of genes and chromosomal loci from complex genomes in yeast

Transformation-associated recombination (TAR) pro-tocol allowing the selective isolation of full-length genes complete with their distal enhancer regions and entire genomic loci with sizes up to 250 kb from complex genomes in yeast S. cerevisiae has been developed more than a decade ago. However, its wide spread usage has been impeded by a low effi-ciency (0.5–2%) of chromosomal region capture dur-ing yeast transformants which in turn requires a time-consuming screen of hundreds of colonies. Here, we demonstrate that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies (up to 32%). As only a dozen or less yeast transformants need to be screened to obtain a clone with the desired chromosomal region, extensive ex-perience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of hu-man genes, each represented by a genomic copy containing its native regulatory elements, that would lead to a significant advance in functional, structural and comparative genomics, in diagnostics, gene re-placement, generation of animal models for human diseases and has a potential for gene therapy