Akt is a mediator of artery specification during zebrafish development.

The dorsal aorta (DA) is the first major blood vessel to develop in the embryonic cardiovascular system. Its formation is governed by a coordinated process involving the migration, specification, and arrangement of angioblasts into arterial and venous lineages, a process conserved across species. Although vascular endothelial growth factor a (VEGF-A) is known to drive DA specification and formation, the kinases involved in this process remain ambiguous. Thus, we investigated the role of protein kinase B (Akt) in zebrafish by generating a quadruple mutant (aktΔ/Δ), in which expression and activity of all Akt genes - akt1, -2, -3a and -3b - are strongly decreased. Live imaging of developing aktΔ/Δ DA uncovers early arteriovenous malformations. Single-cell RNA-sequencing analysis of aktΔ/Δ endothelial cells corroborates the impairment of arterial, yet not venous, cell specification. Notably, endothelial specific expression of ligand-independent activation of Notch or constitutively active Akt1 were sufficient to re-establish normal arterial specification in aktΔ/Δ. The Akt loss-of-function mutant unveils that Akt kinase can act upstream of Notch in arterial endothelial cells, and is involved in proper embryonic artery specification. This sheds light on cardiovascular development, revealing a mechanism behind congenital malformations.

In silico analysis of potential off-target sites to gene editing for Mucopolysaccharidosis type I using the CRISPR/Cas9 system: Implications for population-specific treatments.

Mucopolysaccharidosis type I (MPS I) is caused by alpha-L-iduronidase deficiency encoded by the IDUA gene. Therapy with CRISPR/Cas9 is being developed for treatment, however a detailed investigation of off-target effects must be performed. This study aims to evaluate possible off-targets for a sgRNA aiming to correct the most common variant found in MPS I patients (p.Trp402*). A total of 272 potential off-target sequences was obtained and 84 polymorphic sites were identified in these sequences with a frequency equal to or greater than 1% in at least one of the populations. In the majority of cases, polymorphic sites decrease the chance of off-target cleavage and a new PAM was created, which indicates the importance of such analysis. This study highlights the importance of screening off-targets in a population-specific context using Mucopolysaccharidosis type I as an example of a problem that concerns all therapeutic treatments. Our results can have broader applications for other targets already clinically in use, as they could affect CRISPR/Cas9 safety and efficiency.