Encyclopedia of Experiments: Immunology
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Transcript
Begin with a suspension of T cells and treat them with CAR lentiviruses carrying a gene for the chimeric antigen receptor or CAR and CRISPR lentiviruses.
The CRISPR lentivirus contains genes for the guide RNA and Cas9 nuclease as well as an antibiotic resistance gene.
Both the CAR and CRISPR lentiviruses interact with T cells and release their RNA.
These RNAs are reverse-transcribed and converted into corresponding DNA, which integrates into the T cell DNA.
This enables the synthesis of CAR proteins, guide RNAs, and Cas9 proteins.
The guide RNA forms a complex with Cas9, which specifically recognizes and binds to the target gene on the T cell DNA.
Meanwhile, Cas9 cleaves the DNA, creating a double-stranded break.
This break is repaired via non-homologous end joining, an error-prone repair mechanism, resulting in target gene modification.
This generates genetically modified T cells with chimeric antigen receptors.
Treat the cells with antibiotics to eliminate the unmodified T cells and selectively isolate the genetically modified CAR T cells.
