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Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing

Published on Dec 1, 2017in Nature Communications11.88
· DOI :10.1038/s41467-017-01875-9
Jared Carlson-Stevermer5
Estimated H-index: 5
(UW: University of Wisconsin-Madison),
Amr A. Abdeen11
Estimated H-index: 11
(UW: University of Wisconsin-Madison)
+ 4 AuthorsKrishanu Saha21
Estimated H-index: 21
(UW: University of Wisconsin-Madison)
Cite
Abstract
Writing specific DNA sequences into the human genome is challenging with non-viral gene-editing reagents, since most of the edited sequences contain various imprecise insertions or deletions. We developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to complex CRISPR-Cas9 ribonucleoproteins with a nucleic acid donor template, as well as other biotinylated molecules such as quantum dots. In human cells, tailored S1mplexes increase the ratio of precisely edited to imprecisely edited alleles up to 18-fold higher than standard gene-editing methods, and enrich cell populations containing multiplexed precise edits up to 42-fold. These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro or ex vivo gene-editing applications in precision medicine and drug discovery and aid in the development of increased and serial dosing regimens for somatic gene editing in vivo.
  • References (55)
  • Citations (18)
Cite
References55
Newest
Published on Aug 21, 2017in bioRxiv
Han Li9
Estimated H-index: 9
(UCSF: University of California, San Francisco),
Kyle Beckman3
Estimated H-index: 3
(UCSF: University of California, San Francisco)
+ 3 AuthorsManuel D. Leonetti5
Estimated H-index: 5
(UCSF: University of California, San Francisco)
CRISPR/Cas technologies have transformed our ability to manipulate genomes for research and gene-based therapy. In particular, homology-directed repair after genomic cleavage allows for precise modification of genes using exogenous donor sequences as templates. While both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) forms of donors have been used as repair templates, a systematic comparison of the performance and specificity of repair using ssDNA versus dsDNA donors is still lacki...
Published on Aug 1, 2017in Nature43.07
Hong Ma27
Estimated H-index: 27
(Oregon National Primate Research Center),
Nuria Marti-Gutierrez1
Estimated H-index: 1
(OHSU: Oregon Health & Science University)
+ 28 AuthorsRiffat Ahmed10
Estimated H-index: 10
(OHSU: Oregon Health & Science University)
CRISPR–Cas9 genome editing is used to induce a DNA repair response and correct a disease-causing heterozygous mutation in human embryos with reduced mosaicism and preferential repair using the wild-type copy of the gene.
Published on Aug 1, 2017in Cell36.22
Xin Liu22
Estimated H-index: 22
(UTSW: University of Texas Southwestern Medical Center),
Yuannyu Zhang6
Estimated H-index: 6
(UTSW: University of Texas Southwestern Medical Center)
+ 14 AuthorsZhimin Gu2
Estimated H-index: 2
(UTSW: University of Texas Southwestern Medical Center)
Summary Cis -regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here, we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show...
Published on Jun 20, 2017in Nucleic Acids Research11.15
Thomas Gaj22
Estimated H-index: 22
(University of California, Berkeley),
Brett T. Staahl13
Estimated H-index: 13
(University of California, Berkeley)
+ 4 AuthorsDavid V. Schaffer60
Estimated H-index: 60
(HWNI: Helen Wills Neuroscience Institute)
Published on Aug 1, 2018in Nature Genetics25.45
Christopher D. Richardson3
Estimated H-index: 3
(University of California, Berkeley),
Katelynn R. Kazane2
Estimated H-index: 2
(University of California, Berkeley)
+ 5 AuthorsJacob E. Corn29
Estimated H-index: 29
(University of California, Berkeley)
CRISPR–Cas genome editing creates targeted DNA double-strand breaks (DSBs) that are processed by cellular repair pathways, including the incorporation of exogenous DNA via single-strand template repair (SSTR). To determine the genetic basis of SSTR in human cells, we developed a coupled inhibition-cutting system capable of interrogating multiple editing outcomes in the context of thousands of individual gene knockdowns. We found that human Cas9-induced SSTR requires the Fanconi anemia (FA) pathw...
Published on May 2, 2017in eLife7.55
Kunwoo Lee7
Estimated H-index: 7
,
Vanessa Mackley3
Estimated H-index: 3
+ 4 AuthorsNiren Murthy43
Estimated H-index: 43
(University of California, Berkeley)
There are several different technologies that can be used to make specific changes to particular genes in cells. These “gene editing” approaches have the potential to help humans in a variety of different ways, for example, to treat diseases that presently have no cure, or to improve the nutritional quality of crop plants. One such gene editing approach is known as CRISPR. To edit a specific gene, a molecule called a guide ribonucleic acid (or guide RNA for short) binds to a section of the gene ...
