RNA-guided gene activation by CRISPR-Cas9–based transcription factors

Pérez-Piñera, Pablo; Koçak, D. Dewran; Vockley, Christopher M.; Adler, Andrew F.; Kabadi, Ami M.; Polstein, Lauren R.; Thakore, Pratiksha I.; Glass, Katherine A.; Ousterout, David G.; Leong, Kam W.; Guilak, Farshid; Crawford, Gregory E.; Reddy, Timothy E.; Gersbach, Charles A.

doi:10.1038/nmeth.2600

Cited by 1,166 publications

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“…CRISPR interference (CRISPRi) is a recently developed tool used to study single guide RNA (gRNA)-mediated sequence-specific repression of transcription in both prokaryotic and eukaryotic cells [1][2][3][4][5]. Transcription initiation and elongation of a gene can be interfered by the presence of gRNA:DNA hetero-duplex/dCas9 (a catalytically inactive form of Cas9) complex in its promoter and exons.…”

Section: Dear Editormentioning

confidence: 99%

“…Transgenic worms carrying Pdpy-5::dCas9 or Pdpy-5::dCas9-KRAB and one of seven dpy-5ts-gRNAs did not exhibit obvious changes in dpy-5 expression and body length ( Figure 1F and Supplementary information, Table S1a). When all seven dpy-5ts-gRNAs (1)(2)(3)(4)(5)(6)(7) were included, suppression of dpy-5 expression and Dpy phenotype occurred ( Figure 1F and Supplementary information, Figure S1aA-S1aC and S1aE). It appeared that Pdpy-5::dCas9-KRAB and dpy5ts-gRNAs (1)(2)(3)(4)(5)(6)(7) combination showed slightly stronger effect on dpy-5 expression than that of Pdpy-5::dCas9 and dpy-5ts-gRNAs(1-7) combination, although both combinations resulted in the Dpy phenotype ( Figure 1F and Supplementary information, Figure S1aA-S1aC, S1aE and Table S1a).…”

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Regulation of transcriptionally active genes via the catalytically inactive Cas9 in C. elegans and D. rerio

et al. 2015

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Dear Editor,CRISPR interference (CRISPRi) is a recently developed tool used to study single guide RNA (gRNA)-mediated sequence-specific repression of transcription in both prokaryotic and eukaryotic cells [1][2][3][4][5]. Transcription initiation and elongation of a gene can be interfered by the presence of gRNA:DNA hetero-duplex/dCas9 (a catalytically inactive form of Cas9) complex in its promoter and exons. If Krüppel associated box (KRAB) domain is fused to dCas9, repression of target gene is more efficient. Likewise, fusion of a transcription activator such as VP16 (CRISPR-on) can increase target gene expression through three to four gene-specific gRNAs (up to seven) that recognize the proximal promoter of a target gene in cultured cells and in vivo [2]. We applied both CRISPRi and CRISPR-on tools in worm and zebrafish, and demonstrated here that our dCas9 fusion systems modify gene expression at or near their endogenous expression location(s) through target-specific gRNAs (ts-gRNAs).We fused dCas9 to the KRAB domain (repressor) or the VP160 domain containing 10 tandem VP16 units (activator; Figure 1A). In C. elegans, dCas9 constructs were driven by tissue-specific promoters while ts-gRNAs were under control of a U6-type RNA polymerase III promoter ( Figure 1B). Different combinations of the DNA constructs were injected into adult hermaphrodite gonads to generate transgenic worms. For zebrafish experiments, dCas9-KRAB and dCas9-VP160 mRNAs and ts-gRNAs were synthesized in vitro and injected into one-cell stage embryos ( Figure 1C), and the resulted embryos were analyzed by real-time RT-PCR and in situ hybridization. Flanking sequences, including the nuclear localization sequences (NLSs) and UTRs, were indicated in Figure 1B and 1C or described previously [6].Decrease in dpy-5 expression levels produces short body and dumpy phenotype, known as Dpy. Dbl-1 is a TGF-β family member expressed primarily in neurons and regulates lon-1 to adjust body length. When dbl-1 is overexpressed, worm body length is elongated (Lon phenotype). We targeted dpy-5 by selecting seven ts-gRNAs that recognize its non-template DNA strand of

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Regulation of transcriptionally active genes via the catalytically inactive Cas9 in C. elegans and D. rerio

et al. 2015

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“…These libraries could be able to capture the full complexity of transcript isoforms instead of expressing genes independently of the endogenous regulatory context. To facilitate Cas‐9‐based gain‐of‐function screening, a synthetic activator could be constructed by fusing dCas9 with transcriptional activation domains, e.g., VP64 or p65 98. It has been shown that the delivery of multiple sgRNAs targeting at the same promoter region can improve target gene activation 99.…”

Section: Resistance Target Screening and Identificationmentioning

confidence: 99%

Application of the CRISPR/Cas9 System to Drug Resistance in Breast Cancer

Chen

Zhang

2018

Advanced Science

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Clinical evidence indicates that drug resistance is a great obstacle in breast cancer therapy. It renders the disease uncontrollable and causes high mortality. Multiple mechanisms contribute to the development of drug resistance, but the underlying cause is usually a shift in the genetic composition of tumor cells. It is increasingly feasible to engineer the genome with the clustered regularly interspaced short palindromic repeats (CRISPR)/associated (Cas)9 technology recently developed, which might be advantageous in overcoming drug resistance. This article discusses how the CRISPR/Cas9 system might revert resistance gene mutations and identify potential resistance targets in drug‐resistant breast cancer. In addition, the challenges that impede the clinical applicability of this technology and highlight the CRISPR/Cas9 systems are presented. The CRISPR/Cas9 system is poised to play an important role in preventing drug resistance in breast cancer therapy and will become an essential tool for personalized medicine.

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“…using two adjacent gRNAs with Cas9n, can efficiently introduce both indel mutations and HR events with a single-stranded DNA oligo-nucleotide donor template in mammalian cells [28,10,80]. Complete disruption of the endonuclease activities (RuvC D10A along with HNH H840A ) results in a catalytically inactive Cas9, or dead-Cas9 (dCas9) [78,79]. This has been exploited to physically block the transcriptional machinery when targeted in the promoter region of a gene of interest, coined CRISPR interference (CRISPRi) [22,34] (Fig.…”

Section: Expanding Cas9 Features Through Enzyme Engineeringmentioning

confidence: 99%

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

Ferreira

David

Nielsen

2018

Journal of Industrial Microbiology and Biotechnology

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Clustered regularly interspaced short palindromic repeats (CRISPR) is poised to become one of the key scientific discoveries of the twenty-first century. Originating from prokaryotic and archaeal immune systems to counter phage invasions, CRISPRbased applications have been tailored for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology.

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RNA-guided gene activation by CRISPR-Cas9–based transcription factors

Cited by 1,166 publications

References 31 publications

Regulation of transcriptionally active genes via the catalytically inactive Cas9 in C. elegans and D. rerio

Regulation of transcriptionally active genes via the catalytically inactive Cas9 in C. elegans and D. rerio

Application of the CRISPR/Cas9 System to Drug Resistance in Breast Cancer

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

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