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The exact protocol for lentiviral production will vary depending on the research aim and applied library. [ 35 ] [ 43 ] [ 44 ] If a two vector-system is used, for example, cells are sequentially transduced with Cas9 and sgRNA in a two-step procedure.
Lentiviral vectors in gene therapy have been pioneered by Luigi Naldini. [3] [4] Structure of a virion of HIV, a type of lentivirus. A membrane with protruding glycoproteins surrounds a capsid containing enzymes and the viral RNA genome. The lentivirus is a retrovirus, meaning it has a single stranded RNA genome with a
Knock-out mutations caused by CRISPR-Cas9 result from the repair of the double-stranded break by means of non-homologous end joining (NHEJ) or POLQ/polymerase theta-mediated end-joining (TMEJ). These end-joining pathways can often result in random deletions or insertions at the repair site, which may disrupt or alter gene functionality.
[1] [2] [3] Perturb-seq combines multiplexed CRISPR mediated gene inactivations with single cell RNA sequencing to assess comprehensive gene expression phenotypes for each perturbation. Inferring a gene’s function by applying genetic perturbations to knock down or knock out a gene and studying the resulting phenotype is known as reverse genetics.
CRISPR-based gene knockout is a powerful tool for understanding the genetic basis of disease and for developing new therapies. It is important to note that CRISPR-based gene knockout, like any genetic engineering technique, has the potential to produce unintended or harmful effects on the organism, so it should be used with caution.
Lentivirus (upper panel) – To produce lentiviruses with the gene of interest as the lentiviral DNA construct, first transfect cells with a packaging plasmid and the envelope vector (VSVG). Adeno Associated Virus (AAV) (lower panel) – To produce AAV, package a gene of interest into the AAV-ITR vector and transfect cells with a Helper vector ...