Published on Apr 1, 2017in Cell Research17.85
Ming Ma1
Estimated H-index: 1
(PKU: Peking University),
Fengfeng Zhuang4
Estimated H-index: 4
+ 4 AuthorsJianzhong Jeff Xi6
Estimated H-index: 6
(PKU: Peking University)
Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system
Published on Mar 1, 2017in Nature Protocols11.33
Lindsey A. Lonowski2
Estimated H-index: 2
,
Yoshiki Narimatsu8
Estimated H-index: 8
+ 10 AuthorsHans H. Wandall6
Estimated H-index: 6
This protocol provides a pipeline for increasing and evaluating the efficiency of genome editing by ZFNs, TALENs or CRISPR–Cas9 that includes enrichment of nuclease-expressing cells by FACS, followed by Indel Detection by Amplicon Analysis (IDAA).
Published on Mar 1, 2017in Nature43.07
Justin Eyquem4
Estimated H-index: 4
,
Jorge Mansilla-Soto7
Estimated H-index: 7
+ 6 AuthorsMichel Sadelain70
Estimated H-index: 70
Introducing chimeric antigen receptors into the endogenous T-cell receptor locus reduces tonic signalling, averts accelerated T-cell differentiation and delays T-cell exhaustion, leading to enhanced function and anti-tumour efficacy compared to random integrations.
Published on Jan 11, 2017in Science Translational Medicine17.16
Suk See De Ravin20
Estimated H-index: 20
(NIH: National Institutes of Health),
Linhong Li9
Estimated H-index: 9
+ 17 AuthorsMary Garofalo11
Estimated H-index: 11
(NIH: National Institutes of Health)
Targeted gene therapy has been hampered by the inability to correct mutations in stem cells that can reconstitute the immune system after transplant into patients. De Ravin et al . now report that CRISPR, a DNA editing technology, corrected blood stem cells from patients with an immunodeficiency disorder (chronic granulomatous disease) caused by mutations in NOX2. CRISPR-repaired human stem cells engrafted in mice after transplant and differentiated into leukocytes with a functional NOX2 protein...
Cited By18
Newest
Published on Jan 3, 2019in Nature Communications11.88
Jichao Sun2
Estimated H-index: 2
(UW: University of Wisconsin-Madison),
Jared Carlson-Stevermer5
Estimated H-index: 5
(UW: University of Wisconsin-Madison)
+ 16 AuthorsDianhua Qiao1
Estimated H-index: 1
(UW: University of Wisconsin-Madison)
CRISPR/Cas9 guided gene-editing is a potential therapeutic tool, however application to neurodegenerative disease models has been limited. Moreover, conventional mutation correction by gene-editing would only be relevant for the small fraction of neurodegenerative cases that are inherited. Here we introduce a CRISPR/Cas9-based strategy in cell and animal models to edit endogenous amyloid precursor protein (APP) at the extreme C-terminus and reciprocally manipulate the amyloid pathway, attenuatin...
Published on 2019in DNA Repair3.71
Sandra Bosshard2
Estimated H-index: 2
(UNIL: University of Lausanne),
Pierre-Olivier Duroy (UNIL: University of Lausanne), Nicolas Mermod30
Estimated H-index: 30
(UNIL: University of Lausanne)
Abstract CRISPR technologies greatly foster genome editing in mammalian cells through site-directed DNA double strand breaks (DSBs). However, precise editing outcomes, as mediated by homologous recombination (HR) repair, are typically infrequent and outnumbered by undesired genome alterations. By using knockdown and overexpression studies in Chinese hamster ovary (CHO) cells as well as characterizing repaired DNA junctions, we found that efficient HR-mediated genome editing depends on alternativ...
Published on Jun 1, 2019in Biomaterials10.27
Weiyu Zhao5
Estimated H-index: 5
(OSU: Ohio State University),
Xucheng Hou1
Estimated H-index: 1
(OSU: Ohio State University)
+ 1 AuthorsYizhou Dong22
Estimated H-index: 22
Abstract Genetic and rare diseases (GARDs) affect more than 350 million patients worldwide and remain a significant challenge in the clinic. Hence, continuous efforts have been made to bridge the significant gap between the supply and demand of effective treatments for GARDs. Recent decades have witnessed the impressive progress in the fight against GARDs, with an improved understanding of the genetic origins of rare diseases and the rapid development in gene therapy providing a new avenue for G...
