Abstract
Intellectual disability (ID) disorders are a group of neurodevelopmental disorders defined by limitations in adaptive behavior and cognitive abilities. One to three percent of the global population are estimated to have some degree of ID. The X chromosome accounts for about 15 percent of currently known ID-associated genes. X-linked intellectual disabilities (XLID) are caused by mutations in genes on the X chromosome. In female somatic cells, one X chromosome becomes randomly transcriptionally inactive in each cell, ensuring an X-linked gene dosage compensation between females and males, known as X chromosome inactivation (XCI). This causes a mosaic expression of neurons, where half of the cells express the healthy copy of the gene and the other half express the mutant copy. Interestingly, an estimated 15-30% of X-linked genes escape XCI. These escape genes are associated with specific epigenetic signatures linked to active chromatin. Due to random XCI in females, the presence of the healthy allele in cells presents an opportunity to reactivate the healthy allele from the inactive X chromosome with targeted epigenetic editing.
The overall purpose of this study is to expand an already developed epigenetic editing platform to modify the expression of several XLID genes. A CRISPR activation (CRISPRa) library for nine XLID genes, containing ten sgRNAs per gene, was constructed with the intention of achieving higher throughput in targeting multiple gene loci. It was constructed by cloning individual Weismann CRISPRa single guide RNAs (sgRNAs), with a single Golden Gate cloning reaction, into U6 expression plasmids and sequence validated by amplicon sequencing. The XLID CRISPRa library was then packaged in lentiviral particles. Upon the construction of a CRISPRa library, target genes could be further analyzed by transducing HEK293T cells and probing for gene regulation using RT-qPCR. Following the library screen, target genes and sgRNAs could be selected for deeper assessment of reactivation.
As a proof of concept, we screened sgRNAs targeting the XLID gene, CASK with different epigenetic effector domains. If successful, this approach would lead to targeted reactivation of the wildtype CASK allele from the inactive X chromosome in heterozygous females. De novo loss of function mutations in the X-linked gene CASK disrupt brain development and cause severe symptoms such as severe learning impairments, abnormal repetitive behaviors, and recurrent seizures. The initial CASK sgRNA screen was followed by the identification of sgRNA combinations to increase efficacy of gene regulation. Split dCas9 plasmids on the AAV2 backbone expressing C terminal (C-) MPH and N-terminal (N-) VP64-p65-Rta (VPR) tripartite activator construct (MPH/VPR) were co-transfected in HEK293T cells with individual CASK sgRNAs. qRT-PCR was used to determine increased CASK expression. sgRNA 1 significantly increased CASK expression when coupled with MPH/VPR. To assess if combinations of sgRNA would lead to synergistic effects for gene regulation, combinations of up to three sgRNAs were transfected with MPH/VPR in HEK293T cells. sgRNA combinations were semi-randomly selected equally tiled across the promoter. Two combinations consisting of three sgRNAs each, resulted in a significant increase in CASK expression.
The lead individual sgRNA(s) and sgRNA combination(s) displaying an increase in CASK expression were further tested using C-terminal TET1 catalytic domain (TET1CD) and N- terminal VPR split dCas9 constructs to assess how TET1, a demethylating agent, combined with a strong trans-activator such as VPR (TET1/VPR) affects CASK gene expression and to further assess DNA methylation editing of the CASK gene promoter. A combination consisting of three sgRNAs that displayed a significant increase in CASK expression when co-transfected with MPH/VPR in HEK293T cells, also resulted in a significant increase in CASK expression with TET1/VPR.
In summary, we have shown that the XLID-associated gene CASK can be regulated with CRISPR/dCas9. sgRNAs for CASK will be further tested for epigenetic editing using C-TET1CD and N-VPR split dCas9 constructs for targeted demethylation. Future studies will use CASK variant containing iPSC-derived neuronal stem cells and patient-derived cells to assess reactivation and functional recovery.