Gene cassette

In biology, a gene cassette is a type of mobile genetic element that contains a gene and a recombination site. Each cassette usually contains a single gene and tends to be very small; on the order of 500–1000 base pairs. They may exist incorporated into an integron or freely as circular DNA.[1] Gene cassettes can move around within an organism's genome or be transferred to another organism in the environment via horizontal gene transfer. These cassettes often carry antibiotic resistance genes. An example would be the kanMX cassette which confers kanamycin (an antibiotic) resistance upon bacteria.

Integrons
Main article: Integron

Integrons are genetic structures in bacteria which express and are capable of acquiring and exchanging gene cassettes. The integron consists of a promoter, an attachment site, and an integrase gene that encodes a site-specific recombinase[2] There are three classes of integrons described.[1] The mobile units that insert into integrons are gene cassettes.[2] For cassettes that carry a single gene without a promoter, the entire series of cassettes is transcribed from an adjacent promoter within the integron.[3] The gene cassettes are speculated to be inserted and excised via a circular intermediate.[4] This would involve recombination between short sequences found at their termini and known as 59 base elements (59-be)—which may not be 59 bases long. The 59-be are a diverse family of sequences that function as recognition sites for the site-specific integrase (enzyme responsible for integrating the gene cassette into an integron) that occur downstream from the gene coding sequence.[5]

Diversity and prevalence

The ability of genetic elements like gene cassettes to excise and insert into genomes results in highly similar gene regions appearing in distantly related organisms. The three classes of integrons are similar in structure and are identified by where the insertions occur and what systems they coincide with. Class 1 integrons are seen in a diverse group of bacterial genomes and likely are all descendant from one common ancestor. The prevalence of the integron has shaped bacterial evolution by allowing rapid transfer of genes that are novel to an organism, such as antibiotic resistance genes.[6]

Genetic engineering

In genetic engineering, a gene cassette is a manipulable fragment of DNA carrying, and capable of expressing, one or more genes of interest between one or more sets of restriction sites. It can be transferred from one DNA sequence (usually on a vector) to another by 'cutting' the fragment out using restriction enzymes and 'pasting' it back into the new context. The vectors containing the gene of interest typically also carry an antibiotic resistance gene called a selectable marker to easily identify cells that have successfully integrated the vector into their genome.

To introduce a vector into a target cell, a state of competence must be inferred on the cell. This state is induced in the lab by incubating cells with calcium chloride before a brief heat shock, or by electroporation. This makes the cells more susceptible to the plasmid that is being inserted. Once the plasmid has been added, the cells are grown in the presence of an antibiotic to confirm the uptake and expression of the new genetic elements.

The usage of CRISPR/Cas9 systems has shown success in inserting genes into eukaryotic genomes.[7] While CRISPR modification is still in its infancy, there is significant evidence for usage in combination with other techniques to produce high throughput (HTP) genome editing systems.[8] Genetic engineering of bacteria for production of a variety of industrial products, including biofuels and specialty chemicals/nutraceuticals is a major area of research.[9]

Horizontal gene transfer
Main article: Horizontal gene transfer

Horizontal gene transfer (HGT) is the transfer of genetic elements between cells other than parental inheritance. HGT is responsible for much of the spread of antibiotic resistance among bacteria.[10] Gene cassettes containing antibiotic resistance genes, or other virulence factors such as exotoxins, can be transferred from cell to cell via phage, transduction, taken up from the environment, transformation,[11] or by bacterial conjugation.[12] The ability to transfer gene cassettes between organisms has played a large role in the evolution of prokaryotes. Many commensal organisms, such as E. coli, regularly harbor one or more gene cassettes that convey antibiotic resistance.[13] Horizontal transfer of genetic elements from non-pathogenic commensals to unrelated species results in highly virulent pathogens that can carry multiple antibiotic resistance genes. The increasing prevalence of resistance creates challenging questions for researchers and physicians.

