"Nothing resembles the joy of when a gel showing correctly assembled DNA."
To make a functional test, both the Cas13a and Cas12a proteins had to be synthesized which was carried out in E. coli. The specific genes for cas13a and cas12a that we used were derived from Leptotrichia wadei and Acidaminococcus sp., respectively. We codon optimized the genes for expression in E. coli with IDT’s codon optimizing tool and included features that allowed for the inducible gene expression. Both the cas13a (BBa_K3602001) and the cas12a (BBa_K3602015) genes contain an IPTG inducible promoter region (BBa_R0011), a ribosomal binding site (BBa_0034), and a terminator (BBa_B1006). Additionally, both genes have a 6x-histidine and SUMO-tag (Small Ubiquitin-like modification protein) N-terminally, for subsequent protein purification and TEV cleavage.
Figure 1. An overview of the significant features of our Cas13a (left) and Cas12a (right) biobricks designed with SnapGene.
Due to its high complexity, IDT was unable to synthesize the entire cas13a gene. So, we divided the original Cas13a construct into three parts: Cas13a-1 (approx. 1400 bp), Cas13a-2 (approx. 2000 bp) and Cas13a-3 (approx. 700 bp).
Moreover, we were unable to order the cas12a gene from IDT, as shipment of the gene outside of the United States required an export license. The research unit “PhyLife”, from the University of Southern Denmark was, however, working with the gene and kindly offered us a plasmid containing the cas12a gene. This gene was not codon optimized for E. coli and did not contain the desired promoter region or terminator sequence, see Parts.
Figure 2. Assembly of Cas proteins by way of primer overhangs. Above, Cas13a is seen assembled in 3 parts, with a promoter region belonging to Cas13a-1. This was amplified from the gene fragment and assembled onto Cas12a from PhyLife. Primer and gene fragment length not to scale. Created with BioRender.com
We assembled Cas13a using PCR amplification, by designed primers that generated overhang between the separate gene parts. The forward overhang primer for Cas13-1 and the reverse Cas13a-3 overhang primer were designed to include an EcoRI-site and SpeI-site, respectively. These restriction sites allowed for digestion and ligation of the gene into the backbone of choice.
As previously mentioned, the obtained Cas12a did not have our desired promoter region. Instead we designed primers to amplify these features from the Cas13a-1 gene and attaching them to Cas12a with PCR. Finally, we cloned the cas genes into pSB1C3, which carries chloramphenicol resistance for selection of successful transformants. The newly synthesized plasmids, encoding Cas13a and Cas12a were transformed into the ER2566 strain of E. coli, making the genes suitable for protein expression.
Figure 3. Agarose gel run with backbone + terminator and gene inserts, Cas13a and Cas12a. Bands at 2000 bp indicate linear backbone + terminator, bands at 4000 bp indicate linear Cas13a or Cas12a genes and bands at 6000 indicate linear part backbone + terminator with Cas13a or Cas12a inserted. Lane 1) Undigested backbone + terminator Lane 2) Backbone + terminator digested with FastDigest restriction enzyme EcoRI. Lane 3) Backbone + terminator digested with FastDigest restriction enzymes, EcoRI, and SpeI. Lane 4) Undigested backbone + terminator with Cas13a gene inserted. Lane 5) Backbone + terminator with Cas13a gene inserted, digested with FastDigest restriction enzyme EcoRI. Lane 6) Backbone + terminator with Cas13a gene inserted, digested with FastDigest restriction enzyme EcoRI and SpeI. Lane 7) Undigested backbone + terminator with Cas12a gene inserted. Lane 8) Backbone + terminator with Cas12a gene inserted, digested with FastDigest restriction enzyme EcoRI. Lane 9) Backbone + terminator with Cas13a gene inserted, digested with FastDigest restriction enzyme EcoRI and SpeI.
Once the proteins had been purified the unpurified and purified protein was run SDS-PAGE along with some wash buffers. The SDS-PAGE shows that the Cas proteins are successfully expressed and that the His-tag purification is efficient. For more information on the function of the Cas proteins in complex with sgRNAs can be found on our Proof of Concept page.
Figure 4. Successful protein purification of Cas12a. The first lane displays a sample of lysed E. coli ‘ER2536 + Cas12a plasmid’ flow-through before induction of IPTG. The second lane displays a sample of lysed E. coli ‘ER2536 + Cas12a plasmid’ flow-through after induction of IPTG. The third lane displays flow-through from wash-buffer. The fourth lane displays pre-pre-elution-buffer flow-through. The fifth lane displays pre-elution buffer flow-through. The sixth lane displays 0,5 mL elution-buffer flow-through. Lanes 7-12 display 1 mL elution-buffer fractions. In the lines with elution fractions, there is a clear band above 140 kDa correlating well with the molecular weight of Cas12a+SUMO-Histag = 156,31 kDa. Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”; Gel: Bis-Tris 4-12%; Running buffer: MOPS. Figure 5. Successful protein purification of Cas13a. The first lane displays a sample of lysed E. coli ‘ER2536+ Cas13a plasmid’ flow-through before induction with IPTG. The second lane displays a sample of lysed E. coli ‘ER2536 + Cas13a plasmid’ flow-through after induction of IPTG. The third lane displays flow-through from wash-buffer. The four lane displays pre-pre-elution-buffer flow-through. The fifth lane displays pre-elution buffer flow-through. The sixth lane displays 0,5 mL elution-buffer flow-through. Lane 7-12 displays 1 mL elution-buffer fractions.In the lines with elution fractions, there is a clear band above 140 kDa correlating well with the molecular weight of Cas13a+SUMO-Histag = 153,8 kDa. Ladder: “Thermo Fisher PageRuler Prestained Protein Ladder”; Gel: Bis-Tris 4-12%; Running buffer: MOPS.
Additional information about the assembled parts and the in vitro transcribed guide RNA can be found in the parts registry. Functionality of the Cas-guide RNA complex is illustrated on the Proof of Concept page.
