Evidence map›Paper›PMID 25586224›Full record

ArticleNucleic acids research2015

Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

Manuel Kaulich, Yeon J Lee, Peter Lönn, Aaron D Springer, Bryan R Meade, Steven F Dowdy

Open access · goldAbstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Nucleic acids research, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 20 papers.

0numbers the graph read from it
0cells of the map it votes in
20citing papers in PubMed
1.7field-weighted citation impact, top 16% of its field
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

20 citing papers in PubMed, 30 citations in OpenAlex.

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  17. Genome Engineering Using Adeno-associated Virus: Basic and Clinical Research Applications.Molecular therapy : the journal of the American Society of Gene Therapy · 2016
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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

6 authors at 1 institution in 2 countries.

Manuel KaulichDepartment of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Yeon J LeeDepartment of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Peter LönnDepartment of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Aaron D SpringerDepartment of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Bryan R MeadeDepartment of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Steven F DowdyDepartment of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA sdowdy@ucsd.edu.
University of California San Diego · US

Funding

NCI NIH HHS CA185589
6 · The paper itself

Abstract

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms.

Indexed as

AllelesRNA InterferenceBase SequenceCDC2 Protein KinaseCell LineClustered Regularly Interspaced Short Palindromic RepeatsCyclin-Dependent Kinase 2Cyclin-Dependent KinasesDependovirusDNA PrimersGene Knockdown TechniquesHumansMutationPolymerase Chain ReactionRecombination, GeneticCDC2 Protein KinaseCDK1 protein, humanCDK2 protein, humanCyclin-Dependent Kinase 2Cyclin-Dependent KinasesDNA Primers

Identifiers

PMID25586224
PMCPMC4402508
OpenAlexW2129173652

What OpenQuestion holds

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LicenceCC BY
Read underepoch 390

Registered trials

None linked

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.