# CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA

## TL;DR

Scientists used CRISPR/Cas9 to create knockdown mice by inserting artificial microRNA into specific genome regions, offering a faster and more efficient method for studying gene function.

## Contribution

A novel CRISPR/Cas9-based method for generating knockdown mice using amiRNA insertion into intronic regions.

## Key findings

- amiRNA cassettes targeting exogenous and endogenous genes were efficiently inserted into the genome using CRISPR/Cas9.
- The method allows for reliable gene knockdown and can be adapted for conditional models.
- Long single-stranded DNA templates enabled precise insertion without complex constructs.

## Abstract

Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.