Optimizing CRISPR/Cas9 approaches in the polymorphic tunicate Ciona intestinalis

Area of Science:

• * Developmental Biology
• * Molecular Biology
• * Genetics

Background:

• * Ciona intestinalis is a key invertebrate model for vertebrate developmental studies due to shared features and experimental advantages.
• * High genetic polymorphism and mosaic expression in Ciona hinder efficient CRISPR/Cas9 knockout.
• * In vitro approaches are needed to reduce animal use and overcome seasonality challenges in Ciona research.

Purpose of the Study:

• * To optimize CRISPR/Cas9 gene editing protocols in Ciona intestinalis for improved efficiency and broader applicability.
• * To develop in silico, in vitro, and in vivo strategies for more reliable CRISPR knockouts.
• * To enhance the utility of Ciona as a model organism for genetic studies.

Main Methods:

• * In silico: Correcting guide RNA (sgRNA) design by sequencing target regions and aligning with reference genomes to account for subspecies polymorphisms.
• * In vitro: Validating sgRNA efficiency using in vitro assays to reduce in vivo experimentation.
• * In vivo: Comparing Cas9 with Cas9:Geminin for enhanced editing efficiency, utilizing next-generation sequencing (NGS) for genotyping, and employing double CRISPR for large deletions.

Main Results:

• * Optimized sgRNA design and in silico analysis improved accuracy by correcting for Ciona subspecies polymorphisms.
• * In vitro assays showed suitability for sgRNA validation but also highlighted mismatch tolerance.
• * Cas9:Geminin increased in vivo editing efficiency, enabling access to early expressed genes.
• * Next-generation sequencing streamlined the identification of efficient sgRNAs, and double CRISPR generated significant deletions.

Conclusions:

• * The presented improvements significantly enhance CRISPR/Cas9 efficiency and reliability in Ciona intestinalis.
• * These optimized methods facilitate the establishment of CRISPR techniques in new research settings and are transferable to other organisms.
• * The study provides a robust framework for advancing genetic engineering in Ciona and beyond.