Caption for Figures
Figure 1. CRISPR/Cas9 mechanism of action . The type II CRISPR system is armed with CRISPR-associated protein, Cas9, that has two distinct domains named HNH-nuclease and RuvC-like nuclease, which produce double-stranded breaks (DSBs) in DNA of interest. The trans-activating CRISPR RNA (tracrRNA) and crRNA combine and form a single-guided RNA (sgRNA), a 17-20 nucleotide sequence that binds to the target DNA. sgRNA has a PAM sequence after the 3′ end of its sequence, guiding the Cas9 protein to generate DSBs in desired DNA. Next, DSBs undergo two different mechanisms of repairs, homologous directed repair (HDR) suitable for knocking in a specific donor DNA in the target DNA, and the non-homologous end joining (NHEJ) ideal for knocking out the target DNA.
Figure 2. The general workflow for CRISPR Cas9-based CAR T-cell manufacturing . Peripheral blood mononuclear cells (PBMCs) obtain from the patient. Anticoagulants, red blood cells, and platelets contaminate the product would be removed in a washing step. Afterward, enrichment or depletion processes would be done for specific cell subsets. Next, T cells get activated by using different procedures, including monoclonal antibodies with interleukins (IL-2, IL-7, 1L-15), anti-CD3/CD28 antibody-coated magnetic beads, soluble CD3 antibody, artificial antigen-presenting cells (K562 cell lines), plate-bound antibody, and adhesion molecules (CD2). T cell activation pathways in cell culture media provide both the primary and co-stimulatory signals required to activate the desired T cells. In this stage, CAR transgene may be delivered into the activated T cells by different approaches, including viral (lentivirus and retrovirus) and non-viral (electroporation of naked DNA and transposon/transposase) methods or CRISPR/Cas9 system may be applied first into the T cells to target the gene of interest. Subsequently, the activated T cells undergo an expansion process for a certain period (depending on the methods of expansion, such as using static culture bags or dynamic culture vessels or rotating bioreactors). If CAR transgene first introduced into the T cells with no CRISPR/Cas9 transformation, (a), then the modified T-cells will be introduced with CRISPR/Cas9 components, (b), to target the DNA of interest in CAR T cells. There are different approaches to deliver CRISPR/Cas9: (I) transfection with DNA plasmid encoding both Cas9 protein and sgRNA, (II) the viral delivery using lentivirus and retrovirus, and non-integrating viruses such as adenovirus and adenovirus-associated virus (AAV), (III) transfection with mRNA that encodes Cas9 or separate sgRNA, and (IV) CRISPR delivery via Cas9 protein with guide RNA (ribonucleoprotein (RNP) complex). Finally, the prepared modified T-cells are calculated according to the patient’s condition and type of cancer, then ready to introduce the engineered T cells to the patient through IV injection or intratumoral administration.
Figure 3. Representation of CRISPR-edited CAR-T cell . CRISPR/Cas9 genome editing technology improved CAR-T cells performance in various ways, including targeting the Diacylglycerol kinases (DGKs) gene, and knocking out the TCR, HLA, and inhibitory receptors.