CRISPR/Cas9-mediated genome editing in sea urchins

Area of Science:

• * Molecular Biology
• * Developmental Biology
• * Genomics

Background:

• * The CRISPR/Cas9 system facilitates precise genomic modifications in diverse model organisms.
• * Guide RNAs (gRNAs) enable targeted DNA recognition for gene knockouts.
• * Previous research confirms CRISPR/Cas9 efficacy in sea urchin gene targeting.

Purpose of the Study:

• * To provide detailed methodologies for applying CRISPR/Cas9 technology in sea urchin embryos.
• * To establish protocols for gene knockout and single nucleotide editing using modified Cas9 enzymes.
• * To include methods for assessing gene editing efficiency through genotyping.

Main Methods:

• * Design and in vitro synthesis of single guide RNA (sgRNA).
• * Application of CRISPR/Cas9 for targeted gene disruption (knockout).
• * Utilization of Cas9-DA for single nucleotide conversion.
• * Genotyping procedures for evaluating editing outcomes.

Main Results:

• * Demonstrated successful gene knockout in sea urchin embryos.
• * Showcased efficient single nucleotide editing capabilities.
• * Provided validated methods for assessing gene editing efficiency.

Conclusions:

• * The described CRISPR/Cas9 methods are effective for gene editing in sea urchin embryos.
• * These techniques facilitate functional genomics studies in sea urchins.
• * The protocols support both gene knockout and precise nucleotide modification.