Recombineering

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Recombineering (recombination-mediated genetic engineering) [1] is a genetic and molecular biology technique based on homologous recombination systems, as opposed to the older/more common method of using restriction enzymes and ligases to combine DNA sequences in a specified order. Recombineering is widely used for bacterial genetics, in the generation of target vectors for making a conditional mouse knockout, and for modifying DNA of any source often contained on a bacterial artificial chromosome (BAC), among other applications.

Contents

Development

Although developed in bacteria, much of the inspiration for recombineering techniques came from methods first developed in Saccharomyces cerevisiae [2] where a linear plasmid was used to target genes or clone genes off the chromosome. In addition, recombination with single-strand oligonucleotides (oligos) was first shown in Saccharomyces cerevisiae. [3] Recombination was observed to take place with oligonucleotides as short as 20 bases.

Recombineering is based on homologous recombination in Escherichia coli mediated by bacteriophage proteins, either RecE/RecT from Rac prophage [4] or Redαβδ from bacteriophage lambda. [5] [6] The lambda Red recombination system is now most commonly used and the first demonstrations of Red in vivo genetic engineering were independently made by Kenan Murphy [7] and Francis Stewart. [4] [5] However, Murphy's experiments required expression of RecA and also employed long homology arms. Consequently, the implications for a new DNA engineering technology were not obvious. The Stewart lab showed that these homologous recombination systems mediate efficient recombination of linear DNA molecules flanked by homology sequences as short as 30 base pairs (40-50 base pairs are more efficient) into target DNA sequences in the absence of RecA. Now the homology could be provided by oligonucleotides made to order, and standard recA cloning hosts could be used, greatly expanding the utility of recombineering.

Recombineering with dsDNA

Recombineering utilizes linear DNA substrates that are either double-stranded (dsDNA) or single-stranded (ssDNA). Most commonly, dsDNA recombineering has been used to create gene replacements, deletions, insertions, and inversions. Gene cloning [6] [8] and gene/protein tagging (His tags etc., see [9] ) is also common. For gene replacements or deletions, usually a cassette encoding a drug-resistance gene is made by PCR using bi-partite primers. These primers consist of (from 5’→3’) 50 bases of homology to the target region, where the cassette is to be inserted, followed by 20 bases to prime the drug resistant cassette. The exact junction sequence of the final construct is determined by primer design. [10] [11] These events typically occur at a frequency of approximately 104/108cells that survive electroporation. Electroporation is the method used to transform the linear substrate into the recombining cell.

Selection/counterselection technique

In some cases, one desires a deletion with no marker left behind, to make a gene fusion, or to make a point mutant in a gene. This can be done with two rounds of recombination. [12] In the first stage of recombineering, a selection marker on a cassette is introduced to replace the region to be modified. In the second stage, a second counterselection marker (e.g. sacB) on the cassette is selected against following introduction of a target fragment containing the desired modification. Alternatively, the target fragment could be flanked by loxP or FRT sites, which could be removed later simply by the expression of the Cre or FLP recombinases, respectively. A novel selection marker "mFabI" was also developed to increase recombineering efficiency. [13]

Recombineering with ssDNA

Recombineering with ssDNA provided a breakthrough both in the efficiency of the reaction and the ease of making point mutations. [1] This technique was further enhanced by the discovery that by avoiding the methyl-directed mismatch repair system, the frequency of obtaining recombinants can be increased to over 107/108 viable cells. [14] This frequency is high enough that alterations can now be made without selection. With optimized protocols, over 50% of the cells that survive electroporation contain the desired change. Recombineering with ssDNA only requires the Red Beta protein; Exo, Gamma and the host recombination proteins are not required. As proteins homologous to Beta and RecT are found in many bacteria and bacteriophages (>100 as of February 2010), recombineering is likely to work in many different bacteria. [15] Thus, recombineering with ssDNA is expanding the genetic tools available for research in a variety of organisms. To date, recombineering has been performed in E. coli, S. enterica, Y. pseudotuberculosis, S. cerevisiae and M. tuberculosis. [16] [17] [18] [19] [20] [21]

Red-Independent recombination

In the year 2010, it has been demonstrated that ssDNA recombination can occur in the absence of known recombination functions. [22] Recombinants were found at up to 104/108 viable cells. This Red-independent activity has been demonstrated in P. syringae, E. coli, S. enterica serovar typhimurium and S. flexneria.

