Genome editing in sea urchin embryos by using a CRISPR/Cas9 system

Area of Science:

• Developmental Biology
• Genetics
• Molecular Biology

Background:

• Sea urchin embryos are valuable models for studying early development and gene regulatory networks.
• Antisense morpholino oligonucleotides are commonly used but have limitations like off-target effects and toxicity.
• Previous genome editing tools (ZFNs, TALENs) showed unsatisfactory efficiency in sea urchin embryos.

Purpose of the Study:

• To apply the CRISPR-Cas9 system for efficient genome editing in sea urchin embryos.
• To assess the efficiency and specificity of CRISPR-Cas9 in generating gene knockouts.
• To establish a reliable method for linking genotype to phenotype in edited sea urchin embryos.

Main Methods:

• Designed six guide RNAs (gRNAs) targeting the nodal gene in sea urchin embryos.
• Injected CRISPR-Cas9 components into sea urchin embryos.
• Developed a method for genomic DNA isolation from individual embryos for mutation analysis.
• Sequenced targeted regions to determine mutation rates and analyzed potential off-target sites.

Main Results:

• Five out of six gRNAs induced the expected nodal gene phenotype in 60-80% of injected embryos.
• Mutation rates ranged from 67-100% among sequenced clones.
• No off-target effects were detected at the two examined potential off-target sites.
• A simple method for linking phenotype to genotype was established.

Conclusions:

• The CRISPR-Cas9 system is an efficient and specific tool for genome editing in sea urchin embryos.
• This technology overcomes the limitations of morpholino-based gene knockdown.
• The described methods will accelerate the use of CRISPR-Cas9 for genetic studies in this model organism.