Part:BBa_K1179002

Hef1A_Cas9-VP16

This part encodes for the constitutive expression of a Cas9-VP16 fusion protein. The Cas9 has been mutated in such a way as to remove its nuclease activity while retaining its ability to selectively bind DNA mediated by an appropriate guide RNA (gRNA). The Cas9-VP16 complexes with an expressed gRNA and together target a DNA sequence as defined by the gRNA. Once the Cas9-VP16 is bound, the VP16 domain can activate a downstream inducible promoter like minimal CMV.

Overview of Cas9

CRISPR is the defense system of bacteria and some organisms against invading DNAs. Essentially, when an invading DNA enters bacteria, part of its DNA is incorporated in bacterial genome and later gets transcribed as an spacer sequences in an RNA called per-crRNA. In the type II CRISPR system that we used in our study, this primary transcript in the presence of another trans encoded RNA (tracrRNA) gets processed to smaller RNA called crRNA that bares complementary sequences against invading DNA. The combinations of two RNAs bind to a protein called Cas9 which is an RNA binding DNA nuclease and guide Cas9 to the invading DNA to cleave and degrade it. In our study, we mutated Cas9 nuclease and made it catalytically inactive. It can bind to RNA and be targeted to DNA but doesn't cleave it. We also got inspired by recent publications that have used Cas9 for genetic engineering and instead of two required RNAs used one chimeric RNA that contains sequences from both crRNAs and tracrRNA called guide RNAs. The Cas9-gRNA complex then scans the DNA for a sequence complementary to the 5' nucleotides of the complexed guide RNA. Once a complementary region has been found, the Cas9 stops and binds to that location.