Applications and benefits of recombineering

The biggest advantage of recombineering is that it obviates the need for conveniently positioned restriction sites, whereas in conventional genetic engineering, DNA modification is often compromised by the availability of unique restriction sites. In engineering large constructs of >100 kb, such as the Bacterial Artificial Chromosomes (BACs), or chromosomes, recombineering has become a necessity. Recombineering can generate the desired modifications without leaving any 'footprints' behind. It also forgoes multiple cloning stages for generating intermediate vectors and therefore is used to modify DNA constructs in a relatively short time-frame. The homology required is short enough that it can be generated in synthetic oligonucleotides and recombination with short oligonucleotides themselves is incredibly efficient. Recently, recombineering has been developed for high throughput DNA engineering applications termed 'recombineering pipelines'. [23] Recombineering pipelines support the large scale production of BAC transgenes and gene targeting constructs for functional genomics programs such as EUCOMM (European Conditional Mouse Mutagenesis Consortium) and KOMP (Knock-Out Mouse Program). Recombineering has also been automated, a process called "MAGE" -Multiplex Automated Genome Engineering, in the Church lab. [24] With the development of CRISPR technologies, construction of CRISPR interference strains in E. coli requires only one-step oligo recombineering, providing a simple and easy-to-implement tool for gene expression control. [12] [25] "Recombineering tools" and laboratory protocols have also been implemented for a number of plant species. These tools and procedures are customizable, scalable, and freely available to all researchers. [26]

Related Research Articles

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual mode of reproduction in bacteria.

A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.

Chromosomal crossover Cellular process

Chromosomal crossover, or crossing over, is the exchange of genetic material during sexual reproduction between two homologous chromosomes' non-sister chromatids that results in recombinant chromosomes. It is one of the final phases of genetic recombination, which occurs in the pachytene stage of prophase I of meiosis during a process called synapsis. Synapsis begins before the synaptonemal complex develops and is not completed until near the end of prophase I. Crossover usually occurs when matching regions on matching chromosomes break and then reconnect to the other chromosome.

Genetic recombination Production of offspring with combinations of traits that differ from those found in either parent

Genetic recombination is the exchange of genetic material between different organisms which leads to production of offspring with combinations of traits that differ from those found in either parent. In eukaryotes, genetic recombination during meiosis can lead to a novel set of genetic information that can be passed on from the parents to the offspring. Most recombination is naturally occurring.

Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.

Expression vector

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.

Transformation (genetics) transformation

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

RecBCD

RecBCD is an enzyme of the E. coli bacterium that initiates recombinational repair from potentially lethal double strand breaks in DNA which may result from ionizing radiation, replication errors, endonucleases, oxidative damage, and a host of other factors. The RecBCD enzyme is both a helicase that unwinds, or separates the strands of DNA, and a nuclease that makes single-stranded nicks in DNA.

Nucleoid Region within a prokaryotic cell containing genetic material

The nucleoid is an irregularly shaped region within the prokaryotic cell that contains all or most of the genetic material. The chromosome of a prokaryote is circular, and its length is very large compared to the cell dimensions needing it to be compacted in order to fit. In contrast to the nucleus of a eukaryotic cell, it is not surrounded by a nuclear membrane. Instead, the nucleoid forms by condensation and functional arrangement with the help of chromosomal architectural proteins and RNA molecules as well as DNA supercoiling. The length of a genome widely varies and a cell may contain multiple copies of it.

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

RecA DNA repair protein

RecA is a 38 kilodalton protein essential for the repair and maintenance of DNA. A RecA structural and functional homolog has been found in every species in which one has been seriously sought and serves as an archetype for this class of homologous DNA repair proteins. The homologous protein is called RAD51 in eukaryotes and RadA in archaea.

Synteny

In classical genetics, synteny describes the physical co-localization of genetic loci on the same chromosome within an individual or species. Today, however, biologists usually refer to synteny as the conservation of blocks of order within two sets of chromosomes that are being compared with each other. This concept can also be referred to as shared synteny.

Homologous recombination genetic recombination between identical or highly similar strands of genetic material

Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids. It is widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks (DSB), in a process called homologous recombinational repair (HRR). Homologous recombination also produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make gamete cells, like sperm and egg cells in animals. These new combinations of DNA represent genetic variation in offspring, which in turn enables populations to adapt during the course of evolution. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses.

Synthetic genomics is a nascent field of synthetic biology that uses aspects of genetic modification on pre-existing life forms, or artificial gene synthesis to create new DNA or entire lifeforms.

Artificial gene synthesis Group of methods in synthetic biology

Artificial gene synthesis, or gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides de novo. Unlike DNA synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is solid-phase DNA synthesis, sometimes known as DNA printing. This produces oligonucleotide fragments that are generally under 200 base pairs. The second step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.

Mutagenesis (molecular biology technique)

In molecular biology, mutagenesis is an important laboratory technique whereby DNA mutations are deliberately engineered to produce libraries of mutant genes, proteins, strains of bacteria, or other genetically modified organisms. The various constituents of a gene, as well as its regulatory elements and its gene products, may be mutated so that the functioning of a genetic locus, process, or product can be examined in detail. The mutation may produce mutant proteins with interesting properties or enhanced or novel functions that may be of commercial use. Mutant strains may also be produced that have practical application or allow the molecular basis of a particular cell function to be investigated.

