Data establish multi-site reproducibility of SACF and GILA in the MCF10A-PTPN12 model evaluated here and support their use as animal-free, development-stage characterization tools contributing to tumorigenicity evidence generation.
Abstract
Tumorigenicity is a key safety concern for CRISPR/Cas9-based gene therapies, yet its assessment remains challenging due to the lack of relevant and sensitive in vivo models. This HESI Global multi-site study evaluated the Soft Agar Colony Formation (SACF) assay and the Growth in Low Attachment (GILA) assay for in vitro transformation testing of CRISPR/Cas9-edited cells as part of tumorigenicity risk assessment. Across four laboratories, MCF10A cells were spiked with varying concentrations of cells carrying a CRISPR/Cas9-mediated knockout of the known tumor suppressor PTPN12 to determine the assays' limit of detection (LOD), robustness and reproducibility. Both assays demonstrated a consistent LOD of 0.8% at most sites, with SACF showing a broader dynamic range and slightly stronger correlation across laboratories. GILA exhibited weaker correlation due to plateauing at high spike-in concentrations (>12.5%), and excluding these conditions improved inter-laboratory correlations. Additionally, the study identified key determinants of assay success, including the selection of appropriate cell lines and lots, and ensuring sufficient recovery time post-thaw. Together, these data establish multi-site reproducibility of SACF and GILA in the MCF10A-PTPN12 model evaluated here and support their use as animal-free, development-stage characterization tools contributing to tumorigenicity evidence generation.
Lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH) are reported, a system that enables PHH expansion in liver-humanized mice and enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.
Ansgar F. Stenzel, Antonis Athanasiadis, Georgios Dangas et al.· bioRxiv· 0 citations
It is reported that the choice of sgRNA target site affects its targeted cleavage activity and show that the combination of guideRNA with SpCas9 can effectively delete the exonic fragment of the SLC25A38 gene in HEK293T cells.
S. Yazdanparast, Hamid Galehdari, S. Khatami et al.· Journal of Applied Genetics· 0 citations
Triple-negative breast cancer (TNBC) is characterized by the absence of hormone receptor and HER2 expression, constituting a subtype with high biological aggressiveness and limited therapeutic options. In this context, the BIRC5 gene stands out for encoding the survivin protein, whose overexpression is directly associated with apoptosis inhibition, tumor progression, and resistance to conventional treatments. From this perspective, the present study aimed to analyze the potential of the CRISPR-Cas9 system as a therapeutic tool in TNBC, with emphasis on BIRC5 modulation. This is a descriptive and analytical literature review based on scientific articles published between 2014 and 2025, retrieved from the PubMed, Scopus, and Google Scholar databases. The literature analysis demonstrates that CRISPR-Cas9-mediated gene editing has a significant impact on reducing tumor cell viability, in addition to enhancing the sensitivity of neoplastic cells to conventional therapies. However, the clinical applicability of this approach is still limited by relevant technical challenges, particularly those related to off-target effects and the efficiency of delivery systems. Therefore, although CRISPR-Cas9 has emerged as a promising strategy in the context of precision oncology, its incorporation into clinical practice depends on improvements in safety, specificity, and therapeutic feasibility, as well as on integration with complementary approaches.
Júlia Do Carmo Barros Lobo, Diogo Nery Maciel· Revista de Estudos Interdisc...· 0 citations
BACKGROUND & AIMS
Radiotherapy confers clinical benefits to hepatocellular carcinoma (HCC) patients across all stages, yet its clinical efficacy is limited by radioresistance. This study aimed to identify key regulators of HCC radiosensitivity through genome-wide functional screening.
METHODS
A genome-wide CRISPR-Cas9 screen in Huh7 cells identified radiosensitivity regulators, with SRSF6 validated by siRNA knockdown and γ-H2AX assessment. Stable shRNA-mediated SRSF6 knockdown was established in Huh7 and HepG2 cells, followed by clonogenic, EdU incorporation, apoptosis, micronucleus, and comet assays. Mechanistically, RNA-seq, western blotting, mRNA stability assays, RIP-qPCR, and RAD51 overexpression rescue assays were performed. The therapeutic potential of the SRSF6 inhibitor indacaterol was evaluated using MTS assays, HCC xenograft mouse models (BALB/c-nu/nu, N=28), and HCC patient-derived organoids (PDOs) (N=3). In addition, SRSF6 expression and its correlation with patient survival were analyzed using data from The Cancer Genome Atlas and a tissue microarray (N=14 HCC and 14 paired adjacent non-tumorous liver samples).
