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Original Title: Nature | Can Electroporation Improve the Efficiency of CRISPR-Cas9 Gene Editing? The editor recently noticed an article titled "Reprogramming human T cell function and specificity with non-viral genome" published in Nature in July Targeting (non-viral vector-targeted genome editing to reprogram the function and specificity of human T cells), this article reports the advantages of a T cell gene editing technology that does not require viral vectors in the treatment of cancer cells. 1 Research background The use of recombinant viral vectors to reprogram T cells for cell therapy has been studied for decades, and the need for viral vectors has slowed down the research and clinical application of tumor cell therapy to a certain extent, because they can not target specific sites, and the operation is cumbersome and expensive. The emergence of CRISPR gene editing technology can insert large fragments of genes specifically and efficiently by using HDR repair mechanism in vivo. In this study, the authors used the CRISPR-Cas9 gene editing system to achieve rapid and efficient insertion of long DNA fragments at specific genomic sites in T cells without using viral vectors, while maintaining the growth and function of T cells. First, the authors used this technique to repair IL2RA pathogenic mutations in cells from a monogenic autoimmune disease, demonstrating an improved cellular signaling mechanism. The authors then used this technique to replace the endogenous T-cell receptor (TCR) locus with a new TCR cancer cell antigen. TCR modified T cells can specifically recognize cancer cell antigens, and its anticancer function has been verified in vivo and in vitro. Cytotoxicity of DNA is a major obstacle to targeting the T cell genome using nonviral methods. Although no significant T cell toxicity was found in cells introduced with short strand oligodeoxynucleotides (ssODN) as a homologous recombination repair template, studies have shown that larger double-stranded DNA templates have significant cytotoxicity at high concentrations. However, in the experiment,retractable tape measure sewing, contrary to the expected results, the introduction of CRISPR-Cas9 ribosomal protein complex (RNP) and double-stranded DNA templates longer than 1 kb into human primary T cells by co-electroporation was less toxic than the introduction of double-stranded DNA templates alone. 2 Research process 1. First, RNP and a HDR repair template were electrotransfected into T cells, and the housekeeping gene RAB11a was knocked into the N fragment of GFP fusion gene. About 50% of CD4 + and CD8 + T cells expressed GFP, indicating that T cells could still maintain good proliferation ability and obtain a high efficiency of gene knockin. This result was confirmed by repeated experiments in T cells from different sources (fig. 1). Expand the full text Figure 1 2. The author's next step is to expand the scope and verify whether this gene editing technology is feasible at different locations in the whole genome. As a result,personalised tailor tape, it was confirmed that gene knock-in could be performed using a non-viral vector method at different selected sites (fig. 2). Figure 2 3. Of course, when gene editing is used in clinical gene therapy and cell therapy, safety is the first risk factor to be considered, the most important of which is miss the target risk, that is, the gene is required to be integrated into a specific site in the genome, while other gene sites will not be damaged. In the next step, the authors used targeted site sequencing (TLA) analysis to find no evidence of miss the target.
Further studies at the single-cell level found that there was a rare phenomenon of miss the target, because dsDNA could produce miss the target effect through the free integration of "non-dependent homologous recombination integration (Non-HDR)". This may occur at the site of naturally occurring or induced double-stranded DNA breaks in the cell (e.g., bespoken tape measure ,custom tape measure, off-site cleavage of RNP), but ssDNA can greatly reduce this risk (fig. 3). Figure 3 At the single-cell level, there is a rare proportion of miss the target cells (top), and dsDNA can be inserted into non-specific sites by non-HDR repair, with a significantly reduced off-target rate for ssDNA templates compared to dsDNA templates (bottom right). 4. Next, the author uses this technology to study two treatment methods: (1) repair of T cell gene mutation; (2) T cell reprogramming-TCR gene knock-in. (1) Repair of T cell gene mutation Disease background: Primary monogenic autoimmune disease with recessive inactivating mutations in the IL-2RA gene, c.530A > G and c.800delA; Gene repair methods: electroporation RNP, dsDNA template, ssDNA template; Results: Genes were repaired, T cell function was improved, there was no risk of miss the target, and ssDNA efficiency was higher. Figure 4 (2) T cell reprogramming-TCR gene knock-in (fig. 5) Background Using the CRISPR-Cas9 system and adeno-associated virus (AAV) as a repair template to make chimeric antigen receptors (CARs) encode TCR genes can improve the efficiency of tumor immune cell therapy. Knocking in TCR is a more difficult challenge, because T cells must express both TCR-α and TCR-β to produce functional receptors; Method Electroporation, 1.5-kb DNA cassette, knock-in T cells, retroviral and lentiviral vectors as controls, in vitro cell co-culture (melanoma cells), in vivo anti-tumor cell therapy (NSG mice); Result Both in vitro and in vitro have anti-tumor effect, and that in vivo anti-tumor effect is superior to that of a lentiviral vector. 3 Conclusion of the study Compared with the use of viral vectors, non-viral T cell gene editing has the advantages of short time, low cost, high efficiency, low off-target rate and high safety, which is the trend of future clinical cell therapy. Finally, in fact, Jieyi Biology has been using the CRISPR-Cas9 gene editing technology of "electroporation" to introduce plasmids or RNP to complete hundreds of gene editing (gene knockout, point mutation/repair and gene knock-in) services for ordinary cell lines (such as tumor cells) and induced pluripotent stem cells (iPSC). Similar to the conclusion in the article, according to our experience, gene editing using "non-viral vector" method can achieve the advantages of "stability,custom tailor tape, efficiency, safety and accuracy" through professional personalized program design. Teachers in need can contact us. Welcome to consult. Source: Marketing Center Jerry Original works of Jieyi, please indicate the source for reprinting Source: https://www.nature.com/articles/s41586-018-0326-5. · END · Return to Sohu to see more Responsible Editor:. tape-measure.com