Lentivirus Production For CRISPER-Cas9 Delivery

Introduction

The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA.

 CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in the human body. 

Lentivirus-mediated delivery of CRISPR-Cas9

Lentivirus-mediated delivery of CRISPR-Cas9 is a robust tool to perform function-based screening in mammalian cells and generate knockout animal models. In general, a pool of sgRNA-expressing lentiviruses is used to generate a library of knockout cells for screening under positive and negative selections 

Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes of interest driven by promoter sequences contained within the vector.

 This technology can be used as a standard method for the production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system.

 Lentivirus-mediated CRISPR-Cas9 system eliminates the HIV-1 DNA from CD4+ T cells in host patients, preventing the re-emergence of HIV. In addition, lentivirus-mediated CRISPR-Cas9 gene therapy demonstrated promising results in the treatment of chronic hepatitis B virus (HBV). Because the persistence of viral episomal DNA leads to HBV infection, the main strategy is to cleave viral episomal DNA and inhibit HBV replication by lentivirus-mediated delivery of the CRISPR-Cas9 system.

 Engineered lentiviral particles can deliver Cas9 RNP complexes for genome editing, either tracelessly or while simultaneously integrating a lentiviral-encoded transgene (Cas9-VLPs) in immortalized cell lines and primary human T cells. 

Treatment of primary human T cells with Cas9-VLPs packaging a lentiviral-encoded CAR resulted in the simultaneous knockout of genetic targets relevant to allergenic T cell production while effectively mediating CAR expression, an approach that was amenable to multiplexing. 

In addition, the treatment of T cells with broadly transducing Cas9-VLPs resulted in a targeted genetic knockout in CD4+ and CD8+ T cells, while treatment with Cas9-VLPs pseudo typed with the CD4-tropic HIV-1 envelope glycoprotein drove the exclusive transduction and genome editing of CD4+ T cells within a mixed cell population. 

Engineering lentivirus-based VLPs for the controlled delivery of Cas9 RNP complexes

➢Lentiviral production involves the co-transfection of producer cells with plasmids encoding the viral structural components, viral glycoprotein, and lentiviral transfer plasmid with a transgenic sequence flanked by long terminal repeat (LTR) sequences. 

➢To promote the packaging of Cas9 protein in HIV-1 VLPs (Cas9-VLPs), we constructed a plasmid to express Streptococcus pyogenes Cas9 fused to the C terminus of the Gag polypeptide and included this during lentiviral production. 

➢To promote the separation of Cas9 from Gag during proteolytic virion maturation, we inserted an HIV-1 protease-cleavable linker between Gag and Cas9.

➢A component of the Gag polypeptide, capsid (CA), was used for quantifying Cas9-VLP production by ELISA. 

Advantages

•High-efficient Cas9 expression delivery with markers

•No need for tedious cloning work or vector construction

•Best nuclear penetrating for Cas9 enzyme

•Allow multiple gene editing at the same time

Features of Our Services

•Fast delivery

•Flexible modification approaches available

•Comprehensive understanding of peroxisomes proteins manufacturing; 

•Quick completion of any project.

•Good quality, yields, concentrations, and purity.

•Client satisfaction

•Confirmation of the accuracy

•A more reasonable cost

Deliverables

○The modified CRISPER-Cas9 is also shipped to our customers for them to test internally.

○The customer is sent the entire modified CRISPER-Cas9 in some phases.

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