See also

References
  1. Hall, RM; Collis, CM (1995). "Mobile gene cassettes and integrons: Capture and spread of genes by site-specific recombination". Molecular Microbiology. 15 (4): 593–600. doi:10.1111/j.1365-2958.1995.tb02368.x. PMID 7783631.
  2. Rapa RA, Labbate M (2013). "The function of integron-associated gene cassettes in Vibrio species: the tip of the iceberg". Frontiers in Microbiology. 4: 385. doi:10.3389/fmicb.2013.00385. PMC 3856429. PMID 24367362.
  3. Collis, C. M.; Hall, R. M. (1995-01-01). "Expression of antibiotic resistance genes in the integrated cassettes of integrons". Antimicrobial Agents and Chemotherapy. 39 (1): 155–162. doi:10.1128/aac.39.1.155. ISSN 0066-4804. PMC 162502. PMID 7695299.
  4. Collis, Christina M.; Hall, Ruth M. (1992-10-01). "Gene cassettes from the insert region of integrons are excised as covalently closed circles". Molecular Microbiology. 6 (19): 2875–2885. doi:10.1111/j.1365-2958.1992.tb01467.x. ISSN 1365-2958. PMID 1331702. S2CID 24986780.
  5. Hall, R. M.; Brookes, D. E.; Stokes, H. W. (1991-08-01). "Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point". Molecular Microbiology. 5 (8): 1941–1959. doi:10.1111/j.1365-2958.1991.tb00817.x. ISSN 0950-382X. PMID 1662753. S2CID 23447071.
  6. Gillings, Michael (Jan 2017). "Class 1 Integrons as Invasive Species". Current Opinion in Microbiology. 38: 10–15. doi:10.1016/j.mib.2017.03.002. PMID 28414952.
  7. Broad Institute
  8. Aida, Tomomi; Nakade, Shota; Sakuma, Tetsushi; Izu, Yayoi; Oishi, Ayu; Mochida, Keiji; Ishikubo, Harumi; Usami, Takako; Aizawa, Hidenori (2016-01-01). "Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ". BMC Genomics. 17 (1): 979. doi:10.1186/s12864-016-3331-9. ISSN 1471-2164. PMC 5126809. PMID 27894274.
  9. Chakraborty, Debkumar; Gupta, Gaganjot; Kaur, Baljinder (2016-12-01). "Metabolic engineering of E. coli top 10 for production of vanillin through FA catabolic pathway and bioprocess optimization using RSM". Protein Expression and Purification. 128: 123–133. doi:10.1016/j.pep.2016.08.015. PMID 27591788.
  10. Gyles, C.; Boerlin, P. (2014-03-01). "Horizontally transferred genetic elements and their role in pathogenesis of bacterial disease". Veterinary Pathology. 51 (2): 328–340. doi:10.1177/0300985813511131. ISSN 1544-2217. PMID 24318976. S2CID 206510894.
  11. Domingues, Sara; Harms, Klaus; Fricke, W. Florian; Johnsen, Pål J.; Silva, Gabriela J. da; Nielsen, Kaare Magne (2012-08-02). "Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species". PLOS Pathogens. 8 (8): e1002837. doi:10.1371/journal.ppat.1002837. ISSN 1553-7374. PMC 3410848. PMID 22876180.
  12. Sun, J (1 Aug 2010). "Original Article: Characterization of Two Novel Gene Cassettes, Dfra27 and Aada16, in a Non-O1, Non-O139 Vibrio Cholerae Isolate from China". Clinical Microbiology and Infection. 16 (8): 1125–1129. doi:10.1111/j.1469-0691.2009.03060.x. PMID 19906273.
  13. Kheiri, Roohollah; Akhtari, Leili (2016-01-01). "Antimicrobial resistance and integron gene cassette arrays in commensal Escherichia coli from human and animal sources in IRI". Gut Pathogens. 8 (1): 40. doi:10.1186/s13099-016-0123-3. ISSN 1757-4749. PMC 5006490. PMID 27582900.
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