RESULTS
We identified the RNA-binding protein SRSF6 as a driver of HCC radioresistance. SRSF6 depletion enhanced the radiosensitivity of HCC cells (p<0.05-0.0001) by post-transcriptionally destabilizing the mRNAs of critical DNA repair genes (p<0.05-0.0001), thereby impairing radiation-induced DNA damage repair. The radiosensitizing effect of SRSF6 depletion was partially abrogated by ectopic overexpression of the core DNA repair protein RAD51 (p<0.05-0.001). Indacaterol exhibited cytotoxic effects on HCC cells (p<0.05-0.0001) and enhanced the antitumor efficacy of radiation in vivo (p<0.05-0.0001), as further validated across multiple HCC patient-derived organoids (p<0.05-0.0001).
CONCLUSIONS
SRSF6 is a key regulator of HCC radioresistance through its post-transcriptional control of DNA repair capacity, and represents a novel therapeutic target to sensitize HCC to radiotherapy.
IMPACT AND IMPLICATIONS
In this study, we performed a genome-wide CRISPR-Cas9 knockout library screen to dissect the molecular determinants governing HCC radiosensitivity, and identified RNA-binding protein SRSF6 as a driver of HCC radioresistance. We demonstrate that SRSF6 depletion disrupts the post-transcriptional stability of key DNA repair gene mRNAs and enhances HCC radiosensitivity. These findings are important for radiation oncologists and translational researchers, as they identify SRSF6-dependent RNA regulation as a critical determinant of radiotherapy response in HCC. Practically, we show that the clinically approved bronchodilator indacaterol suppresses SRSF6 function and enhances the antitumor efficacy of radiotherapy, offering a readily repurposable pharmacological strategy to overcome radioresistance. These implications are based on preclinical evidence across multiple models; however, future clinical trials are needed to validate the safety and efficacy of indacaterol-based radiosensitization in patients with HCC.
Wenyi Jiang, Xin Sui, D. Dong et al.· JHEP Reports· 0 citations
It is shown that off-target editing and translocations vary widely between individual cells and organs, motivating the development of more sensitive and organ-specific safety assays for CRISPR therapies.
Alexandra Madsen, Niklas Selfjord, M. Martinez-Lage et al.· Nature Communications· 0 citations
Human induced pluripotent stem cells (hiPSCs) represent a powerful platform for disease modeling, especially in monogenic diseases as they preserve the donor’s genetic background while enabling directed differentiation into disease-relevant cell types. This makes them highly suitable for studying disease mechanisms in a patient-specific and physiologically relevant context. Although CRISPR/Cas9 is widely applied for genome editing, precise correction of pathogenic variants in hiPSCs remains challenging due to the lack of standardized CRISPR component selection and experimental design. Here, we describe an optimized CRISPR-based strategy for correcting a heterozygous HNF1A frameshift mutation (c.235_236insG; p.Glu79Glyfs*16) in HNF1A-MODY patient-derived hiPSCs. Using electroporation, we efficiently delivered CRISPR components, including a ribonucleoprotein complex of Cas9 and single-guide RNA, along with a single-stranded oligodeoxynucleotide repair template. Corrected hiPSC lines were validated for pluripotency, absence of exogenous reprogramming factors, and off-target effects. Additionally, we discuss key technical challenges encountered during the editing process and provide practical recommendations that may improve the generation of mutation-corrected hiPSC lines. These guidelines could serve as a useful reference for researchers employing CRISPR-based strategies for generation of reliable disease modelling tools.
D. Skoczek, Jerzy Hohendorff, Maciej T. Małecki et al.· Human Genetics· 0 citations
