text: Targeted gene knockout using CRISPR/Cas9 requires the use of a delivery system to introduce the sgRNA and Cas9 into the cell. Although a number of different delivery systems are potentially available for CRISPR, genome-wide loss-of-function screens are predominantly carried out using third generation lentiviral vectors. These lentiviral vectors are able to efficiently transduce a broad range of cell types and stably integrate into the genome of dividing and non-dividing cells.
  Third generation lentiviral particles are produced by co-transfecting 293T human embryonic kidney (HEK) cells with:
  # two packaging plasmids, one encoding Rev and the other Gag and Pol;
  # an interchangeable envelope plasmid that encodes for an envelope glycoprotein of another virus (most commonly the G protein of vesicular stomatitis virus (VSV-G)); 
  # one or two (depending on the applied library) transfer plasmids, encoding for Cas9 and sgRNA, as well as selection markers.
  The lentiviral particle-containing supernatant is harvested, concentrated and subsequently used to infect the target cells. The exact protocol for lentiviral production will vary depending on the research aim and applied library. If a two vector-system is used, for example, cells are sequentially transduced with Cas9 and sgRNA in a two-step procedure. Although more complex, this has the advantage of a higher titre for the sgRNA library virus.