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.