Part:BBa_K3201005

S.mansoni Type 1 Hammerhead Ribozyme, Cpf1 Optimised

This is part BBa_K3201002 with the alteration of some bases so that it is compatible with the Aptamer Basic Cassette part. It can be recognised by the Cpf1 Cas12 as a part of our CIDER mutagenesis system.

Usage and Biology

This part is a modified hammerhead ribozyme (HHR) with the addition of a T in the DNA sequence which creates a PAM for Cpf1. We have computationally modelled the molecule to determine its structure. The structure of the Cpf1 optimised HHR is shown in Figure 1 below.

Figure 1: Cpf1 optimised HHR.

We also compared its structure to the original Hammerhead ribozyme (BBa_K3201002) through superimposing the two structures. The result is shown in Figure 2 below.

Figure 2: Superimposed Cpf1 optimised HHR and original HHR. Blue line: Cpf1 optimised HHR. White/brown line: Original HHR

As it can be seen, the structures do not vary significantly. This suggested that the Cpf1 optimised HHR should retain its catalytic function.

We assessed this through an in vitro HHR cleavage assay. First, we produced sufficient amounts of the ribozymes through in vitro transcription. We incorporated a T7 promoter and used the in vitro transcription kit from NEB (T2050S). The in vitro transcription products were run in a 1% agarose gel and the result is shown in Figure 3.

Figure 3: In vitro transcribed Cpf1 optimised HHR. Lane 1: Ladder 1kb Plus (NEB). Lane 2: In vitro transcribed product

Then we prepared the in vitro cleavage assay. In an eppendorf tube the following were added: 0.5 μL of in vitro transcribed HHR, 0.5 μL of 1 M Tris-HCl (final concentration 50 mM), 0.3 μL of 1 M MgCl2 (final concentration 30 mM), and 8.7 μL of nuclease-free water. The mixture was incubated in an incubator at 37 degrees Celsius for 1 hour. After 1 hour, the reaction was stopped using 10 μL of 6X loading dye. The mixture was then heated at 70 degrees Celsius for 5 minutes and then loaded on a gradient (4-20%) polyacrylamide gel. Before electrophoresis, the gel was prerun for 30 mins at 150V. The PAGE was done using 1X TBE as the running buffer and at a constant voltage of 150 V. The gel was stained overnight in Ethidium Bromide. The result is shown in Figure 4.

Figure 4: In vitro cleavage assay. The Cpf1 optimised HHR behaves similarly to the original HHR.

As it can be seen, the addition of a single base does not affect the function of the HHR and therefore it can be used in order to target Cpf1 to this locus.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]