title: Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast.
abstract: Transformation-associated recombination (TAR) protocol 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 efficiency (0.5-2%) of chromosomal region capture during 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 experience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of human 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 replacement, generation of animal models for human diseases and has a potential for gene therapy.
authors.affiliation: Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Nicholas C O
authors.initials: NC
authors.lastname: Lee
authors.affiliation: Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Larionov
authors.affiliation: Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, USA kouprinn@mail.nih.gov.
authors.forename: Natalay
authors.initials: N
authors.lastname: Kouprina
mesh_terms: D064113:CRISPR-Cas Systems; D018797:Cell Cycle Proteins / Q000235:genetics; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005796:Genes; D056426:Genetic Loci; D016681:Genome, Fungal; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics; D012313:RNA / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D012313:RNA; D004706:Endodeoxyribonucleases