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Bi-FoRe: an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent
Bingzhou Han1, Yage Zhang1, Xuetong Bi1
1Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, Peking University Genome Editing Research Center, College of Life Sciences, Peking University, Beijing, 100871, China.
Protein & Cell
|July 19, 2020
Summary
Researchers developed novel CRISPR-Cas tools in zebrafish for precise gene function studies. These tools enable conditional gene knockout and fluorescent labeling, facilitating efficient genotype discrimination in live embryos.
Area of Science:
- Developmental Biology
- Genetics
- Molecular Biology
Background:
- Precise gene function analysis requires methods for both labeling gene expression and conditionally manipulating gene activity.
- Existing techniques lack efficient methods for generating dual-function knockin alleles that simultaneously enable conditional gene manipulation and genetic tagging.
Purpose of the Study:
- To develop a novel CRISPR-Cas-based system in zebrafish for generating conditional knockout alleles with fluorescent reporter tagging.
- To engineer an improved system for one-step bidirectional knockin, creating paired conditional alleles with distinct fluorescent reporters for parallel manipulation and discrimination.
Main Methods:
- Utilized a CRISPR/Cas system with a novel flipping donor (FoRe) for NHEJ-mediated unidirectional targeted insertion in zebrafish.
- Developed an improved bidirectional donor (Bi-FoRe) for efficient one-step knockin of paired alleles with differential fluorescent reporters.
- Employed minicircle DNA technology to eliminate bacterial backbones from donor constructs.
Main Results:
- Successfully generated conditional knockout alleles with fluorescent labeling at sox10 and isl1 loci, enabling Cre-induced gene inactivation and lineage tracing.
- Demonstrated the Bi-FoRe system's efficiency in generating differentially fluorescently tagged positive and negative alleles at the sox10 locus.
- Achieved real-time discrimination of live embryos with different genotypes prior to phenotypic manifestation.
Conclusions:
- The developed FoRe and Bi-FoRe systems provide powerful tools for conditional gene manipulation and fluorescent labeling in zebrafish.
- These systems allow for parallel conditional knockout and gene restoration, coupled with efficient genotype identification via differential fluorescent reporters.
- The technology is expandable to other genes and organisms, offering significant potential for advancing gene function studies and single-cell sequencing applications.
