Genetic loci underlying variation in traits with agronomic importance or genetic risk factors in human diseases have been identified by linkage analysis and genome-wide association studies.However, narrowing down the mapping to the individual causal genes and variations within these is much more challenging, and so is the ability to break linkage drag between beneficial and unfavourable loci in crop breeding. We developed RECAS9 as a transgene-free approach for precisely targeting recombination events by delivering CRISPR/Cas9 ribonucleotide protein (RNP) complex into heterozygous mitotic cells for the barley (Hordeum vulgare) Heat3.1 locus. A wild species (H. spontaneum) introgression in this region carries the agronomical unfavourable tough rachis phenotype (non-brittle) allele linked with a circadian clock accelerating QTL near GIGANTEA gene. We delivered RNP, which was targeted between two single nucleotide polymorphism (SNPs), to mitotic calli cells by particle bombardment. We estimated recombination events by next generation sequencing (NGS) and droplet digital PCR (ddPCR). While NGS analysis grieved from confounding effects of PCR recombination, ddPCR analysis allowed us to associate RNP treatment on heterozygous individuals with significant increase of homologous directed repair (HDR) between cultivated and wild alleles, with recombination rate ranging between zero to 57%. These results show for the first time in plants a directed and transgene free mitotic recombination driven by Cas9 RNP, and provide a starting point for precise breeding and fine scale mapping of beneficial alleles from crop wild relatives. Amunugama R, Fishel R (2012) Homologous recombination in eukaryotes. Prog. Mol. Biol. Transl. Sci. Elsevier B.V., pp 155-206 Barcelo P, Lazzeri PA (1995) Transformation of cereals by microprojectile bombardment of immature inflorescence and scutellum tissues. Methods Mol Biol. Bloom JS, Boocock J, Treusch S, Sadhu MJ, Day L, Oates-Barker H, Kruglyak L (2019) Rare variants contribute disproportionately to quantitative trait variation in yeast. Elife 8: 8-10 Brachi B, Faure N, Horton M, Flahauw E, Vazquez A, Nordborg M, Bergelson J, Cuguen J, Roux F (2010) Linkage and association mapping of Arabidopsis thaliana flowering time in nature. PLoS Genet 6: 40 Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kühn R (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. Edwards KD, Lynn JR, Gyula P, Nagy F, Millar AJ (2005) Natural allelic variation in the temperature-compensation mechanisms of the Arabidopsis thaliana circadian clock. Genetics 170: 387-400 Fernandes JB, Séguéla-Arnaud M, Larchevêque C, Lloyd AH, Mercier R (2018) Unleashing meiotic crossovers in hybrid plants. Proc Natl Acad Sci U S A 115: 2431-2436 Fernández-Calleja M, Casas AM, Pérez-Torres A, Gracia MP, Igartua E (2019) Rachis brittleness in a hybrid-parent barley (Hordeum vulgare) breeding germplasm with different combinations at the non-brittle...