Abstract
The field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients' samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene.Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant , which should result in the accurate recapitulation of the splicing defect.
Highlights
Splicing defects account for up to one-third of human diseasecausing variants, according to the current estimates [1–3]
We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant, which should result in the accurate recapitulation of the splicing defect
Based on a naturally employed bacterial defense mechanism [14, 15], clustered-regulatory interspaced short palindromic repeats (CRISPR)/Cas9 technology was developed as a programmable system of genetic editing that commonly uses the Cas9 nuclease from Streptococcus pyogenes and a RNA duplex comprised of a sequence-specific CRISPR RNA and a generic trans-activating CRISPR RNA that directs the nuclease to a cut site point, three base pairs upstream of the protospacer adjacent motif or PAM
Summary
Splicing defects account for up to one-third of human diseasecausing variants, according to the current estimates [1–3]. The first requirement for the accurate design and testing of antisense splice correction therapy is the availability of relevant experimental models in which to dissect the underlying molecular mechanisms of pathogenic variants and to test candidate molecules In this sense, the development of clustered-regulatory interspaced short palindromic repeats (CRISPR)-CRISPR associated nuclease (Cas) genome editing has paved the way to the rapid and easy generation of new and improved cell/animal models of disease. We use as example the CRISPR/Cas9-mediated introduction of the recently characterized PAH intronic variant, c.1199 + 20G > C, that causes exon skipping due to disruption of a splicing regulatory element [18] This variant creates a PshAI restriction site, which is used to screen for gene edition in the transfected cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
