“Overall, our results describe a workflow to elaborate robust protocols for gene editing and expression in primary tumor cells,” lead study author Federica Nardi, of the University of Siena and Fondazione Toscana Life Sciences, in Siena, Italy, and coauthors wrote in the poster.
Patient-derived cancer cells can be used to study the biology of a disease and are favorable to immortalized cell lines because they are more like in vivo environments. However, primary cells are challenging to work with; therefore, the usefulness of conventional methods for gene editing and expression is limited, which has created a need for improved means for gene editing and expression.
To that end, investigators first created optimal electroporation conditions for CRISPR/Cas9-based gene knockout and mRNA-driven gene overexpression in B cells that were newly taken from the peripheral blood or bone marrow of patients with CLL.
“[A] CRISPR/Cas9 ribonucleoprotein system allows to easily screen tens to hundreds [of] genes in parallel in 96-well format (cost per gene around $50 to $100). The same applies for mRNA-based system (cost per gene around $25),” wrote the authors.
Green fluorescent protein mRNA was electroporated into primary CLL B cells using 12 different electroporation settings. B-cell survival and green fluorescent protein signal were assessed by cytofluorimetry. Pulsing parameters giving the best results were chosen and used for further study.
Next, gene function screening was performed based on efficient CRISPR/Cas9 gene knockout. Ribonucleoprotein complexes were created by incubating Cas9 ribonucleoprotein with single-guide RNAs and used to electroporate resting CLL B cells. B-cell proliferation was subsequently stimulated with CD40L, IL-4 and IL-21. The efficiency of gene knockout was assessed by cytofluorimetry.
“Advantages of the developed system include the ability to manipulate B cells isolated from CLL patients, and it is also applicable to primary B cells of other origins,” wrote the authors.
A platform was then developed for gene function screening based on high-throughput in vitro mRNA synthesis and overexpression. Genes of interest were amplified from complementary DNA using specific primers (PCR1). PCR products were cloned into a vector bearing the T7 promoter by Gibson assembly. The region spanning from the T7 promoter to the 3 prime untranslated region was amplified by PCR 2, then used for in vitro synthesis of mRNA. Obtained mRNA was introduced into CLL B cells by electroporation.
The results confirmed that electroporation conditions can be created that only mildly affect CLL cell viability. Specifically, more than 70% to 80% of homozygous knockout of a given gene can be achieved therein by electroporating CLL cells with ribonucleoprotein complexes that harbor recombinant Cas9 nuclease and synthetic guides.
Additional results showed that electroporation with in vitro transcribed mRNA led to more 90% electroporation efficiency and enabled finely modulated gene expression levels in CLL cells. Moreover, gene overexpression persisted for at least 4 days, enabling the use of long-term in vitro assays.
“Electroporation with RNPs or mRNA does not impact cell viability and does not require prior expansion, which is particularly relevant for studies that aim to assess function of genes regulating B-cell proliferation and differentiation,” concluded the authors.
- Nardi F, Di Rita A, Pezzella L, et al. CRISPR/Cas9 and mRNA-based gene editing and expression in chronic lymphocytic leukemia cells. Presented at: 2021 International Workshop on CLL; September 16-20, 2021; virtual. Abstract 1084281.
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