A multitude of genetically inherited and acquired diseases can be traced back to faulty or altered mRNA expression in eukaryotic cells. (1) To be able to treat these diseases and develop efficient therapeutic agents, the exact molecular mechanisms underlying faulty mRNA synthesis and export must be elucidated. Furthermore, studies of healthy cell proliferation and differentiation also indicate a degree of subcellular localization, which yet has to be investigated further to be explained and understood.(2) Our CRISPR/dCas13a construct functions as an “mRNA localization device” to investigate the fate of mRNA in vivo.
Cas13a is a RNA guided endonuclease which degrades RNAs based on alignment of its CRISPR derived crRNA. We used dead Cas13a with four mutations, two in HEPN1 and two in the HEPN2 domains, which abort its nuclease activity, but maintain its helicase properties, resulting instead in attachment of dCAS13a to the targeted RNA sequence.
Simultaneously, guide RNAs constructs (for crRNA expression) destined for incorporation into the bacterial genome within CRISP repeats was designed. These constructs contain a U6 promoter followed by coding sequences for human β-Actin, pkp4, inpp-1, and Rab13 (individually) and terminated by an oligo (T) repeat. The protein sequences are chosen based on susceptibility to mutations and expression in disease states.
With the aim to investigate and track mRNA localization in vivo, we engineered a gene construct containing a GFP attached via a linker sequence, allowing conformational flexibility, to a dCas13a coding gene. Additionally, two Nuclear localization sequences are on either side of the construct, which navigate the fusion protein back to the cell nucleus after protein synthesis in the ER is completed. In the nucleus, the protein construct recognizes the CRISPR incorporated sequence and attaches to it. When the targeted mRNA is consequently exported, the GFP+dCas13a tags along, allowing the ‘tracking’ of mRNA up until its translation in the ER. The unbound fusion protein constructs are retained in the nucleus and do not localize within the cell cytoplasm, reducing background noise.