Commonly used Vector Systems Copy

Transcript:

Current advances in genome editing have accelerated research in biotechnology, genetics, therapeutics, and plant biology. With a relatively easy and straight-forward approach, CRISPR/Cas9 has become a popular genome editing tool. The key step of genome editing tools such as CRISPR/Cas9 is the introduction of double strand breaks (DSBs) in the DNA of the cells where editing is to be performed. To achieve this, the Cas9 along with the sgRNA has to be inserted into the target cells. One of the commonly used methods to insert the CRISPR/Cas9 components involves the use of viral vectors such as retroviral, lentiviral, and adeno-associated viral vectors.

Viruses have naturally evolved with robust capabilities of transducing cells efficiently with foreign nucleic acid molecules. This capability has attracted the attention of researchers to use viruses as vectors for gene delivery. The genome of the wild type virus is replaced with the engineered transgene of interest. This is usually achieved by co-transfecting cells with several plasmids. One of the plasmids contains the transgene of interest to be delivered by the vector. The others contain genes encoding virion proteins such as reverse transcriptase and capsid that are essential for vector formation.

Upon entering the host cells through virus-specific mechanisms, viruses such as retroviruses and lentiviruses initiate reverse transcription reaction to synthesise the proviral or cDNA which gets incorporated into the host cell genome. Thereafter, the cDNA undergoes replication along with the host DNA and gets passed onto the progeny of the cell. 

Lentiviruses such as HIV1 are capable of importing the viral genetic material directly across the host cell membrane by exploiting the host-protein machinery. HIV-based vectors are capable of transducing most cell types with high efficiency. They can also transduce non-dividing cells and those that have terminally differentiated. Such viral vectors can sustain stable and long-lasting transgene expression.

Adeno-associated viral (AAVs) vectors are also commonly used vectors in genome editing. The recombinant AAVs are derived from the wild type AAVs containing genomes that encode eight proteins (Rep proteins, long Rep78/68 isoforms, short Rep 52/40 isoforms, and structural capsid proteins). Of them, the long Rep78/68 isoforms are responsible for the replication and integration of the viral genome into the host cell. The AAV vector enters the host cell through receptor-mediated endocytosis.

Both lentivirus and AAV have a relatively low risk of adverse effects arising from insertional mutagenesis and are known to cause only mild immune response in humans. However, retroviruses have been associated with adverse effects such as T-cell leukaemia in retroviral vector-based gene therapy. The risk of carcinogenesis and immune response, along with limited packaging size and difficulty in large-scale production has triggered the development of non-integrating lentiviral vectors and non-viral vectors such as peptide-based nanocomplexes, lipid, and polymer-based nanocarriers.