CRISPR

Clustered regularly interspaced short palindromic repeats (CRISPR) is quickly becoming the most popular tool in the genome editing arena. CRISPR has been adapted for use in mammalian cells from bacterial anti-bacteriophage immune systems.

The TriLink CRISPR tool kit includes two components: the first is a CRISPR nuclease encoded by a Cas9 or Cas12a mRNA, and the second is a guide RNA. In nature, the Cas9 guide consists of a CRISPR RNA (crRNA), as well as a trans-acting CRISPR RNA (tracrRNA). For simplicity and increased potency, researchers fused these two RNAs into a single RNA of ~100 nucleotides, which they called a single guide RNA (sgRNA). In contrast to Cas9, Cas12a utilizes a single guide RNA of ~42 nucleotides.

When Cas9 or Cas12a mRNA and a sgRNA are co-transfected into cells, the guide strand directs the Cas9 or Cas12a protein to a specific location in the genome, where Cas9 then creates a double stranded break. Cas9 Nickase mRNA encodes Cas9 with a D10A mutation, which causes a single stranded nick instead of a double stranded break. This may result in fewer off-target effects.

Chimeric antigen receptor (CAR) T cells have been used for 30 years as one of the Adoptive T cell therapy (ACT) immunotherapy methods for clinical treatment of hematological cancers. More recently, they have also been applied to solid tumors.

A CAR construct typically has been modified to contain a single chain antibody fragment (scFv) binding domain, a transmembrane domain, and the ability to activate T cells through intracellular signaling domains independent of Major Histocompatibility Complexes (MHCs). The modified T cells express CARs on the cell surface that recognize tumor-associated antigens during treatment.

The source material for most CAR T cells is collected from the patient to be treated (autologous transplant). Harvested cells are expanded and modified to express the CAR. CRISPR is often used to site specifically insert at the TRAC (T-cell receptor α constant) locus, replacing the endogenous T-cell receptor. While autologous transplants avoid rejection and graft-versus-host disease, these cells are often from very ill patients and may thus be less potent than cells harvested from healthy donors. An alternative universal, off-the-shelf solution uses modified cells from healthy, allogeneic donors. This offers a pathway to overcome prohibitive costs, minimize manufacturing complexities, shorten the timeline to treatment, and address patient health and biosafety issues.

However, to prevent rejection of these foreign cells, extensive gene deletions via CRSIPR are required. CRISPR-Cas engineering offers next-generation CAR T cell solutions that can silence or disrupt any desired genomic locus, minimize graft-versus-host disease (GvHD) and rejection issues, knock out checkpoint receptors, enhance antitumor responses, and more. Today’s CRISPR-Cas engineered CAR T cells have therapeutic potential in treating infectious disease, autoimmune disease, and promoting transplantation tolerance.

TriLink offers several mRNAs for CRISPR that are unmodified or modified with 5-methoxyuridine (5moU) to reduce innate immune responses. With the help of leading researchers in the field, these have been sequence optimized for optimal expression. TriLink also manufactures zinc-finger nuclease, and Transcription activator-like effector nuclease (TALEN) mRNAs, which are used to produce CAR T-cell therapies. When you are ready to move your research to the clinic, TriLink offers cGMP synthesis of guide strands and nuclease mRNAs in our cGMP facility.

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