Published on Jul 12, 2019in Nucleic Acids Research11.15
Pin Lyu (Anhui Normal University), Parisa Javidi-Parsijani1
Estimated H-index: 1
(Wake Forest Institute for Regenerative Medicine)
+ 1 AuthorsBaisong Lu13
Estimated H-index: 13
(Wake Forest Institute for Regenerative Medicine)
Published on Jul 1, 2019in Current Gene Therapy2.22
Cia-Hin Lau (CityU: City University of Hong Kong), Chung Tin (CityU: City University of Hong Kong)
Published on Sep 9, 2019in Nature Nanotechnology33.41
Guojun Chen15
Estimated H-index: 15
(UW: University of Wisconsin-Madison),
Amr A. Abdeen11
Estimated H-index: 11
(UW: University of Wisconsin-Madison)
+ 7 AuthorsShaoqin Gong51
Estimated H-index: 51
Delivery technologies for the CRISPR-Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing system often require viral vectors, which pose safety concerns for therapeutic genome editing1. Alternatively, cationic liposomal components or polymers can be used to encapsulate multiple CRISPR components into large particles (typically >100 nm diameter); however, such systems are limited by variability in the loading of the cargo. Here, we report the design of customizabl...
Published on 2019in Applied Microbiology and Biotechnology3.67
Quan Le (UB: University at Buffalo), Vyncent P. Nguyen (UB: University at Buffalo), Sheldon Park14
Estimated H-index: 14
(UB: University at Buffalo)
Streptavidin (SA), and other related proteins, has been isolated from a wide range of organisms, including bacteria, fungi, frogs, fish, and birds. Although their original function is not well understood, they have found an important place in biotechnology based on their unique ability to bind biotin molecules with high affinity and specificity. The SA–biotin interaction is robust and easy to incorporate into different designs, and as such, it is used when reliable molecule interaction is needed...
Published on Sep 1, 2019in Biotechnology Advances12.83
Mu-Nung Hsu4
Estimated H-index: 4
(NTHU: National Tsing Hua University),
ChangYu-Han29
Estimated H-index: 29
(CGU: Chang Gung University)
+ 3 AuthorsHuYu-Chen42
Estimated H-index: 42
(NTHU: National Tsing Hua University)
Abstract CRISPR/Cas9 system exploits the concerted action of Cas9 nuclease and programmable single guide RNA (sgRNA), and has been widely used for genome editing. The Cas9 nuclease activity can be abolished by mutation to yield the catalytically deactivated Cas9 (dCas9). Coupling with the customizable sgRNA for targeting, dCas9 can be fused with transcription repressors to inhibit specific gene expression (CRISPR interference, CRISPRi) or fused with transcription activators to activate the expre...
Published on Sep 1, 2019in MethodsX
Kyriel M. Pineault (IGC: Instituto Gulbenkian de Ciência), Ana Nóvoa11
Estimated H-index: 11
(IGC: Instituto Gulbenkian de Ciência)
+ 2 AuthorsMoisés Mallo31
Estimated H-index: 31
(IGC: Instituto Gulbenkian de Ciência)
Abstract Genetically modified model organisms are valuable tools for probing gene function, dissecting complex signaling networks, studying human disease, and more. CRISPR/Cas9 technology has significantly democratized and reduced the time and cost of generating genetically modified models to the point that small gene edits are now routinely and efficiently generated in as little as two months. However, generation of larger and more sophisticated gene-modifications continues to be inefficient. A...
Published on Jul 15, 2019in Nature plants13.30
Yingxiao Zhang3
Estimated H-index: 3
(UMD: University of Maryland, College Park),
Aimee Malzahn7
Estimated H-index: 7
(UMD: University of Maryland, College Park)
+ 1 AuthorsYiping Qi22
Estimated H-index: 22
(UMD: University of Maryland, College Park)
The application of clustered regularly interspaced short palindromic repeats (CRISPR) for genetic manipulation has revolutionized life science over the past few years. CRISPR was first discovered as an adaptive immune system in bacteria and archaea, and then engineered to generate targeted DNA breaks in living cells and organisms. During the cellular DNA repair process, various DNA changes can be introduced. The diverse and expanding CRISPR toolbox allows programmable genome editing, epigenome e...
View next paperHigh-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects