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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 Communications11.878
· DOI :10.1038/s41467-017-01875-9
Jared Carlson-Stevermer6
Estimated H-index: 6
(UW: University of Wisconsin-Madison),
Amr A. Abdeen11
Estimated H-index: 11
(UW: University of Wisconsin-Madison)
+ 4 AuthorsKrishanu Saha23
Estimated H-index: 23
(UW: University of Wisconsin-Madison)
Sources
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.
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  • References (56)
  • Citations (33)
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References56
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#2Jason Cheng (Harvard University)H-Index: 5
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Duchenne muscular dystrophy (DMD) is a devastating, degenerative muscle disease that affects ~1 in every 3500 male births. DMD arises from mutations in the DMD gene that prevent expression of its encoded protein, Dystrophin (Burghes et al. Nature 328:434–437, 1987). Interestingly, patients with Dmd mutations that delete certain segments of the Dystrophin coding region, but maintain protein reading frame, have a much milder form of the disease, known as Becker Muscular Dystrophy (BMD). This obser...
1 CitationsSource
#1Han Li (UCSF: University of California, San Francisco)H-Index: 9
#2Kyle Beckman (UCSF: University of California, San Francisco)H-Index: 3
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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...
17 CitationsSource
#1Hong Ma (Oregon National Primate Research Center)H-Index: 27
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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.
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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...
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Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA...
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CRISPR-Cas9 genome editing creates targeted double strand breaks (DSBs) in eukaryotic cells that are processed by cellular DNA repair pathways. Co-administration of single stranded oligonucleotide donor DNA (ssODN) during editing can result in high-efficiency (>20%) incorporation of ssODN sequences into the break site. This process is commonly referred to as homology directed repair (HDR) and here referred to as single stranded template repair (SSTR) to distinguish it from repair using a double ...
14 CitationsSource
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#2Vanessa A. MackleyH-Index: 3
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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 ...
41 CitationsSource
#1Ming Ma (PKU: Peking University)H-Index: 1
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Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system
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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).
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Abstract Efficient delivery of hydrophilic drugs, nucleic acids, proteins, and any combination thereof is essential for various biomedical applications. Herein, we report a straightforward, yet versatile approach to efficiently encapsulate and deliver various hydrophilic payloads using a pH-responsive silica–metal–organic framework hybrid nanoparticle (SMOF NP) consisting of both silica and zeolitic imidazole framework (ZIF). This unique SMOF NP offers a high loading content and efficiency, exce...
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Site-specific chemical conjugation of proteins can enhance their therapeutic and diagnostic utility but has seldom been applied to CRISPR-Cas9, which is a rapidly growing field with great therapeutic potential. The low efficiency of homology-directed repair remains a major hurdle in CRISPR-Cas9–mediated precise genome editing, which is limited by low concentration of donor DNA template at the cleavage site. In this study, we have developed methodology to site-specifically conjugate oligonucleoti...
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Abstract The strong molecular interaction between biotin and streptavidin is widely used in the growing field of nucleic acid nanotechnology. Several biotin labeled oligonucleotide tools have been developed for the detection of biological molecules as well as for protein purification. For these reasons, biotinylation can be considered one of the main chemical reactions for nucleic acid labeling. However, despite its widespread application and the presence on the market of many reagents for the c...
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