A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a ‘self-cleaving’ GFP-expression plasmid
| Autor: | Sarah Laura Krausz, Adrienn Borsy, Ervin Welker, Petra Bencsura, Péter István Kulcsár, Elfrieda Fodor, Antal Nyeste, Ádám Sturm, Zoltán Ligeti, András Tálas, István Vida, Nóra Weinhardt, Bianka Gordos, Kornélia Szebényi, Krisztina Huszár, Orsolya Ivett Hoffmann, Eszter Tóth |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
DNA End-Joining Repair Green Fluorescent Proteins Computational biology Biology 03 medical and health sciences Mice Plasmid Genome editing Genetics CRISPR Animals Humans Guide RNA Cas9 Molecular Biology Gene gRNA testing Gene Editing Reporter gene Expression vector NHEJ-cloning reporter assay General Medicine Genomics Full Papers 030104 developmental biology HEK293 Cells NIH 3T3 Cells CRISPR-Cas Systems HeLa Cells Plasmids RNA Guide Kinetoplastida |
| Zdroj: | DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes |
| ISSN: | 1756-1663 1340-2838 |
| Popis: | The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell. The use of 'self- cleaving' GFP-plasmids containing universal gRNAs and corresponding targets alleviates cloning burdens when this method is applied. These universal gRNAs mediate efficient plasmid cleavage and are designed to avoid genomic targets in several model species. The method combines the advantages of the straightforward FACS detection provided by applying fluorescent reporter systems and of the PCR-based approaches being capable of testing targets in their genomic context, without necessitating any extra cloning steps. Additionally, we show that NHEJ-cloning can also be used in mammalian cells for targeted integration of donor plasmids up to 10 kb in size, with up to 30% efficiency, without any selection or enrichment. |
| Databáze: | OpenAIRE |
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