25 March 2020

March 2020

Effective CRISPR interference of an endogenous gene via a single transgene in mice

Mouse models are used in biomedical research to interrogate gene function often mimicking genetic diseases found in humans. Roughly the models can be grouped in to either constitutive or conditional knock-outs where the gene of interest (GOI) is permanently knocked-out in the entire organism or where the gene can be knocked-out in a spatial and/or temporal manner, respectively. Conditional gene knock-out models rely on the Cre-LoxP recombination system, a potent two-component system used in laboratories worldwide. The system bypasses barriers such as embryonic lethality and provides cell type specific loss of function information. However, this system has been shown to produce off-target recombination in additional cell types, making it unreliable. Furthermore, the system requires many breeding cycles for one gene knock-out alone, making it time consuming especially if several genes are to be eliminated.

The CRISPR/Cas9 system revolutionized genome editing due to its dynamic nature and ease of use. The ribonucleoprotein complex consisting of an endonuclease, Cas9, together with an RNA guide for specificity can be programmed to target thousand of genes and is today the go-to method for producing mouse model strains. In this study the authors investigated the potential use of the CRISPR interference (CRISPRi) system for producing a conditional mouse model. The CRISPRi system utilizes a dead Cas9 (dCas9) targeted within 150 bp of the transcription start site (TSS) of the GOI to sterically block its transcription. The system has been further improved by tethering the repressive Kruppel-associated box (KRAB) domain to the dCas9. The dCas9-KRAB complex can block the transcription from both alleles simultaneously and be introduced in the form of a single transcript. This would allow for a conditional knock-out mouse in a single generation as opposed to a minimum of two generations using the Cre-LoxP system. In addition, it has lower off-target effects in non-targeted cell types as lower levels of the dCas9-KRAB are not sufficient to cause repression. Furthermore, this system allows for more than one gene to be repressed by simply including additional gRNAs in to the transgene, giving it an additional advantage over the canonical Cre-LoxP system.

The authors sought to examine if a single transgene encoding dCas9-KRAB and sgRNA would be enough to repress the well characterized Tnfsf11 gene in mice. This gene is necessary for osteoclast formation and a null mutation for this gene leads to a phenomenon called osteopetrosis characterized by misshapen bones, failure of tooth eruption and retention of mineralized cartilage beneath growth plates additionally Tnfsf11 mice fail to develop lymph nodes. The transgene to cause Tnsfs11 repression consisted of the CAG promoter for driving the dCas9-KRAB expression and the U6 promoter in the opposite direction driving the sgRNA transcript and was inserted in a safe harbor locus via pronuclear microinjections in zygotes. Seven mice harbored the transgene and the phenotypes varied from a normal profile to phenotypes similar to those found in Tnfsf11 null mice, suggesting mosaicism amongst the founder mice. The mouse that recapitulated the full phenotype thus proved that CRISPRi can be used to successfully cause loss of function mutations in mice. The authors did not investigate spatial and temporal specificity but they did prove that CRISPRi causes lasting gene repression in different cell types, shown by the lack of lymph nodes and bone deformations recapitulated in one of the founder mice. In future experiments the system could be placed under a tissue specific promoter for spatial specificity. To address the temporal specificity the system could be placed under a tetracycline-regulated system.

 In conclusion this study showed that the adapted CRISPRi system can be employed to cause a lasting gene knock-down in vivo in various cell types. Furthermore, by selecting tissue specific promoters for transgene design and pairing it with a tetracycline-regulated system it could pose as an alternative to the Cre-LoxP system.