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Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

ABSTRACT: Genome editing typically involves recombination between donor nucleic acids and genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, amongst other deleterious by-products, DSBs yield translocations, off-target mutations and, most pervasively, unpredictable on-target allelic disruptions. Remarkably, hitherto, the untoward phenotypic consequences of on-target disruptions at allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness due to on-target DSBs and report that simultaneous single-stranded DNA break formation at donor and target DNA by CRISPR-Cas9 “nickases” overcomes, to a great extent, such genotype-phenotype disrupting events. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments (i.e. live-cell reporter tags) into essential and multiple-copy genomic sequences while circumventing most of the allelic and non-allelic collateral mutations and chromosomal rearrangements characteristic of nuclease-dependent gene editing procedures.

ORGANISM(S): Homo sapiens

PROVIDER: GSE135064 | GEO | 2019/12/04

REPOSITORIES: GEO

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Project description:DNA double strand breaks (DSBs) in repetitive sequences are a potent source of genomic instability, due to the possibility of non-allelic homologous recombination (NAHR). Repetitive sequences are especially at risk during meiosis, when numerous programmed DSBs are introduced into the genome to initiate meiotic recombination 1. Within the budding yeast repetitive ribosomal (r)DNA array, meiotic DSB formation is prevented in part through Sir2-dependent heterochromatin 2,3. Here, we demonstrate that the edges of the rDNA array are exceptionally susceptible to meiotic DSBs, revealing an inherent heterogeneity within the rDNA array. We find that this localised DSB susceptibility necessitates a border-specific protection system consisting of the meiotic ATPase Pch2 and the origin recognition complex subunit Orc1. Upon disruption of these factors, DSB formation and recombination specifically increased in the outermost rDNA repeats, leading to NAHR and rDNA instability. Strikingly, the Sir2-dependent heterochromatin of the rDNA itself was responsible for the induction of DSBs at the rDNA borders in pch2? cells. Thus, while Sir2 activity globally prevents meiotic DSBs within the rDNA, it creates a highly permissive environment for DSB formation at the heterochromatin/euchromatin junctions. Heterochromatinised repetitive DNA arrays are abundantly present in most eukaryotic genomes. Our data define the borders of such chromatin domains as distinct high-risk regions for meiotic NAHR, whose protection may be a universal requirement to prevent meiotic genome rearrangements associated with genomic diseases and birth defects. This SuperSeries is composed of the following subset Series: GSE30071: ssDNA mapping in dmc1 strains GSE30072: ChIP-chip of DSB factors in wild type and pch2 strains Two types of study were undertaken to understand the regulation of meiotic DSB formation close to repetitive DNA elements in yeast. First, DSBs were mapped using ssDNA enrichment in strains isogenic for a dmc1 mutation, and also including pch2 delete, orc1-161, rdna delete and a reciprocal translocation between chromosomes 2 and 12 (trans2to12). Second, the association of the DSB factors Hop1, Rec114, Mer2, and Mre1, as well as total histone H3 and H3K4-trimethylation were measured by ChIP-chip analysis in wild-type and pch2 delete strains.

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Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

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