Efficient targeted integration directed by short homology in zebrafish and mammalian cells

Autor:	Jacklyn Levey, Merrina Lan, Carla M. Mann, Maura McGrail, Maira P. Almeida, Beau R. Webber, Kristen M. Kwan, Dennis A. Webster, Trevor J. Weiss, Jeffrey J. Essner, Darius Balciunas, Wesley A. Wierson, Karl J. Clark, Zhitao Ming, Jeffrey A. Haltom, Alec Wehmeier, Stephen C. Ekker, Melanie E. Torrie, Christopher S. Mikelson, Stacy L. Solin, Sekhar Kambakam, Daniel F. Carlson, Macy K. Vollbrecht, Branden S. Moriarity, Jordan M. Welker, Chi Bin Chien, Drena Dobbs, Kenna C. McKeighan
Jazyk:	angličtina
Rok vydání:	2020
Předmět:	0301 basic medicine CRISPR-Associated Proteins Sus scrofa Animals Genetically Modified end joining 0302 clinical medicine Genes Reporter CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Gene Knock-In Techniques Biology (General) Zebrafish Transcription activator-like effector nuclease biology General Neuroscience Gene targeting General Medicine Medicine RNA Guide Kinetoplastida Research Article pig fibroblasts QH301-705.5 Science Green Fluorescent Proteins Computational biology knock-in General Biochemistry Genetics and Molecular Biology Genome engineering 03 medical and health sciences Leukemia Myelogenous Chronic BCR-ABL Positive Sequence Homology Nucleic Acid Transcription Activator-Like Effector Nucleases Gene knockin Animals Humans human Gene CRISPR/Cas9 Reporter gene General Immunology and Microbiology Recombinational DNA Repair Genetics and Genomics Fibroblasts biology.organism_classification 030104 developmental biology Gene Expression Regulation targeted integration CRISPR-Cas Systems K562 Cells 030217 neurology & neurosurgery Developmental Biology
Zdroj:	eLife, Cambridge : eLife Sciences Publications, Ltd, 2020, vol. 9, art. no. e53968, p. [1-25] eLife, Vol 9 (2020) eLife
ISSN:	2050-084X
Popis:	Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24–48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22–100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_dedup___::bfdaa4c2ba4444b21a6e2fac41b3b74c https://repository.vu.lt/VU:ELABAPDB141054958&prefLang=en_US Zobrazit plný text záznamu