Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus.
Publication Year:
2023
PubMed ID:
37127707
Public Summary:
In the field of genetic engineering, the efficient and stable insertion of large genetic payloads into primary human cells including human induced pluripotent stem cells (iPSC) remains a formidable challenge. Conventional methods, like retroviral vectors, face limitations due to semi-random insertion and gene silencing. To address these limitations, our team has developed a new method, termed CLIP (CRISPR for Long-fragment Integration via Pseudovirus). This approach allows for the precise and stable integration of large genetic payloads into specific sites within the genome, with minimal cytotoxicity.
CLIP combines the specificity of CRISPR-Cas9 technology with the delivery efficiency of an integrase-deficient lentivirus (IDLV). The IDLV carries the genetic payload, flanked by homology arms and specific 'cut sites', allowing integration upstream and in-frame with an essential endogenous gene. This strategic placement ensures consistent expression of the inserted gene. Additionally, the CRISPR-associated ribonucleoprotein complex, delivered via electroporation, further enhances the precision and efficiency of this process.
Our research demonstrates that CLIP can effectively integrate large genetic elements, as well as simultaneously insert two genes at different genomic loci. This capability was showcased by successfully integrating two complex viral antigens into primary T cells. Remarkably, CLIP achieves this with significantly lower levels of cytotoxicity compared to existing methods, making it a safer alternative for cell engineering. The CLIP method has implications on the field of gene therapy and biotechnology. It offers a scalable and efficient solution for manufacturing genetically engineered primary cells, paving the way for advanced therapeutic interventions. The ability to reliably insert large genetic payloads into specific genomic locations opens new avenues for treating diseases and advancing our understanding of genetic functions.
In summary, the CLIP method represents a major step forward in genetic engineering. Its precision, efficiency, and reduced cytotoxicity make it a promising tool for a wide range of applications in medicine and biotechnology. Our research not only showcases the potential of CLIP but also sets the stage for further innovations in genome engineering.
Scientific Abstract:
The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.