BMJ · 2004 · 15 citations · 0 references
Life SciencesGeneticsMolecular BiologyGenetic EngineeringGenome EditingGenome EngineeringCrispr-based EngineeringCrispr-cas SystemsGene Knock-in StrategiesGenome Integration StrategiesHomologous RecombinationHomology-directed RepairConditional Knock-in AllelesConditional Lethal AllelesCrispr-mediated Embryo EditingGenome Editing By Homology-directed RepairHomologyIn Vivo Genome EditingLarge Fragment Knock-inPrecise Genome Editing
<h3>ABSTRACT</h3> CRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as concatemers. Here, we show that injecting 5’biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient single-copy HDR (scHDR) alleles. Our dedicated genotyping strategy showed that these alleles occurred with a frequency of 19%, 20%, and 26%, respectively, in three independent gene loci, indicating that scHDR is dramatically boosted by 5’biotinylation. Thus, we suggest that a combination of a 5’biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins into the mouse genome.