Part:BBa_K2807002

PspCas13b

PspCas13b is a CRISPR-associated protein originating from Prevotella sp and can be expressed in mammalian cells to target and cleave messenger RNA with compatible guide RNA. The sequence also contains a nuclear export signal (NES) and a 3x hemagglutinin (HA) tag.

Expression of Cas13b in mammalian cells

To verify the expression of the Cas13b part in the mammalian system, HEK293T cells were transfected with px330A-PspCas13b using Lipofectamine 2000. Cas13b is able to target and cleave messenger RNA strands with the aid of a compatible guide RNA, unlike in dCas13b, whereby the nuclease activity 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 __: 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 3, the appearance of a band between 150 kDa and 100 kDa showed that both Cas13b and dCas13b were 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2884
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1787
  Illegal BamHI site found at 839
  Illegal BamHI site found at 3271
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1520
  Illegal AgeI site found at 2396
  
• 1000
  COMPATIBLE WITH RFC[1000]