# Stage- and tissue-specific gene editing using 4-OHT–inducible Cas9 in whole organism

## TL;DR

Scientists developed a new method to edit genes in specific tissues and stages in zebrafish using a 4-OHT–inducible Cas9 system.

## Contribution

The novel system enables spatiotemporal gene editing in zebrafish with inducible nCas9ERT2 and gRNAs.

## Key findings

- The nCas9ERT2 system allows gene editing in primordial germ cells and adult germ cells upon 4-OHT treatment.
- The system is effective in both zebrafish embryos and early mouse embryos.
- This approach provides a new tool for tissue- and stage-specific gene editing in whole organisms.

## Abstract

Li et al. report a novel Cas9-based spatiotemporal gene-editing approach in zebrafish. Given the limited germline transmission efficiency of knock-in alleles in zebrafish, this system, featuring germline-specific nCas9ERT2 expression driven by piwil1 promoter together with ubiquitously expressed gRNAs, enables rapid and high-throughput genetic screening in vivo.

Vertebrate genes function in specific tissues and stages, so their functional studies require conditional knockout or editing. In zebrafish, spatiotemporally inducible genome editing, particularly during early embryogenesis, remains challenging. Here, we establish inducible Cas9-based editing in defined cell types and stages. The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos. As a proof of concept, we demonstrate that genes in primordial germ cells in embryos and germ cells in adult ovaries from a transgenic line with stable expression of nCas9ERT2 and gRNAs can be mutated by 4-OHT induction. The system also works in early mouse embryos. Thus, this inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level, expanding the tissue- and stage-specific gene-editing toolkit.