PMID- 28800587
OWN - NLM
STAT- MEDLINE
DCOM- 20171004
LR  - 20240104
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 12
IP  - 8
DP  - 2017
TI  - Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary 
      cells.
PG  - e0182974
LID - 10.1371/journal.pone.0182974 [doi]
LID - e0182974
AB  - Phenotypic assays using human primary cells are highly valuable tools for target 
      discovery and validation in drug discovery. Expression knockdown (KD) of such 
      targets in these assays allows the investigation of their role in models of 
      disease processes. Therefore, efficient and fast modes of protein KD in 
      phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a 
      versatile and efficient means of gene inactivation in immortalized cell lines. 
      Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient 
      gene inactivation in two human primary cell types, normal human lung fibroblasts 
      and human bronchial epithelial cells. The effects of gene inactivation were 
      studied in the TGF-beta-induced fibroblast to myofibroblast transition assay (FMT) 
      and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 
      dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction 
      (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and 
      efficient genome editing judged by insertion/deletion (indel) formation, as well 
      as significant reduction of SMAD3 protein expression and nuclear translocation. 
      This led to phenotypic changes downstream of SMAD3 inhibition, including 
      substantially decreased alpha smooth muscle actin and fibronectin 1 expression, 
      which are markers for FMT and EMT, respectively. A direct comparison between 
      co-TD of separate Cas9 and gRNA AdV, versus TD with a single "all-in-one" 
      Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel 
      formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient 
      tool for protein KD in human primary cell phenotypic assays. The use of AdV 
      CRISPR/Cas9 may offer significant advantages over the current existing tools and 
      should enhance target discovery and validation opportunities.
FAU - Voets, Olaf
AU  - Voets O
AUID- ORCID: 0000-0002-7827-9211
AD  - Galapagos BV, CL Leiden, The Netherlands.
FAU - Tielen, Frans
AU  - Tielen F
AD  - Galapagos BV, CL Leiden, The Netherlands.
FAU - Elstak, Edo
AU  - Elstak E
AD  - Galapagos BV, CL Leiden, The Netherlands.
FAU - Benschop, Julian
AU  - Benschop J
AD  - Galapagos BV, CL Leiden, The Netherlands.
FAU - Grimbergen, Max
AU  - Grimbergen M
AD  - Galapagos BV, CL Leiden, The Netherlands.
FAU - Stallen, Jan
AU  - Stallen J
AD  - Galapagos BV, CL Leiden, The Netherlands.
FAU - Janssen, Richard
AU  - Janssen R
AD  - Galapagos BV, CL Leiden, The Netherlands.
FAU - van Marle, Andre
AU  - van Marle A
AD  - Galapagos BV, CL Leiden, The Netherlands.
FAU - Essrich, Christian
AU  - Essrich C
AD  - Galapagos BV, CL Leiden, The Netherlands.
LA  - eng
PT  - Journal Article
DEP - 20170811
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (ACTA2 protein, human)
RN  - 0 (Actins)
RN  - 0 (FN1 protein, human)
RN  - 0 (Fibronectins)
RN  - 0 (RNA, Guide, CRISPR-Cas Systems)
RN  - 0 (SMAD3 protein, human)
RN  - 0 (Smad3 Protein)
RN  - 0 (Transforming Growth Factor beta)
RN  - EC 3.1.- (Endonucleases)
SB  - IM
MH  - Actins/genetics/metabolism
MH  - Adenoviridae/genetics/metabolism
MH  - Base Sequence
MH  - Bronchi/cytology/metabolism
MH  - *CRISPR-Cas Systems
MH  - *Clustered Regularly Interspaced Short Palindromic Repeats
MH  - Endonucleases/genetics/metabolism
MH  - Epithelial Cells/cytology/drug effects/metabolism
MH  - Epithelial-Mesenchymal Transition/drug effects/genetics
MH  - Fibroblasts/cytology/drug effects/metabolism
MH  - Fibronectins/genetics/metabolism
MH  - *Gene Editing
MH  - Gene Expression Regulation
MH  - *Gene Silencing
MH  - Genetic Vectors/chemistry/metabolism
MH  - Genome, Human
MH  - Humans
MH  - INDEL Mutation
MH  - Primary Cell Culture
MH  - Protein Transport
MH  - RNA, Guide, CRISPR-Cas Systems/*genetics/metabolism
MH  - Smad3 Protein/*antagonists & inhibitors/genetics/metabolism
MH  - Transduction, Genetic
MH  - Transforming Growth Factor beta/pharmacology
PMC - PMC5553774
COIS- Competing Interests: All authors were supported in the form of salary by 
      Galapagos B.V. There are no patents, products in development or marketed products 
      to declare. This does not alter our adherence to PLOS ONE policies on sharing 
      data and materials.
EDAT- 2017/08/12 06:00
MHDA- 2017/10/05 06:00
PMCR- 2017/08/11
CRDT- 2017/08/12 06:00
PHST- 2016/12/23 00:00 [received]
PHST- 2017/07/27 00:00 [accepted]
PHST- 2017/08/12 06:00 [entrez]
PHST- 2017/08/12 06:00 [pubmed]
PHST- 2017/10/05 06:00 [medline]
PHST- 2017/08/11 00:00 [pmc-release]
AID - PONE-D-16-50797 [pii]
AID - 10.1371/journal.pone.0182974 [doi]
PST - epublish
SO  - PLoS One. 2017 Aug 11;12(8):e0182974. doi: 10.1371/journal.pone.0182974. 
      eCollection 2017.