Knockknock clip3/2/2024 ![]() By targeting CLIP to ACTB, an essential gene encoding a component of the cellular cytoskeleton, the transgene took on the expression characteristics of the ACTB locus and its expression remained stable over time. With CLIP, we were able to efficiently integrate large genetic payloads into an essential gene of primary human T cells, which achieved an improved duration of gene expression compared with traditional lentivirus vectors.ĬLIP doubles the number of targeted integration events compared with unmodified IDLV vectors and was non-toxic in primary human T cells, unlike naked DNA donors. The CLIP (CRISPR for long-fragment integration via pseudovirus) method was tested in cell lines and primary human T cells to measure knock-in efficiency and toxicity compared with unmodified IDLV vectors and naked DNA donors. ![]() Cas9 ribonucleoprotein electroporated into cells 24 hours after transduction not only creates a double-stranded break in the genome that induces HDR, but also enables linearization and processing of the reverse-transcribed DNA template, which can greatly improve transgene knock-in efficiency. We engineered the IDLV genome to include CRISPR ‘cut sites’ that match the intended genomic cut site (Fig. Integrase-deficient lentivirus (IDLV) enables reverse transcription of an encoded payload from an RNA molecule into a DNA template, which creates a useful yet low-efficiency donor for HDR. However, the most widely utilized DNA donors - adeno-associated virus (AAV) vectors 3 and naked DNA templates 4 - are restricted by limited payload size or high cytotoxicity in primary cells, which limits their use for synthetic biology and manufacture of cell-based therapies. Homology-directed repair (HDR) could be leveraged to overcome gene silencing through targeted knock-in of payloads into endogenous genomic loci whose expression is essential to cell survival (termed essential genes), thereby using the natural genomic context to stabilize transgene expression. However, this methodology often succumbs to gene silencing (an umbrella term for multiple cellular processes that result in the loss of transgene expression over time) 2. Engineered pseudovirus vectors based on lentivirus or gamma retrovirus are leveraged for semi-random insertion of genetic payloads into genomes to enable transgene expression over multiple generations. Technologies such as engineered receptors, clustered regularly interspaced short palindromic repeats activation (CRISPRa) or CRISPR inhibition (CRISPRi) systems, gene circuits, and cell-based vaccines have created a need for stable, long-term expression of large transgenes in primary human cells 1.
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