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Gene editing modify specific sites within the genome through gene deletions, insertions or conversions to study functionally unknown genes or conduct gene therapy. It is also used to change the biological traits of organisms to establish new varieties. Gene editing techniques include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas 9) (CRISPR/Cas9).

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

The inducible CreER/CreERT2 system is based on a fusion between Cre recombinase and a modified estrogen receptor ligand-binding domain that retains Cre in the cytoplasm under basal conditions and allows nuclear translocation upon tamoxifen binding, enabling temporal control of site-specific recombination at loxP-flanked genomic loci in vivo or in vitro. Upon tamoxifen administration, CreER translocates to the nucleus and catalyzes recombination between loxP sites, resulting in excision or inversion of floxed DNA segments and enabling temporally defined gene knockout in specific tissues depending on promoter-driven CreER expression. This system has been widely used for inducible gene deletion and lineage tracing in mice, including validation of efficient temporal recombination in developmental and adult tissues using tamoxifen induction paradigms.

The Cre-lox conditional knockout system is a site-specific recombination technology in which Cre recombinase recognizes loxP sites flanking a genomic sequence, enabling excision, inversion, or rearrangement of the intervening DNA in a spatially and/or temporally controlled manner, allowing tissue-specific gene inactivation in vivo when Cre is expressed under defined promoters. This system is widely used to overcome limitations of conventional germline knockouts, particularly embryonic lethality or systemic effects that obscure tissue-specific gene function, by restricting recombination to selected cell types or developmental stages. Cre-mediated recombination efficiency and specificity depend on factors such as Cre driver expression pattern, loxP configuration, and genomic context, which can lead to mosaic recombination and variable knockout outcomes. The system is typically validated using reporter alleles or floxed target genes to confirm recombination at DNA and protein levels.

Cre-lox lineage tracing labels cells that express Cre recombinase by excising a loxP-flanked STOP cassette in a reporter allele, causing permanent reporter expression in the recombined cell and its descendants. Inducible CreERT2 models add temporal control because tamoxifen activates CreERT2-dependent recombination, allowing cells expressing a selected promoter at a chosen time point to be fate-mapped during development, homeostasis, injury, or disease. Reporter readouts include β-galactosidase, EYFP/ECFP, tdTomato, membrane GFP, and multicolor fluorescent proteins; these readouts detect recombined cells by histology, fluorescence microscopy, flow cytometry, or tissue imaging.

CRISPR-Cas9 editing of human pluripotent stem cells uses a guide RNA to direct Cas9 to a genomic target, where Cas9 creates a double-strand break that is repaired mainly by non-homologous end joining for knockout mutations or by homology-directed repair when a donor template is supplied for precise knock-in or sequence correction. The readout is generated by genotyping edited bulk populations or single-cell-derived clones, using PCR, sequencing, restriction-based assays, reporter fluorescence, or allele-specific analysis to distinguish unedited alleles, indels, precise donor-mediated edits, biallelic deletions, and unwanted on-target lesions.