Part:BBa_K2807001
dPspCas13b
Usage and Biology
dPspCas13b is a CRISPR-associated protein originating from Prevotella sp and can be expressed in mammalian cells to target messenger RNA with compatible guide RNA. RNAse activity of dPspCas13b is inactivated through a mutation of catalytic histidine residues (CAC to GCA/histidine to alanine mutations) in the HEPN domains (Tambe et al., 2018). To note, dPspCas13b is also a novel part in the iGEM Biobrick Parts Registry. The sequence also contains a nuclear export signal (NES) and a 3x hemagglutinin (HA) tag.
Characterisation
Expression of dCas13b in mammalian cells
To verify the expression of the dCas13b part in the mammalian system, HEK293T cells were transfected with px330A-dPspCas13b using Lipofectamine 2000. Unlike Cas13b that is able to target and cleave messenger RNA strands with the aid of a compatible guide RNA, the nuclease activity in dCas13b has been inactivated through the mutation of the enzymatic catalytic sites. An inactivated form of Cas13b is important for our RESCUE editor as we aim to make a site-directed change rather than cleave the target strand.
Transfected cells were lysed and cell extract was separated on an SDS-PAGE gel. As our PspCas13b and dPspCas13b includes an HA epitope tag at the C-terminus, we were able to detect for either Cas13b and dCas13b protein using a monoclonal HA antibody. As Cas13b and dCas13b are 3402 base pairs long, we expect a protein size of approximately 124 kDa to be expressed.
Figure 1: Western blot of PspCas13b and dPspCas13b. Both Cas13b and dCas13b are approximately 124 kDa and can be identified as a band between 150 kDa and 100 kDa.
From Figure 1, the appearance of a band between 150 kDa and 100 kDa showed that dCas13b was expressed at the right molecular weight. However, we note that the different intensities in protein expression across the three replicate samples may be attributed to variation in transfection efficiency across each sample.
References
Tambe, A., East-Seletsky, A., Knott, G. J., Doudna, J. A., & O’Connell, M. R. (2018). RNA-binding and HEPN-nuclease activation are decoupled in CRISPR-Cas13a. Cell Reports, 24(4), 1025–1036. http://doi.org/10.1016/j.celrep.2018.06.105
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2884
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1787
Illegal BamHI site found at 839
Illegal BamHI site found at 3271 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1520
Illegal AgeI site found at 2396 - 1000COMPATIBLE WITH RFC[1000]
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