Gene editing, also known as genome editing or genome engineering, is a new and more accurate genetic engineering technology or process that can modify specific target genes in the genome of organisms.
Early genetic engineering technology can only randomly insert exogenous or endogenous genetic material into the host genome, while gene editing can edit the desired genes at fixed points. Gene editing relies on nucleic acid enzymes that have been genetically engineered, also known as "molecular scissors", to produce site-specific double-strand breaks (DSBs) at specific locations in the genome, inducing organisms to repair DSBs through non-homologous end joining (NHEJ) or homologous recombination (HR), because this repair process is prone to errors, leading to targeted mutations.

In gene editing technology, CRISPR-cas9, allows us to use gRNA to specify almost all available genes. At the same time, CRISPR-cas9 causes a double-strand break of specific target sequences of the genome in millions to hundreds of millions of bases. Simultaneous cutting of multiple targets greatly increases the efficiency of gene editing. In addition, CRISPR-cas9 can screen and identify genes, Therefore, it still has great prospects in major diseases, but after tens of thousands of experiments, we gradually found the defects of this technology.
CRISPR-cas9 could cut a genome at specific sites, but in this process, genes are likely to mutate. In view of this situation, we need improved tools. Casponson, which takes advantage of the self-copying and pasting advantages of transposons, further improves the experimental efficiency.

Casponson is a new archaeal and bacterial mobile element superfamily, which not only has the advantages of general transposons: including terminal reverse repeats and B-group DNA polymerase genes but also contains CASL endonuclease in this transposon, which is an enzyme that can integrate elements into the host genome. With this enzyme, genes can be integrated, and CASL can be used for integration and excision. This new casposase is the first self-synthesized transposon family found in prokaryotes. The casposase we used gave rise to the adaptation module of CRISPR-Cas systems.

After protein binding, the transposon will recognize the TSD end of our target vector pUC19 and insert the gene into the target region.

In this project, we developed an in vitro gene editing system. First, we introduced the TIR sequence into the pUC19 plasmid by PCR. After verification of the plasmid by Sanger sequencing, we extracted the plasmid as one of the components of our in vitro reaction system. Next, we amplified different lengths of target genes containing the Kanamycin gene and extracted these fragments as other components of our in vitro reaction system.
Then, we mixed the purified casposase protein, the target genes, and the plasmid with the reaction buffer, the gene editing process was taken in a metal bath at 37℃ for 1h. The edited plasmids in the reaction system were obtained and transformed into DH10b competent cells and coated on LB solid medium with corresponding antibiotics. Finally, the function of casposons was verified by calculating the number of colonies. What’s more, we used this system to insert the Ampicillin DNA fragment into pET28a plasmid special region.

1. Successfully construct pUC19-TSD plasmid and amplify the target genes.
2. Achieve gene editing through our in vitro gene-editing system named casposons.
3. Insert the Ampicillin gene fragment into the pET28a plasmid with casposons.

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.