutations cause epilepsy in heterozygous females but spare hemizygous males. The cellular mechanism responsible for this unique pattern of X-linked inheritance is unknown. We show that PCDH19 contributes to adhesion specificity in a combinatorial manner such that mosaic expression of Pcdh19 in heterozygous female mice leads to striking sorting between cells expressing wild-type (WT) PCDH19 and null PCDH19 in the developing cortex, correlating with altered network activity. Complete deletion of PCDH19 in heterozygous mice abolishes abnormal cell sorting and restores normal network activity. Furthermore, we identify variable cortical malformations in PCDH19 epilepsy patients. Our results highlight the role of PCDH19 in determining cell adhesion affinities during cortical development and the way segregation of WT and null PCDH19 cells is associated with the unique X-linked inheritance of PCDH19 epilepsy.

Protocadherin 19 (Pcdh19) is an X-linked gene belonging to the protocadherin superfamily, whose members are predominantly expressed in the central nervous system and have been implicated in cell-cell adhesion, axon guidance and dendrite self-avoidance. Heterozygous loss-of-function mutations in humans result in the childhood epilepsy disorder PCDH19 Girls Clustering Epilepsy (PCDH19 GCE) indicating that PCDH19 is required for brain development. However, understanding PCDH19 function in vivo has proven challenging and has not been studied in mammalian models. Here, we validate a murine Pcdh19 null allele in which a β-Geo reporter cassette is expressed under the control of the endogenous promoter. Analysis of β-Geo reporter activity revealed widespread but restricted expression of PCDH19 in embryonic, postnatal and adult brains. No gross morphological defects were identified in Pcdh19+/β-Geo and Pcdh19Y/β-Geo brains and the location of Pcdh19 null cells was normal. However, in vitro migration assays revealed that the motility of Pcdh19 null neurons was significantly elevated, potentially contributing to pathogenesis in patients with PCDH19 mutations. Overall our initial characterization of Pcdh19+/β-Geo, Pcdh19β-Geo/β-Geo and Pcdh19Y/β-Geomice reveals that despite widespread expression of Pcdh19 in the CNS, and its role in human epilepsy, its function in mice is not essential for brain development.

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis , an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models. Electronic supplementary material The online version of this article (10.1186/s13059-019-1776-2) contains supplementary material, which is available to authorized users.