Abstract
TP63 encodes TAp63, which is functionally similar to the tumor suppressor TP53, and ΔNp63, which lacks the transcription-activating domain of TAp63 and appears potently oncogenic in squamous cell carcinomas (SCCs). In this study, we developed an integrated CRISPR interference (CRISPRi) system to selectively suppress ΔNp63 (CRISPRiΔNp63). We engineered this CRISPRi using tandemized guide RNA expression cassettes that targeted the 50 to 100 bp downstream of the transcription start site of ΔNp63 in combination with inactivated Cas9 linked to the transcription repression module Krüppel-associated box repressor domain. The plasmid vector harboring CRISPRiΔNp63 repressed ΔNp63 transcription in lung and esophageal SCC cells. Likewise, Ad-CRISPRiΔNp63, an all-in-one adenoviral vector containing the tandemized gRNAs and dCas9/KRAB expression cassette suppressed ΔNp63 expression in SCC cells. Ad-CRISPRiΔNp63 also effectively decreased cell proliferation and colony formation and induced apoptosis in lung and esophageal SCC cells in vitro and significantly inhibited tumor growth in a mouse lung SCC xenograft model in vivo. These results indicate that ΔNp63 suppression using CRISPRiΔNp63 may be an effective strategy for treating lung and esophageal SCC.
Highlights
The development of gene editing methods using engineered zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) enabled precise genetic modification through induction of targeted DNA double-strand breaks [1, 2]
These results suggest ΔNp63 is commonly expressed in human lung and esophageal squamous cell carcinomas (SCCs) and that molecular targeting of ΔNp63 may be a useful approach to treating SCC
We evaluated whether ΔNp63 expression could be suppressed in SCC cells and keratinocytes using an all-in-one adenoviral vector containing double gRNA expression cassettes and a dCas9/KRAB expression cassette to target ΔNp63 (Ad-CRISPRiΔNp63) (Figure 4A)
Summary
The development of gene editing methods using engineered zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) enabled precise genetic modification through induction of targeted DNA double-strand breaks [1, 2]. The difficulty of creating the ZFN or TALEN constructs was a limitation of using these chimeric nucleases to facilitate gene targeting. The most widely used version is CRISPR/Cas from Streptococcus pyogenes [3], which has two important advantages over ZFNs and TALENs. First, the design and construction of a CRISPR/Cas system is easier than systems using ZFNs and TALENs. Second, by introducing multiple gRNAs, CRISPR/Cas can be used to target several genes or sequences at the same time [4]. Catalytically inactive Cas (dCas9) fusion protein guides that use gRNAs have been developed to target selected DNA sequences to inhibit (CRISPRi) or activate (CRISPRa) transcription of target genes [5]
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