Dataset Information

Efficient and flexible tagging of endogenous genes by homology-independent intron targeting.

ABSTRACT: Genome editing tools have simplified the generation of knock-in gene fusions, yet the prevalent use of gene-specific homology-directed repair (HDR) templates still hinders scalability. Consequently, realization of large-scale gene tagging requires further development of approaches to generate knock-in protein fusions via generic donors that do not require locus-specific homology sequences. Here, we combine intron-based protein trapping with homology-independent repair-based integration of a generic donor and demonstrate precise, scalable, and efficient gene tagging. Because editing is performed in introns using a synthetic exon, this approach tolerates mutations in the unedited allele, indels at the integration site, and the addition of resistance genes that do not disrupt the target gene coding sequence, resulting in easy and flexible gene tagging.

SUBMITTER: Serebrenik YV

PROVIDER: S-EPMC6673721 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Efficient and flexible tagging of endogenous genes by homology-independent intron targeting.

Serebrenik Yevgeniy V YV Sansbury Stephanie E SE Kumar Saranya Santhosh SS Henao-Mejia Jorge J Shalem Ophir O

Genome research 20190625 8

Genome editing tools have simplified the generation of knock-in gene fusions, yet the prevalent use of gene-specific homology-directed repair (HDR) templates still hinders scalability. Consequently, realization of large-scale gene tagging requires further development of approaches to generate knock-in protein fusions via generic donors that do not require locus-specific homology sequences. Here, we combine intron-based protein trapping with homology-independent repair-based integration of a gene ...[more]

PMID: 31239279

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Project description:ABSTRACT: Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional 70 and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited. Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group 75 of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3M-bM-^@M-2 UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr- siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements 80 through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially in other eukaryotes. Analysis of smRNA populations in Brachypodium plants challenged by abiotic stresses: To profile the populations of smRNAs in the model monocot Brachypodium distachyon and examine their regulation in response to abiotic stresses, we conducted high-throughput sequencing of small RNAs from plants exposed to four different abiotic stress conditions, cold, heat (air), heat (water immersion), and salt, in the wild type Brachypodium cultivar Bd21. For our experiments we used information from the literature to select two time-points for stress durations, short and long, which differed for each stress: cold (6 and 24 hours), heat-air (1 and 3 hours), heat-water (1 and 3 hours), and salt (48 hours). We generated small RNA libraries for Illumina sequencing (GAII) from the leaves of Brachypodium plants subjected to stresses and selected smRNAs between 15 and 40 nt in length, which we mapped to the Brachypodium genome.

Dataset Information

Efficient and flexible tagging of endogenous genes by homology-independent intron targeting.

Publications

Efficient and flexible tagging of endogenous genes by homology-independent intron targeting.

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