Part:BBa_K2429011
pENTR L. shahii NL-dCas13a-NL
This part includes the deactivated Leptotrichia shahii Cas13a protein coding region. This basic part is a pENTR vector that serves as an intermediate in the production process of a final expression vector. The final expression vector produces a deactivated version of the CRISPR protein Cas13a, which normally binds and cuts mRNA. With the deactivation sequence, the protein becomes catalytically inactive, and unable to cut the mRNA; however, it can still bind to the mRNA molecule. Without the deactivation sequence, a CRISPR protein known as Cas13a would be produced, which binds and cuts mRNA. Upon recognition of the mRNA, this protein exhibits "promiscuous" ribonuclease activity, not only cutting at base pairs along the targeted RNA molecule, but other nearby RNA molecules as well.
Flanking the Cas13a coding sequence are a pair of nuclear localization sequences (NLSs) The sequences code for signaling peptides that indicates that the remaining portion of the translated protein will remain in the cell (specifically in the nucleus) rather then get excreted by the cell. The translated amino acid sequence of the NLS is PKKKRRV and classifies as a classic nuclear localization sequence (cNLS). Such sequences are considered the best characterized transport signal [1].
Typically, during the process of alternative splicing in eukaryotic cells, specific RNA binding proteins will bind to sequences or motifs on a pre-mRNA strand and eventually come together to form a spliceosome protein. This resulting protein will cleave the mRNA strand to exclude portions of the pre-mRNA (called introns) and the remaining sequences in the mature mRNA are known as exons.
Our team used this protein in an attempt to control what exons would be included in an mRNA transcript by targeting the motifs in an intron, thus blocking splicing factors from binding and retaining an exon. Furthermore, our team tested variations of this protein (e.g. catalytically deactivated, additional domains) to see whether such variations would affect the splicing capabilities. This specific variation of the protein is catalytically active in bacteria; however, since we used it in mammalian cells, the protein's endonuclease activity isn't expected to be seen. Additionally, the focus is on the protein's binding and blocking ability rather than the catalytic activity. The nuclear localization sequence is to ensure the resulting Cas13a protein will exist in the nucleus, and increase the chances of interacting with mRNA transcripts.
[1] Lange, et al. "Classical Nuclear Localization Signals: Definition, Function, and Interaction with Importin α*"
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 4422
Illegal PstI site found at 1903
Illegal PstI site found at 2272
Illegal PstI site found at 3001 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4422
Illegal PstI site found at 1903
Illegal PstI site found at 2272
Illegal PstI site found at 3001 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4422
Illegal BglII site found at 647
Illegal BglII site found at 1271
Illegal BglII site found at 1667
Illegal BglII site found at 1958
Illegal BglII site found at 2048
Illegal BglII site found at 3209
Illegal BglII site found at 3296
Illegal BamHI site found at 1
Illegal BamHI site found at 70
Illegal BamHI site found at 4291 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 4422
Illegal PstI site found at 1903
Illegal PstI site found at 2272
Illegal PstI site found at 3001 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 4422
Illegal PstI site found at 1903
Illegal PstI site found at 2272
Illegal PstI site found at 3001
Illegal NgoMIV site found at 4248
Illegal NgoMIV site found at 4267 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 633
Illegal SapI.rc site found at 739
Illegal SapI.rc site found at 1609
Illegal SapI.rc site found at 2443
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