Large-scale F0 CRISPR screens in vivo using MIC-Drop

Area of Science:

• Genetics
• Developmental Biology
• Molecular Biology

Background:

• Zebrafish are a key model for genetic disease and development studies.
• CRISPR-Cas9 is efficient for zebrafish gene editing but scaling is challenging.
• High-throughput in vivo functional genetics requires efficient screening methods.

Purpose of the Study:

• To present a protocol for Multiplexed Intermixed CRISPR Droplets (MIC-Drop) for large-scale CRISPR screening in zebrafish.
• To enable high-throughput in vivo functional genetic screens in a vertebrate model.
• To facilitate target identification for chemical biology studies.

Main Methods:

• MIC-Drop utilizes multiplexed single-guide RNAs for biallelic mutations in zebrafish embryos.
• Microfluidics and DNA barcoding allow simultaneous targeting of numerous genes.
• Screens are performed in F0 animals, enabling rapid genotype-phenotype correlation.

Main Results:

• MIC-Drop facilitates large-scale CRISPR screening in zebrafish.
• Simultaneous targeting of hundreds of genes is possible from a single injection.
• A typical screen of 100 genes can be completed by one user in 2-3 weeks.

Conclusions:

• MIC-Drop overcomes scalability challenges for CRISPR screening in zebrafish.
• This method accelerates functional genetic screens in vertebrate models.
• MIC-Drop is valuable for developmental biologists, geneticists, and chemical biologists.