TIGIT CRISPR/Cas9 Lentivirus (Non-Integrating)

Catalog #

78065

$995 *

Size: 500 µl x 2

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Bulk/Custom

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Description

TIGIT (T-cell immunoreceptor with Ig and ITIM domains; VSTM3; VSIG9) is a co-inhibitory receptor that is highly expressed in Natural Killer (NK) cells and activated CD4+, CD8+, and regulatory T-cells. Interaction with the Poliovirus Receptor (PVR; CD155) on antigen presenting cells, such as dendritic cells, recruits either the Src homology (SH) domain-containing tyrosine phosphatases SHP1 and SHP2, or the Inositol phosphatase SHIP1 and SHIP2, to the TIGIT ITIM domain. This increases IL-10 release and suppresses NF-κB and NFAT T-cell receptor (TCR) signaling, which blocks T-cell proliferation and cytokine production. TIGIT also serves as a competitive inhibitor of CD226, a costimulatory receptor for CD155. TIGIT-targeting antibodies which block this T-cell intrinsic inhibitory effect have shown enhanced anti-tumor and anti-viral functions in preclinical studies.

The TIGIT CRISPR Lentiviruses are replication incompetent, HIV-based VSV-G pseudo-typed lentiviral particles that are ready to be transduced into almost all types of mammalian cells, including primary and non-dividing cells. The particles contain a CRISPR/Cas9 gene driven by an EF1A promoter, along with 4 sgRNA (single guide RNA) targeting human TIGIT (GenBank Accession #NM_173799) driven by a U6 promoter (Figures 1 and 2).

Figure 1. Schematic of the Lenti-vector used to generate the TIGIT CRISPR/Cas9 Lentivirus.

Figure 1. Schematic of the Lenti-vector used to generate the TIGIT CRISPR/Cas9 Lentivirus.

Gene Target:	Primer ID:	sgRNA Sequence
TIGIT	TIGIT-1	CATCTGCACAGCAGTCATCG
TIGIT	TIGIT-2	CAGGCACAATAGAAACAACG
TIGIT	TIGIT-3	GCTGACCGTGAACGATACAG
TIGIT	TIGIT-4	ACCCTGATGGGACGTACACT

Figure 2. List of sgRNA Sequences in the TIGIT CRISPR/Cas9 Lentivirus.

Note: unlike human TIGIT CRISPR/Cas9 Lentivirus (Integrating) (BPS Bioscience, #78058), the human TIGIT CRISPR/Cas9 Lentivirus (Non-Integrating) is made with a mutated Integrase, resulting in only transient expression of the Cas9 and TIGIT targeting sgRNA. While this may minimize potential off-targeting risks due to either prolonged expression or integration of the Cas9, puromycin selection should not be used for more than 48 hours post-transduction, which may lower knockout efficiency.

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Synonyms

VSTM3 lentivirus, VSIG9 CRISPR

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Product Info

Storage and Usage

Citations

Species

Replication incompetent HIV

Supplied As

Two vials (500 μl x 2) of lentivirus at a titer ≥1 x 10⁶ TU/ml. The titer will vary with each lot; the exact value is provided with each shipment.

Formulation

The lentiviruses were produced from HEK293T cells in medium containing 90% DMEM + 10% FBS.

Storage/Stability

Lentiviruses are shipped with dry ice. For long-term storage, it is recommended to store the lentiviruses at -80°C for up to 12 months from date of receipt. Avoid repeated freeze/thaw cycles. Titers can drop significantly with each freeze/thaw cycle.

Instructions for Use

See assay protocol for detailed instructions.

Applications

1. Transient knock-down of TIGIT in a target cell pool.
2. Generation of stable TIGIT knock-out cells using transient puromycin selection (48h maximum) followed by limited dilution.

Shipping Temperature

-80°C

Notes

The CRISPR/CAS9 technology is covered under numerous patents, including U.S. Patent Nos. 8,697,359 and 8,771,945, as well as corresponding foreign patents applications, and patent rights.

Warnings

Biosafety
None of the HIV genes (gag, pol, rev) will be expressed in the transduced cells. Although the pseudotyped lentiviruses are replication-incompetent, they do require the use of a Biosafety Level 2 facility. BPS recommends following all federal, state, local, and institutional regulations and using all appropriate safety precautions.

Scientific Category

Immunotherapy

Regulatory Status

Research Use Only

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TIGIT CRISPR/Cas9 Lentivirus (Non-Integrating)

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