Gene editing is a new and more accurate genetic engineering technology that can modify specific target genes in the genome of organisms. The gene-editing technology has led to innovations in medicine, evolution, and agriculture — and raised profound ethical questions about altering human DNA. However, CRISPR/Cas9 may induce double-strand breaks (DSBs) that are resolved by the classical non-homologous end joining pathway (c-NHEJ) and typically result in insertions or deletions (indels) of relatively small DNA sequences at both on-target and off-target sites, leading to disruption of the target sequence. So it is important to develop new gene editing tools. As early as 2019, the iGEM19_Nanjing_High_School team provided a CRISPR-associated protein-Cas1 (BBa_K3134005) which encodes the only universally conserved protein component of CRISPR immune systems, yet its function is unknown.
In our project, we employed a recombinase (casposase, BBa_K4411032) homologous to the Cas1 endonuclease, which can recognize TSD and TIR elements to implement gene insertion and develop an in vitro gene editing system. Based on the existing literature, we conducted experiments and studied the subject under the best conditions available, and strive to make a breakthrough in this technology. In order to test the function of the gene-editing system and measure the parameter of gene length to insert by casposons, we implied different lengths of the genes containing Kanamycin as a reporter system and calculated the number of colonies. As a result, we successfully detected the gene insertion indicating that our gene-editing system was successfully developed.

The casposon is a member of a distinct superfamily of archaeal and bacterial self-synthesizing transposons that employ a recombinase (casposase) homologous to the Cas1 endonuclease. It has a strong sequence preference in the presence of a proper target site, so we use the Aciduliprofundum boonei casposase to catalyze casposons integration into specific regions.

In order to insert the TIR sequence into the pUC19 plasmid (Figure 1A), we use PCR to amplify the target DNA fragment and inserted the amplicons into DH10b competent cells. Inoculated a single colony into LB (Amp) culture medium, extracted the plasmid, and send to the company for Sanger sequencing (Figure 1B). As shown in the sequencing result, we successfully constructed the plasmid.

In order to obtain our target genes, we amplified different lengths of the target genes containing the Kanamycin gene fragment from the pUC19-DONER plasmid (Figure 2A). In order to successfully amplify the genes, we use different annealing temperatures, such as 59℃, 61℃, and 63℃ (Figure 2B).

In figure2, a clear and single DNA band at 1kp can be seen, indicating that we successfully amplified our target genes. We extracted the DNA fragments and stored them at -20℃ for future use.

To verify whether the long fragment gene with TIR sequence could be inserted into the TSD sequence effectively and correctly, the protein casposase was added for reaction, and the reaction products were recovered. Mixed components according to the table below, reacted in a metal bath at 37°C for 1h. Add PK enzyme at 37°C for 30min, then 95°C for 10min to terminate the reaction, added isopropanol into the reaction system and discard the supernatant, and resuspend the pellet with sterile water.

We transformed the recycled plasmids pool into E. coli DH10b competent cells, and coat on the LB solid medium plate containing both Kanamycin and Ampicillin antibiotics, incubated at 37℃ overnight. The next day, we calculated the number of colonies on the plate (Figure 3).

Because the colonies on the plate are too intensive to calculate, we resuspend the colonies with LB culture medium, incubated at 37℃ for 30min, diluted 8 times, and coated on the LB (Kana+Amp) solid medium plates and incubated at 37℃ overnight (Figure 4). The next day, we calculated 1/4 area of the plate of the number of colonies (Figure 5).

As a result, we can find that when the length of inserted gene is around 3.5k, we still achieved gene-editing with casposons. what’s more, as the length of the inserted gene increased, the number of colonies decreased. However, casposons is still an excellent tool we could use in future research for gene editing.

We inoculate the single colony in the LB liquid culture medium (Kana+Amp), extracted plasmids, amplified the target-gene-containing fragments, and send the company for Sanger sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 6), and the plasmids were successfully constructed. So far, we have successfully developed our gene editing system.

Casposons is a transposons that could be used to recognize TSD sequences and insert target genes with TIRs into the specific region. From the result, compared with the negative control, we can find that with casposase in the reaction system we successfully inserted the different lengths of target genes into the pUC19-TSD plasmid so that the strain could grow on the plate, indicating that our in vitro gene-editing system works well and could be used for future researches.