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Marker-free coselection for CRISPR-driven genome editing in human cells

Published on Jun 1, 2017in Nature Methods28.467
· DOI :10.1038/nmeth.4265
D. Agudelo15
Estimated H-index: 15
,
Alexis Duringer1
Estimated H-index: 1
+ 9 AuthorsYannick Doyon24
Estimated H-index: 24
Abstract
A gain-of-function mutation in a sodium/potassium pump renders cells resistant to a small-molecule drug and provides an efficient coselection strategy to enrich for CRISPR-induced mutations at an independent locus.
  • References (49)
  • Citations (43)
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References49
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#1D. AgudeloH-Index: 15
#2Alexis DuringerH-Index: 1
Last. Yannick DoyonH-Index: 24
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1 CitationsSource
#1Katie A. Mitzelfelt (UofU: University of Utah)H-Index: 3
#2Chris McDermott-Roe (MCW: Medical College of Wisconsin)H-Index: 4
Last. Aron M. Geurts (MCW: Medical College of Wisconsin)H-Index: 29
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Summary Genome editing in induced pluripotent stem cells is currently hampered by the laborious and expensive nature of identifying homology-directed repair (HDR)-modified cells. We present an approach where isolation of cells bearing a selectable, HDR-mediated editing event at one locus enriches for HDR-mediated edits at additional loci. This strategy, called co-targeting with selection, improves the probability of isolating cells bearing HDR-mediated variants and accelerates the production of ...
13 CitationsSource
#1Hui K. Kim (Yonsei University)H-Index: 1
#2Myungjae Song (Hanyang University)H-Index: 3
Last. Hyongbum Kim (Yonsei University)H-Index: 16
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A lentiviral library expressing Cpf1 guide RNAs and containing target sequences allows high-throughput profiling of highly active guide RNAs and is the basis for cindel, a webtool to predict the activity at any given target sequence.
102 CitationsSource
#1Bernd Zetsche (MIT: Massachusetts Institute of Technology)H-Index: 1
#2Matthias Heidenreich (MIT: Massachusetts Institute of Technology)H-Index: 1
Last. Feng Zhang (MIT: Massachusetts Institute of Technology)H-Index: 115
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Multiplexed genome editing is simplified by harnessing the ability of Cpf1 to process its own pre-crRNA.
242 CitationsSource
#1Nanci S. Kane (RU: Rutgers University)H-Index: 1
#2Mehul Vora (RU: Rutgers University)H-Index: 5
Last. Richard W. Padgett (RU: Rutgers University)H-Index: 33
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Genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease (Cas9) enables specific genetic modifications, including deletions, insertions, and substitutions in numerous organisms, such as the fruit fly Drosophila melanogaster . One challenge of the CRISPR/Cas9 system can be the laborious and time-consuming screening required to find CRISPR-induced modifications due to a lack of an obvious phenotype and low frequency after editing. Here we ...
14 CitationsSource
#1Daniel P. DeverH-Index: 12
#2Rasmus O. BakH-Index: 20
Last. Matthew H. PorteusH-Index: 46
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These preclinical studies outline a CRISPR-based methodology for correcting β-globin gene mutations in haematopoietic stem cells to advance the development of next-generation therapies for β-haemoglobinopathies.
247 CitationsSource
#1Mark A. DeWitt (University of California, Berkeley)H-Index: 10
#2Wendy Magis (Children's Hospital Oakland Research Institute)H-Index: 4
Last. Jacob E. Corn (University of California, Berkeley)H-Index: 32
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Sickle cell disease is a genetic disorder caused by a mutation in one of the hemoglobin genes, which causes deformation of red blood cells and results in occlusion of blood vessels, severe pain crises, and progressive organ injury. To correct the mutation that causes this disease, DeWitt et al . modified hematopoietic stem cells from sickle cell disease patients using a CRISPR/Cas9 gene editing approach. The authors showed that the corrected cells successfully engrafted in a mouse model and prod...
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#1Daniel Tianfang Ge (UMMS: University of Massachusetts Medical School)H-Index: 3
#2Cindy Tipping (UMMS: University of Massachusetts Medical School)H-Index: 4
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Adoption of a streamlined version of the bacterial clustered regular interspersed short palindromic repeat (CRISPR)/Cas9 defense system has accelerated targeted genome engineering. The Streptococcus pyogenes Cas9 protein, directed by a simplified, CRISPR-like single-guide RNA, catalyzes a double-stranded DNA break at a specific genomic site; subsequent repair by end joining can introduce mutagenic insertions or deletions, while repair by homologous recombination using an exogenous DNA template c...
