Part:BBa_K2429183
L. shahii Cas13a BioBrick
This is the BioBrick version of another part (BBa_K2429006) that includes the Leptotrichia shahii Cas13a protein coding region. The part produces a CRISPR protein known as Cas13a, 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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 572
Illegal BglII site found at 1196
Illegal BglII site found at 1592
Illegal BglII site found at 1883
Illegal BglII site found at 1973
Illegal BglII site found at 3134
Illegal BglII site found at 3221
Illegal BamHI site found at 4216 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4173
Illegal NgoMIV site found at 4192 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 558
Illegal SapI.rc site found at 664
Illegal SapI.rc site found at 1534
Illegal SapI.rc site found at 2368
| None |