Optimization of electroporation conditions in acute myeloid leukemia cell lines for CRISPR-Cas9-mediated gene knockout
CRISPR-Cas9 is a powerful tool for targeted genome editing and is widely used to investigate gene function in cancer models. In acute myeloid leukemia (AML), efficient gene knockout in relevant cell lines is essential for functional studies and the identification of therapeutic targets. Here, we optimized electroporation parameters for three AML cell lines, Molm13, OCI-AML5, and TF-1, and applied these conditions for CRISPR-Cas9-mediated gene editing. Electroporation using a 150 V, 5 millisecond square-wave pulse provided an optimal balance between cell viability and mRNA delivery. Furthermore, higher doses of Cas9 and sgRNA improved knockout efficiency, with the combination of 37.5 pmol Cas9 protein and 75 pmol sgRNA achieving the maximal protein depletion across all tested cell lines, without evidence of increased off-target effects as shown by NGS sequencing. This work establishes a reproducible optimized platform for gene editing in AML cell lines, facilitating functional studies and potential therapeutic applications.
CRISPR-Cas9 is a powerful tool for targeted genome editing and is widely used to investigate gene function in cancer models. In acute myeloid leukemia (AML), efficient gene knockout in relevant cell lines is essential for functional studies and the identification of therapeutic targets. Here, we optimized electroporation parameters for three AML cell lines, Molm13, OCI-AML5, and TF-1, and applied these conditions for CRISPR-Cas9-mediated gene editing. Electroporation using a 150 V, 5 millisecond square-wave pulse provided an optimal balance between cell viability and mRNA delivery. Furthermore, higher doses of Cas9 and sgRNA improved knockout efficiency, with the combination of 37.5 pmol Cas9 protein and 75 pmol sgRNA achieving the maximal protein depletion across all tested cell lines, without evidence of increased off-target effects as shown by NGS sequencing. This work establishes a reproducible optimized platform for gene editing in AML cell lines, facilitating functional studies and potential therapeutic applications.
leibniz institute of virology