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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.
The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated nucleases) system was originally discovered to be an acquired immune response mechanism used by archaea and bacteria. It has since been adopted for use as a tool in the genetic engineering of higher organisms.
Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses CRISPR sequences as a guide to recognize and open up specific strands of DNA that are complementary to the CRISPR sequence. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within living organisms.
CRISPR-Cas9 genome editing techniques have many potential applications. The use of the CRISPR-Cas9-gRNA complex for genome editing [10] was the AAAS's choice for Breakthrough of the Year in 2015. [11] Many bioethical concerns have been raised about the prospect of using CRISPR for germline editing, especially in human embryos. [12]
Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic engineering applications.
Another challenge associated with this protocol is the creation of large scale CRISPR libraries. The preparation of these extensive libraries depends upon a comparative increase in the resources required to culture the massive numbers of cells that are needed to achieve a successful screen of many perturbations.
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.
CRISPR/Cas9 edits rely on non-homologous end joining (NHEJ) or homology-directed repair (HDR) to fix DNA breaks, while the prime editing system employs DNA mismatch repair. This is an important feature of this technology given that DNA repair mechanisms such as NHEJ and HDR, generate unwanted, random insertions or deletions (INDELs).
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