Part:BBa_K3134005
E.coli Cas1
E.coli Cas1 is a special protein that can form a complex with E.coli Cas2. The composite can be used as a protein that can record fragments when they invaded the cell.
Improvement by 2022 SHBS_BANZ
Compared to the old part BBa_K3134005, a CRISPR-associated protein-Cas1, we design a new part BBa_K4411032, which is composed of casposase, TIR DNA sequence, and TSD DNA fragment.
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.
Ab-Caposon
Profile
Name: Ab-Casposon
Base Pairs: 1227 bp
Origin: Aciduliprofundum boonei, genome
Properties: A self-synthesizing transposon
Usage and Biology
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, which can recognize TSD and TIR elements to implement gene insertion and develop an in vitro gene editing system. It has a strong sequence preference in the presence of a proper target site, so we use the Aciduliprofundum boonei casposase to form casposons to integrate target genes into specific regions [1-6].
Experimental approach
1. Target DNA fragment Electrophoresis
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. To successfully amplify the genes, we use different annealing temperatures, such as 59℃, 61℃, and 63℃ (Figure 2). 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.
This step is used to obtain the target DNA fragments we used to verify if the casposons work well in vitro reaction platform.
Proof of function
1. In vitro casposons gene-editing system
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.
2. Screen for TIR-Kan plasmids
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.
3. Sanger sequencing to amplify the recombinant plasmids
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 transposon 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.
References
1.Hickman AB, Dyda F. The casposon-encoded Cas1 protein from Aciduliprofundum boonei is a DNA integrase that generates target site duplications. Nucleic Acids Res. 2015 Dec 15;43(22):10576-87. doi: 10.1093/nar/gkv1180. PMID: 26573596
2.Krupovic M, Shmakov S, Makarova KS, Forterre P, Koonin EV. Recent Mobility of Casposons, Self-Synthesizing Transposons at the Origin of the CRISPR-Cas Immunity. Genome Biol Evol. 2016 Jan 13;8(2):375-86. doi:10.1093/gbe/evw006. PMID: 26764427; PMCID: PMC4779613.
3.Béguin P, Charpin N, Koonin EV, Forterre P, Krupovic M. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems. Nucleic Acids Res. 2016 Dec 1;44(21):10367-10376. doi: 10.1093/nar/gkw821. PMID: 27655632; PMCID: PMC5137440.
4.Krupovic M, Béguin P, Koonin EV. Casposons: mobile genetic elements that gave rise to the CRISPR-Cas adaptation machinery. Curr Opin Microbiol. 2017 Aug; 38:36-43. doi: 10.1016/j.mib.2017.04.004. PMID: 28472712; PMCID: PMC5665730.
5.Béguin P, Chekli Y, Sezonov G, Forterre P, Krupovic M. Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei. Nucleic Acids Res. 2019 Jul 9;47(12):6386-6395.doi:10.1093/nar/gkz447.PMID:31114911; PMCID: PMC6614799.
6.Wang X, Yuan Q, Zhang W, Ji S, Lv Y, Ren K, Lu M, Xiao Y. Sequence specific integration by the family 1 casposase from Candidatus Nitrosopumilus koreensis AR1. Nucleic Acids Res. 2021 Sep 27;49(17):9938-9952. doi: 10.1093/nar/gkab725. PMID: 34428286; PMCID: PMC8464041.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 116
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 837
- 1000COMPATIBLE WITH RFC[1000]
| None |