Evidence map›Paper›PMID 25937281›Full record

ArticleCell reports2015

Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.

Florian T Merkle, Werner M Neuhausser, David Santos, Eivind Valen, James A Gagnon, Kristi Maas, Jackson Sandoe, Alexander F Schier, Kevin Eggan

Abstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Cell reports, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 89 papers.

0numbers the graph read from it
0cells of the map it votes in
89citing papers in PubMed
–field-weighted citation impact
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

89 citing papers in PubMed.

  1. Review
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  12. Enrichment strategies to enhance genome editing.Journal of biomedical science · 2023
    Review
  13. Review
  14. Review
  15. Review
  16. Genome Editing and Pathological Cardiac Hypertrophy.Advances in experimental medicine and biology · 2023
    Article
  17. Review
  18. Review
  19. Article
  20. Review

29 more citing papers are in PubMed but not listed here.

4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

9 authors.

Florian T MerkleDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
Werner M NeuhausserDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA; Boston IVF, Waltham, MA 02451, USA.
David SantosDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
Eivind ValenDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; Computational Biology Unit, Department of Informatics, University of Bergen, 5020 Bergen, Norway.
James A GagnonDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Kristi MaasDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, USA; Boston IVF, Waltham, MA 02451, USA.
Jackson SandoeDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Alexander F SchierDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Center for Brain Science, Harvard University, Cambridge, MA 02138, USA.
Kevin EgganDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; The Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA; Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address: eggan@mcb.harvard.edu.

Funding

Regulatory Roles and Dynamics of Nuclear long-noncoding RNAs in PluripotencyP01GM099117 · NIGMS · HARVARD UNIVERSITY · PI RINN, JOHN LOUIS · 2011 to 2021
$21.2M
Genetics of Sleep in ZebrafishR01HL109525 · NHLBI · HARVARD UNIVERSITY · PI SCHIER, ALEXANDER F · 2011 to 2015
$2.1M
Generation of Hypocretin Neurons from Narcoleptic PatientsR21NS071598 · NINDS · HARVARD UNIVERSITY · PI SCHIER, ALEXANDER F · 2010 to 2011
$457k
Cellular models of hypocretin neuron loss in narcolepsyK99NS083713 · NINDS · HARVARD UNIVERSITY · PI MERKLE, FLORIAN TOBIAS · 2013 to 2014
$178k
Howard Hughes Medical InstituteNHLBI NIH HHS HL109525NHLBI NIH HHS R01 HL109525NIGMS NIH HHS P01 GM099117NINDS NIH HHS 1R21NS071598NINDS NIH HHS 5K99NS083713NINDS NIH HHS K99 NS083713NINDS NIH HHS R21 NS071598
6 · The paper itself

Abstract

The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus.

Indexed as

Gene TargetingGenetic LociBase SequenceClone CellsCRISPR-Cas SystemsGene Knock-In TechniquesHumansINDEL MutationMolecular Sequence DataMutationPluripotent Stem Cells

Identifiers

PMID25937281
PMCPMC5533178

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Registered trials

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.