Evidence map›Paper›PMID 40282329›Full record

ArticleGenes2025

Techniques for Validating CRISPR Changes Using RNA-Sequencing Data.

Susan K Rathe, Tracy A Marko, Elizabeth N Edwards, Paige Hazelton Ridder, Jyotika Varshney, Kyle B Williams, James E Johnson, Branden S Moriarity, David A Largaespada

Abstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Genes, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

0numbers the graph read from it
0cells of the map it votes in
0citing 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

0 citing papers in PubMed.

No citing paper in PubMed yet.

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.

Susan K RatheMasonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
Tracy A MarkoMasonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
Elizabeth N EdwardsMasonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
Paige Hazelton RidderMasonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
Jyotika VarshneyMasonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.ORCID 0000-0003-0275-1521
Kyle B WilliamsMasonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.ORCID 0000-0003-4333-9111
James E JohnsonSupercomputing Institute, University of Minnesota, Minneapolis, MN 55455, USA.
Branden S MoriarityMasonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
David A LargaespadaMasonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.ORCID 0000-0002-3183-0491

Funding

MEDICAL SCIENTIST TRAINING PROGRAMT32GM008244 · NIGMS · UNIVERSITY OF MINNESOTA TWIN CITIES · PI SHIMIZU, YOJI · 1988 to 2024
$13.6M
TRAINING GRANT IN MICROBIOLOGY/CANCER RESEARCHT32CA009138 · NCI · UNIVERSITY OF MINNESOTA TWIN CITIES · PI Scott M. Dehm · 1985 to 2026
$11.3M
Sleeping Beauty for uncovering cancer genes in miceR01CA113636 · NCI · UNIVERSITY OF MINNESOTA · PI LARGAESPADA, DAVID ANDREW · 2006 to 2016
$2.5M
ACS Research Professorship #123939Children's Cancer Research Fund Emerging Scientist Award NAChildren's Tumor Foundation 5T32CA009138-40Children's Tumor Foundation Young Investigator Award NANCI NIH HHS R01 CA113636NCI NIH HHS T32 CA009138NIGMS NIH HHS T32 GM008244NIH HHS MSTP grant T32 GM008224NIH HHS R01-CA113636Zach Sobiech Osteosarcoma Fund of the Children's Cancer Research Fund, Minneapolis MN NA
6 · The paper itself

Abstract

The use of CRISPR to knockdown or knockout genes is a powerful tool for understanding the specific role of a gene in disease development. However, it can cause many unanticipated changes to the transcriptome that are not detected by DNA amplification and Sanger sequencing of the target site. Various RNA-sequencing techniques can be used to identify these changes and effectively gauge the full impact of the CRISPR knockout, thereby providing a means of selecting appropriate clones for further experimentation. BACKGROUND/

objectivesRNA-seq data from 4 CRISPR knockout experiments were analyzed and techniques developed to both confirm the success of the CRISPR modifications and identify potential issues.

methodsA broad-based analysis of RNA-sequencing data identified many CRISPR-based changes not identified by PCR amplification of DNA around the CRISPR target site. These changes included an inter-chromosomal fusion event, exon skipping, chromosomal truncation, and the unintentional transcriptional modification and amplification of a neighboring gene.

conclusionsThe inadvertent modifications identified by the evaluation of 4 CRISPR experiments highlight the value of using RNA-seq to identify transcriptional changes to cells altered by CRISPR, many of which cannot be recognized by evaluating DNA alone. Specific guidelines are presented for designing and analyzing CRISPR experiments using RNA-seq data.

Indexed as

Clustered Regularly Interspaced Short Palindromic RepeatsCRISPR-Cas SystemsRNA-SeqSequence Analysis, RNAGene EditingGene Knockout TechniquesHumansTranscriptomeCRISPRknockout experimentsRNA sequencing

Identifiers

PMID40282329
PMCPMC12027141

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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.