Abstracts

Rationale: Koolen-de Vries Syndrome (KdVS) is an autosomal dominant neurodevelopmental disorder caused by haploinsufficiency of the KANSL1 gene. This syndrome is characterized by developmental delay, intellectual disability, hypotonia, and distinctive facial features, and approximately a third of affected individuals have childhood-onset epilepsy. Most individuals (95%) with KdVS have a de novo deletion of ~500-kb on 17q21.31 that includes the KANSL1 gene, while ~5% of individuals have a de novo pathogenic variant in the KANSL1 gene. Haploinsufficiency of KANSL1 is enough to cause the full spectrum of 17q21.31 microdeletion disorder. Therefore, we hypothesize that increasing the amount KANSL1 protein by targeting the remaining functional copy of KANSL1 gene is a potential therapeutic approach for KdVS. Here, we investigate the ability of the CRISPRa-dCas9-VP64 system to upregulate transcription of KANSL1.

Methods: To test our hypothesis, the St. Jude Center for Advanced Genome Engineering (CAGE) created a heterozygous KANSL1 knockout (KO) SF126 (human glioblastoma) cell line to resemble 95% of patients with one functional copy of KANSL1. We then generated a version of the cell line that is stably expressing dCas9-VP64. VP64 is a strong transcriptional activator and dCas9 has no nuclease activity. The fusion of these two components, in the presence of gRNA, results in CRISPR activation (CRISPRa) of the targeted gene. To investigate whether the CRISPRa system can rescue KANSL1 gene expression, we designed five gRNAs targeting different sites of KANSL1 transcriptional start site (TSS) and tested the efficiency of each gRNA in the KANSL1 haploinsufficient SF126 cell line stably expressing dCas9-VP64. We used RT-qPCR to evaluate changes in RNA expression and western blotting to evaluate protein expression.

Results: We confirmed the presence of the dCas9-VP64 system in the SF126-het-KANSL1-KO cell line using western blotting. Our preliminary result showed that one of the gRNAs can increase KANSL1 gene expression in KANSL1 haploinsufficient SF126 cells stably expressing dCas9-VP64. The data suggests that the CRISPRa system can rescue the haploinsufficiency of KANSL1 in the SF126-het-KANSL1-KO cell line and has a potential to be used for targeted therapy, however more studies need to be done.

Conclusions: Our results show promise that KANSL1 haploinsufficiency can be rescued with CRISPRa, specifically with the dCas9-VP64 system. Currently, we are investigating whether KANSL1 upregulation can rescue cellular phenotypes such as reducing accumulation of autophagosome in our KANSL1 haploinsufficient cell line and increasing synaptic activity in 2D neurons.

Funding: AES BRIDGE Program