Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a powerful gene-editing tool.
• The large size of Streptococcus pyogenes Cas9 (SpCas9) poses challenges for vector delivery.
• There is a need for smaller, more efficient Cas9 systems for gene editing in various organisms, particularly fish models.

Purpose of the Study:

• To evaluate the efficacy of a codon-optimized Staphylococcus aureus Cas9 (SaCas9) system for gene editing in the medaka fish (Oryzias latipes).
• To assess the utility of a transfer RNA (tRNA)-single-guide RNA (sgRNA) system for expressing sgRNA in medaka.
• To determine if SaCas9 and tRNA-sgRNA can be combined for efficient gene editing in medaka, targeting tyrosinase (tyr), oculocutaneous albinism II (oca2), and paired box 6.1 (pax6.1) genes.

Main Methods:

• Utilized a codon-optimized Staphylococcus aureus Cas9 (SaCas9) gene, which is smaller than SpCas9.
• Employed a transfer RNA (tRNA)-single-guide RNA (sgRNA) system for transcription of sgRNA.
• Applied SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems to edit specific genes in the medaka genome.
• Constructed and tested an all-in-one expression cassette (CMV-SaCas9-tRNA-sgRNA-tRNA).

Main Results:

• The SaCas9 system effectively edited the tyrosinase (tyr) gene in medaka.
• Both SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems demonstrated efficient medaka genome editing.
• The protospacer-adjacent motif (PAM) sequence (5'-NNGRRT-3') was crucial for editing efficiency.
• The tRNA component enhanced system flexibility by allowing sgRNA control via promoters like CMV.
• The all-in-one CMV-SaCas9-tRNA-sgRNA-tRNA cassette proved functional for medaka gene editing.

Conclusions:

• The codon-optimized SaCas9 system is a viable and smaller alternative for gene editing in medaka.
• The SaCas9/tRNA-sgRNA system offers an efficient and flexible approach for fish genome engineering.
• This approach has potential applications for editing other fish genomes, facilitating genetic research and manipulation.