Multiplexed CRISPR/Cas9 gene knockout with simple crRNA:tracrRNA co-transfection.

Cell Biosci 2019 20;9:41. Epub 2019 May 20.

1Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD USA.

Background: CRISPR/Cas9 mediated gene knockout is a powerful tool for genome editing with the ability to target multiple genes simultaneously. Establishing an efficient, multiplexed gene knockout system using CRISPR/Cas9 that is both simple and robust in its application would further advance the adoption of CRISPR/Cas9 for genetic studies.

Results: In this study, we present a simple, versatile and highly efficient method to achieve acute gene knockout with CRISPR/Cas9 using chemically synthesized crRNA and tracrRNA oligos. We demonstrate that co-transfection of the crRNA:tracrRNA duplex into Cas9-expressing cells leads to target gene mutation and loss of target protein expression in the majority of the cell population. We also show that delivering three crRNAs targeting EGFP, KRAS and PTEN in the same reaction leads to the simultaneous knockout of all three genes. Direct comparison of multiplexed gene targeting by crRNA:tracrRNA and by siRNA indicates that these two methods are comparable in their efficiency and kinetics of gene silencing.

Conclusions: Our method is a convenient yet powerful tool to enable rapid and scalable gene knockout using CRISPR/Cas9 in mammalian cells.

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13578-019-0304-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6528186PMC
May 2019
7 Reads

Publication Analysis

Top Keywords

gene knockout
20
powerful tool
8
knockout crispr/cas9
8
multiplexed gene
8
gene
8
knockout
6
crispr/cas9
5
demonstrate co-transfection
4
tracrrna oligos
4
crrna tracrrna
4
oligos demonstrate
4
sirna indicates
4
cells leads
4
leads target
4
cas9-expressing cells
4
duplex cas9-expressing
4
indicates methods
4
crrnatracrrna duplex
4
co-transfection crrnatracrrna
4
methods comparable
4

References

(Supplied by CrossRef)

M Jinek et al.
Science 2012

SH Sternberg et al.
Nature 2014

L Cong et al.
Science 2013

A Seki et al.
J Exp Med 2018

J Tan et al.
PLoS ONE 2016

K-C Su et al.
Mol Biol Cell 2018

TL Yuan et al.
Cancer Discov 2014

H Wang et al.
Cell 2013

RO Bak et al.
eLife 2017

P Mali et al.
Science 2013

AM Kabadi et al.
Nucleic Acids Res 2014

Similar Publications