How to perform a CRISPR knockout experiment?

Using CRISPR Cas9 gene editing two types of gene edits can be made, namely knockouts and knock-ins. In a gene knockout experiment, a double-strand break is introduced by the Cas9 nuclease which leads to Non-homologous end joining of the DNA, and hence insertions or deletions of bases can take place. Due to insertions or deletions in knockout systems, frameshift mutations take place and hence lead to the formation of nonfunctional products. In Knock-in experiments the double-strand break by the Cas9 nuclease is repaired by homologous recombination, hence if a homologous DNA with flanking genes is introduced then that gene might get incorporated into the system forming a-knockin.

Steps to do a CRIPSR knockout:

1) Design a gRNA for the target gene sequence.

2) The synthesized gRNA is synthesized and cloned to a gRNA vector. Transfection grade plasmids are generated and cells are cotransfected with gRNA plasmid and cas9 plasmid. The cells are sanger sequenced to determine the number and type of mutations generated by the gRNA. If it is clear that cells are capable of knock out function then cells are taken forward.

3) The cells with knockout function are taken for single clone generation. Each clone is sanger sequenced and ones that contain frameshift mutations are selected forward.