ErCas12a and T5exo-ErCas12a Mediate Simple and Efficient Genome Editing in Zebrafish

Area of Science:

• Molecular Biology
• Genetics
• Zebrafish Model Organisms

Background:

• CRISPR-Cas9 is widely used in zebrafish for gene editing.
• CRISPR-Cas12a systems in zebrafish face limitations including protein delivery and heatshock requirements.
• Existing CRISPR tools in zebrafish necessitate complex procedures for gene editing.

Purpose of the Study:

• To investigate the potential of mRNA-active ErCas12a for simplified genome editing in zebrafish.
• To enhance CRISPR/Cas12a efficiency for large DNA fragment knockin.
• To develop a heatshock-free CRISPR genome editing method in zebrafish.

Main Methods:

• Utilized mRNA-active ErCas12a for gene knockin via microhomology-mediated end joining (MMEJ) and non-homologous end joining (NHEJ) pathways.
• Employed a heatshock protocol for ErCas12a-injected zebrafish embryos.
• Developed and tested a T5 exonuclease-ErCas12a fusion protein (T5exo-ErCas12a) for gene knockout and knockin without heatshock.

Main Results:

• ErCas12a injection with heatshock showed comparable knockin efficiency to Cas9 for large DNA fragments.
• T5exo-ErCas12a fusion enabled high-efficiency gene knockout and knockin.
• The T5exo-ErCas12a system eliminated the need for embryo heatshock, simplifying the genome editing process.

Conclusions:

• ErCas12a-mediated genome manipulation is feasible in zebrafish under simplified conditions.
• T5exo-ErCas12a fusion provides an efficient and heatshock-free method for zebrafish genome editing.
• These advancements expand the genome editing toolbox for diverse applications in zebrafish research.