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#1Brian R. Shy (UIC: University of Illinois at Chicago)H-Index: 4
#2Matthew S. MacDougall (UIC: University of Illinois at Chicago)H-Index: 3
Last. Bradley J. Merrill (UIC: University of Illinois at Chicago)H-Index: 18
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CRISPR/Cas9 nucleases have enabled powerful, new genome editing capabilities; however, the preponderance of non-homologous end joining (NHEJ) mediated repair events over homology directed repair (HDR) in most cell types limits the ability to engineer precise changes in mammalian genomes. Here, we increase the efficiency of isolating precise HDR-mediated events in mouse embryonic stem (ES) cells by more than 20-fold through the use of co-incidental insertion (COIN) of independent donor DNA sequen...
18 CitationsSource
#1Megan D. Hoban (UCLA: University of California, Los Angeles)H-Index: 8
#2Dianne Lumaquin (UCLA: University of California, Los Angeles)H-Index: 4
Last. Donald B. Kohn (UCLA: University of California, Los Angeles)H-Index: 71
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Targeted genome editing technology can correct the sickle cell disease mutation of the β-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-speci...
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The CRISPR-Cas12a is a class II, type V clustered regularly interspaced short palindromic repeat (CRISPR) system with both RNase and DNase activity. Compared to the CRISPR-Cas9 system, it recognizes T-rich PAM sequences and has the advantage of multiplex genomic editing. Here, in fission yeast Schizosaccharomyces pombe, we successfully implemented the CRISPR-Cas12a system for versatile genomic editing and manipulation. In addition to the rrk1 promoter, we used new pol II promoters from endogenou...
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Genome editing in human induced pluripotent stem cells (iPSCs) provides the potential for disease modeling and cell therapy. By generating iPSCs with specific mutations, researchers can differentiate the modified cells to their lineage of interest for further investigation. However, the low efficiency of targeting in iPSCs has hampered the application of genome editing. In this study we used a CRISPR-Cas9 system that introduces a specific point substitution into the sequence of the Na(+)/K(+)-AT...
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Prolonged mitosis often results in apoptosis1. Shortened mitosis causes tumorigenic aneuploidy, but it is unclear whether it also activates the apoptotic machinery2. Separase, a cysteine protease and trigger of all eukaryotic anaphases, has a caspase-like catalytic domain but has not previously been associated with cell death3,4. Here we show that human cells that enter mitosis with already active separase rapidly undergo death in mitosis owing to direct cleavage of anti-apoptotic MCL1 and BCL-X...
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CRISPR/Cas9-mediated homology-directed repair (HDR) can be leveraged to precisely engineer mammalian genomes. However, the inherently low efficiency of HDR often hampers to identify the desired modified cells. Here, we developed a novel Universal Surrogate Reporter system that efficiently enriches for genetically modified cells arising from CRISPR/Cas9-induced HDR events (namely, HDR-USR). This episomally based reporter can be self-cleaved and self-repaired via HDR to create a functional puromyc...
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The H2A.Z histone variant plays major roles in the control of gene expression. In human, H2A.Z is encoded by two genes expressing two isoforms, H2A.Z.1 and H2A.Z.2 differing by three amino acids. Here, we undertook an integrated analysis of their functions in gene expression using endogenously-tagged proteins. RNA-Seq analysis in untransformed cells showed that they can regulate both distinct and overlapping sets of genes positively or negatively in a context-dependent manner. Furthermore, they ...
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Here we describe a simple and time-efficient strategy for endogenous gene tagging in mammalian tissue culture cells. We take advantage of CRISPR-Cas12a-induced double strand breaks and their repair by homologous recombination using heterologous templates with short homology arms. Assembly of these templates and the Cas12a endonuclease is restricted to a single PCR step followed by DNA transfection, which avoids handling of RNAs, recombinant proteins or cloning of plasmids. The method is robust a...
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Summary CRISPR-Cas9 engineered CYBBko THP-1 cell lines display an inflammatory profile with increased IL-1β, IL-6 and TNF-α secretion as consequence of NADPH-induced ROS deficiency. This phenotype is reminiscent of the one observed in phagocytes from CGD patients.
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Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog ...
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