Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells

Wu, Xuebing; Scott, David; Kriz, Andrea J.; Chiu, Anthony; Hsu, Patrick; Dadon, Daniel Benjamin; Cheng, Albert W.; Trevino, Alexandro E.; Konermann, Silvana; Chen, Sidi; Jaenisch, Rudolf; Zhang, Feng; Sharp, Phillip A.

doi:10.1038/nbt.2889

Cited by 862 publications

(928 citation statements)

References 47 publications

(81 reference statements)

Supporting

Mentioning

845

Contrasting

Unclassified

Order By: Relevance

“…We believe that different genome modification(s) in culture cells, i.e., epigenetic states (open chromatin state, CpG methylation, and so on), may account for the discrepancy [10][11]. The strand-specific repression by ts-gRNAs of a gene was explained to be the consequence of steric inhibition of RNA polymerase II activity in prokaryotes [5].…”

mentioning

confidence: 99%

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

et al. 2015

View full text Add to dashboard Cite

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

show abstract

mentioning

confidence: 99%

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

et al. 2015

View full text Add to dashboard Cite

show abstract

“…However, off-target cleavage is a major concern for any nuclease therapy. Single-molecule microscopy (30) and immunoprecipitation assays (31,32) have measured the off-target sites binding behavior of the Cas9 endonuclease. ChiP-seq further allowed unbiased detection of genome-wide Cas9 binding (31,32).…”

Section: Discussionmentioning

confidence: 99%

“…Single-molecule microscopy (30) and immunoprecipitation assays (31,32) have measured the off-target sites binding behavior of the Cas9 endonuclease. ChiP-seq further allowed unbiased detection of genome-wide Cas9 binding (31,32). Together with recent improvements on CRISPR target specificity with dual-guide RNA designs (33)(34)(35), these studies show that off-target effects are not a substantial impediment to human genome-engineering applications and certainly do not appear to be an obstacle for applications that target nonhuman genomes (36).…”

Section: Discussionmentioning

confidence: 99%

RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection

Wang

Quake

2014

Proc. Natl. Acad. Sci. U.S.A.

194

153

View full text Add to dashboard Cite

Latent viral infection is a persistent cause of human disease. Although standard antiviral therapies can suppress active viral replication, no existing treatment can effectively eradicate latent infection and therefore a cure is lacking for many prevalent viral diseases. The prokaryotic immune system clustered regularly interspaced short palindromic repeat (CRISPR)/Cas evolved as a natural response to phage infections, and we demonstrate here that the CRISPR/Cas9 system can be adapted for antiviral treatment in human cells by specifically targeting the genomes of latent viral infections. Patient-derived cells from a Burkitt's lymphoma with latent Epstein-Barr virus infection showed dramatic proliferation arrest and a concomitant decrease in viral load after exposure to a CRISPR/Cas9 vector targeted to the viral genome.genome editing | latency | herpes virus T he herpesviridae virus family consists of some of the most widespread human pathogens in the world. More than 90% of adults have been infected with at least one of the eight subtypes of herpes viruses, and latent infection persists in most people (1). These herpes virus subtypes infect a wide range of cells, including epithelium, neuron, monocyte, and lymphocyte, and the consequences can be either mild (herpes simplex by HSV-1) or severe [cancer by Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpes virus]. HSV infection is also a known risk factor for HIV (2). In its latent state, the viral genome persists within the host cells and it has not been possible to find therapeutic approaches that completely eradicate such infections.Since its discovery 50 y ago, EBV has been a closely studied member of the herpesviridae. As one of the most common human viruses, EBV causes infectious mononucleosis and is associated with certain forms of lymphoma. To date, however, no EBV vaccine or treatment exists. EBV is highly efficient at transforming quiescent human B lymphocytes; the resulting lymphoblastoid cell lines are now commonly used for human genetic studies, and it is possible to use patient-derived cells that propagate directly in culture because of the viral infection and require no other manipulation. The EBV genome encodes about 85 genes, several of which are essential for lytic or latent infection. During latency, the EBV genome circularizes and resides in the cell nucleus as an episome. EBV latency usually progresses through three programs, with protein production decreasing from full sets of EBV nuclear antigens (EBNAs) and latent membrane proteins to just EBNA1. EBNA1 binds to the EBV origin of replication (oriP) to maintain viral episomes; it also regulates expression of other viral genes.Most current antiviral drug development programs are focused on protein targets and are only effective in preventing active viral replication. It has been recognized that it would be useful to target latent infections with viral genome-specific nucleases (3-5), but the challenges of engineering sequence-specific nucleases have hampered progress. Clustered reg...

show abstract

“…Therefore, genome-wide unbiased methods such as GUIDE-seq and Digenome-seq should be harnessed to provide a comprehensive profile of the off-target events. [20][21][22] As off-target genome editing may incur unwanted consequences including malignancies, they should be precisely profiled and reduced to almost nil if applied to human gene therapy. Significant advances have been made recently to reduce the off-target effects, such as double nicking by Cas9 nickases, 23 optimizing sgRNA design 24,25 and reconstruction of Cas9 nuclease.…”

mentioning

confidence: 99%