| Title |
Comparative analysis of antibody-mediated loss-of-function versus gene knock-out and knock-down |
| Authors |
Buck-Wiese, Marie ; Liechocki, Sally ; Erfle, Holger ; Starkuvienė, Vytautė |
| DOI |
10.1016/j.slasd.2025.100283 |
| Full Text |
|
| Is Part of |
SLAS Discovery.. New York : Elsevier; Society for Laboratory Automation and Screening (SLAS). 2025, vol. 37, art. no.100283, p. [1-11].. ISSN 2472-5552. eISSN 2472-5560 |
| Keywords [eng] |
antibody delivery ; cell adhesion ; Kindlin ; Talin ; off-target effect |
| Abstract [eng] |
In this study we compare three methods for manipulating cell function: RNA interference (RNAi), CRISPR-Cas9 gene knock-out, and antibody-mediated loss-of-function. We have focused on analyzing changes in cell-matrix adhesion via targeting two key regulators, Talin1 (TLN1) and Kindlin-2 (KD2). Adhesion-relevant phenotypic assays revealed distinct temporal onset dynamics for each method. RNAi and CRISPR-Cas9 effectively reduced target mRNA and protein levels. In contrast, antibody transfection induced phenotypic changes without altering target expression, suggesting direct intracellular antibody-target interaction. Transcriptome analysis demonstrated that antibody transfection and CRISPR-Cas9 induced fewer deregulated mRNAs than RNAi. Furthermore, transfected antibodies and sgRNAs shared 30 % and 70 % of deregulated transcripts to their negative controls, respectively. Whereas only 10 % of overlap was recorded between targeting and control siRNAs. Our findings emphasize the importance of considering method-specific temporal dynamics of on-target phenotype appearance and off-target manifestation. Additionally, they highlight intracellular delivered antibodies as a valuable alternative for validating and complementing genetic approaches. |
| Published |
New York : Elsevier; Society for Laboratory Automation and Screening (SLAS) |
| Type |
Journal article |
| Language |
English |
| Publication date |
2025 |
| CC license |
|
