NDA@SZTAKI

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.

TĂĄlas, AndrĂĄs

KulcsĂĄr, PĂŠter IstvĂĄn

Weinhardt, NĂłra

Borsy, Adrienn

TĂłth, Eszter

SzebĂŠnyi, KornĂŠlia

Krausz, Sarah Laura

HuszĂĄr, Krisztina

Vida, IstvĂĄn

Sturm, Ádåm

Gordos, Bianka

Hoffmann, Orsolya Ivett

Bencsura, Petra

Nyeste, Antal

Ligeti, ZoltĂĄn

Fodor, Elfrieda

Welker, Ervin

Oxford

2017

QD Chemistry / kĂŠmia

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.

angol

Article

PeerReviewed

info:eu-repo/semantics/article

text

http://real.mtak.hu/75175/1/dsx029.pdf

Tålas, Andrås and Kulcsår, PÊter Istvån and Weinhardt, Nóra and Borsy, Adrienn and Tóth, Eszter and SzebÊnyi, KornÊlia and Krausz, Sarah Laura and Huszår, Krisztina and Vida, Istvån and Sturm, Ádåm and Gordos, Bianka and Hoffmann, Orsolya Ivett and Bencsura, Petra and Nyeste, Antal and Ligeti, Zoltån and Fodor, Elfrieda and Welker, Ervin (2017) 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. DNA RESEARCH, 24 (6). pp. 609-621. ISSN 1340-2838

http://real.mtak.hu/75175/

https://doi.org/10.1093/dnares/dsx029

MTMT:3265579; doi:10.1093/dnares/dsx029