knockins

A knock-in is a genetic modification in which a new sequence is inserted at a specific locus in the genome, resulting in a targeted alteration at that site. Unlike a knockout, which disrupts or removes a gene, a knock-in retains the endogenous locus and introduces an additional element or precise change, such as a reporter gene, a tag, a conditional allele, or a single-nucleotide variant. Knock-ins are used to study gene expression, protein localization, or gene function under controlled conditions.

Methods to generate knock-ins have evolved from classical homologous recombination in embryonic stem cells to more efficient site-specific genome editing. Traditional approaches rely on donor DNA with homology arms to guide integration at the desired locus. Modern methods frequently employ CRISPR/Cas9 or other nucleases to create a targeted break and stimulate homology-directed repair, enabling precise insertion with relative efficiency. Selection markers and subsequent removal strategies are commonly used during cell screening, especially in model organisms, with final animals or cells carrying the modification without the marker.

Common knock-in designs include fluorescent or luminescent reporters to monitor gene expression, tagging of proteins to study localization, humanized or modified genes to model human biology, and conditional alleles that allow controlled expression or inactivation. Point mutations or small sequence changes can also be introduced to model disease-associated variants.

Applications span functional genomics, disease modeling, protein tracking, and the study of regulatory elements. Limitations include potential off-target effects, variable editing efficiency, and the need to confirm physiological relevance. Ethical and regulatory considerations apply particularly to animal models and humanized systems.