Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity

Ran, F. Ann; Hsu, Patrick; Lin, Chie Yu; Gootenberg, Jonathan S.; Konermann, Silvana; Trevino, Alexandro E.; Scott, David; Inoue, Atsuyuki; Matoba, Shogo; Zhang, Yi; Zhang, Feng

doi:10.1016/j.cell.2013.08.021

Cited by 2,863 publications

(2,494 citation statements)

References 40 publications

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“…In order to generate clones containing MS2 sequences at subtelomere 15q, AGS cells were transfected with the MS2 cassette and a vector expressing the Cas9 nickase enzyme [32,33] and guide RNAs designed to specifically promote Cas9 activity within the subtelomere 15q sequence adjacent to the telomeric repeats tract (see Figure S1 and supplementary information section). Neomycin-resistant single clones were selected and screened by PCR using primers annealing within subtelomere 15q and downstream of the MS2 repeats.…”

Section: Resultsmentioning

confidence: 99%

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

et al. 2018

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Telomeres cap the ends of eukaryotic chromosomes, protecting them from degradation and erroneous recombination events which may lead to genome instability. Telomeres are transcribed giving rise to telomeric repeat-containing RNAs, called TERRA. The TERRA long noncoding RNAs have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. While TERRA RNAs are predominantly nuclear and localize at telomeres, little is known about the dynamics and function of TERRA molecules expressed from individual telomeres. Herein, we developed an assay to image endogenous TERRA molecules expressed from a single telomere in living human cancer cells. We show that single-telomere TERRA can be detected as TERRA RNA single particles which freely diffuse within the nucleus. Furthermore, TERRA molecules aggregate forming TERRA clusters. Three-dimensional size distribution and single particle tracking analyses revealed distinct sizes and dynamics for TERRA RNA single particles and clusters. Simultaneous time lapse confocal imaging of TERRA particles and telomeres showed that TERRA clusters transiently co-localize with telomeres. Finally, we used chemically modified antisense oligonucleotides to deplete TERRA molecules expressed from a single telomere. Single-telomere TERRA depletion resulted in increased DNA damage at telomeres and elsewhere in the genome. These results suggest that single-telomere TERRA transcripts participate in the maintenance of genomic integrity in human cancer cells.

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Section: Resultsmentioning

confidence: 99%

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

et al. 2018

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“…1b). In another approach, mutation in one of the nuclease activity domains (RuvC D10A or HNH H840A , Cas9n) was shown to result in a modified Cas9 only capable of performing single-strand DNA breaks (nick) instead of the original blunt DNA break [80]. This feature has been shown to reduce off-targeting and enhance HR in some organisms [12,70].…”

Section: Expanding Cas9 Features Through Enzyme Engineeringmentioning

confidence: 99%

“…By extension, 'paired nickases', i.e. 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].…”

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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“…However, in the absence of the DNA template, the broken DNA could reconnect through a non‐homologous end joining (NHEJ) 11, which may induce a mutation via a deletion or insertion of base pair (s)13. Subsequently, a Cas9 nickase, inducing a nick on double‐stranded DNA, was engineered to increase the number of specifically recognized bases and reduce off‐target cleavage 14.…”

Section: Introductionmentioning

confidence: 99%

Expression of PTEN‐long mediated by CRISPR/Cas9 can repress U87 cell proliferation

Fang

Wang

et al. 2017

J Cellular Molecular Medi

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PTEN is a tumour suppressor that is frequently mutated in a variety of cancers. Hence, PTEN has significant potential as a therapeutic molecule. PTEN‐long is an alternative translation variant, with an additional 173 amino acids added to the N‐terminal of the canonical PTEN when CUG of the mRNA is utilized as the start codon. PTEN‐long is secreted into serum and can re‐enter cells throughout the body. One of the major barriers for gene therapy is to efficiently and specifically deliver DNA or RNA material to target cells. As an alternative approach, if a therapeutic protein can be directly delivered to target cell of interest, it should theoretically function well within the cells, particularly for genes that are deficiently expressed in vivo. Most therapeutic proteins are incapable of efficiently permeating the cell membrane. In this study, we have employed CRISPR/Cas9 gene editing tool combined with single‐stranded template to edit CTG of PTEN‐long to ATG in the genome. Two guide RNAs close to CTG site were found to have similar efficiency in driving PTEN‐long expression. Furthermore, we detected PTEN‐long expression in transfected whole‐cell lysate and in concentrated culture media in Western blot. Interestingly, the culture media of PTEN‐long expression can reduce Akt phosphorylation level and repress U87 cell proliferation compared to wild‐type U87 or control media. Taken together, PTEN‐long driven by CRISPR/Cas9 imports and exports cells and represses nearby cell proliferation, indicating the PTEN‐long generated by CRISPR/Cas9 has potential to be an alternative strategy for PTEN gene therapy.

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Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity

Cited by 2,863 publications

References 40 publications

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

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

Expression of PTEN‐long mediated by CRISPR/Cas9 can repress U87 cell proliferation

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