Synthetic genome is a synthetically-built genome whose formation involves either genetic modification on pre-existing life forms or artificial gene synthesis to create new DNA or entire lifeforms. The field that studies synthetic genomes is called Synthetic Genomics.

SCAR-less genome editing Scarless Cas9 Assisted Recombineering (no-SCAR) is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, in this method, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

Bacterial recombination is a type of genetic recombination in bacteria characterized by DNA transfer from one organism called donor to another organism as recipient. This process occurs in three main ways:

Illegitimate recombination

Illegitimate recombination, or nonhomologous recombination, is the process by which two unrelated double stranded segments of DNA are joined. This insertion of genetic material which is not meant to be adjacent tends to lead to genes being broken causing the protein which they encode to not be properly expressed. One of the primary pathways by which this will occur is the repair mechanism known as non-homologous end joining (NHEJ).

References

  1. 1 2 Ellis H. M., Yu D., DiTizio T., Court D. L. (2001). "High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides". Proceedings of the National Academy of Sciences. 98 (12): 6742–6746. Bibcode:2001PNAS...98.6742E. doi: 10.1073/pnas.121164898 . PMC   34423 . PMID   11381128.CS1 maint: multiple names: authors list (link)
  2. ↑ Orr-Weaver, T. L., J. W. Szostak, et al. (1983) Genetic applications of yeast transformation with linear and gapped plasmids. Methods. Enzymol. 101: 228-245.
  3. ↑ Moerschell R. P., Tsunasawa S.; et al. (1988). "Transformation of yeast with synthetic oligonucleotides". Proceedings of the National Academy of Sciences. 85 (2): 524–528. Bibcode:1988PNAS...85..524M. doi: 10.1073/pnas.85.2.524 . PMC   279583 . PMID   2829192.
  4. 1 2 Zhang Y., Buchholz F., Muyrers J. P., Stewart A. F. (1998). "A new logic for DNA engineering using recombination in Escherichia coli". Nature Genetics. 20 (2): 123–128. doi:10.1038/2417. PMID   9771703. S2CID   21186444.CS1 maint: multiple names: authors list (link)
  5. 1 2 Muyrers J. P., Zhang Y., Testa G., Stewart A. F. (1999). "Rapid modification of bacterial artificial chromosomes by ET- recombination". Nucleic Acids Research. 27 (6): 1555–1557. doi:10.1093/nar/27.6.1555. PMC   148353 . PMID   10037821.CS1 maint: multiple names: authors list (link)
  6. 1 2 Yu D., Ellis H. M.; et al. (2000). "An efficient recombination system for chromosome engineering in Escherichia coli". Proceedings of the National Academy of Sciences. 97 (11): 5978–5983. Bibcode:2000PNAS...97.5978Y. doi: 10.1073/pnas.100127597 . PMC   18544 . PMID   10811905.
  7. ↑ Murphy K. C. (1998). "Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli". Journal of Bacteriology. 180 (8): 2063–2071. doi:10.1128/JB.180.8.2063-2071.1998. PMC   107131 . PMID   9555887.
  8. ↑ Zhang, Y., Muyrers, J.P.P., Testa, G. and Stewart, A.F. (2000) DNA cloning by homologous recombination in Escherichia coli Nature Biotechnology 18, 1314 - 1317
  9. ↑ Poser I, Sarov M, Hutchins JR, Hériché JK, Toyoda Y, Pozniakovsky A, Weigl D, Nitzsche A, Hegemann B, Bird AW, Pelletier L, Kittler R, Hua S, Naumann R, Augsburg M, Sykora MM, Hofemeister H, Zhang Y, Nasmyth K, White KP, Dietzel S, Mechtler K, Durbin R, Stewart AF, Peters JM, Buchholz F, Hyman AA (2008). "BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals". Nature Methods. 5 (5): 409–15. doi:10.1038/nmeth.1199. PMC   2871289 . PMID   18391959.CS1 maint: multiple names: authors list (link)
  10. ↑ Sawitzke J. A., Thomason L. C., Costantino N., Bubunenko M., Datta S., Court D. L. (2007). "Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond". Advanced Bacterial Genetics: Use of Transposons and Phage for Genomic Engineering. Methods in Enzymology. 421. pp. 171–199. doi:10.1016/S0076-6879(06)21015-2. ISBN   9780123737496. PMID   17352923.CS1 maint: multiple names: authors list (link)
  11. ↑ Thomason, L., D. L. Court, M. Bubunenko, N. Costantino, H. Wilson, S. Datta & A. Oppenheim, (2007) Recombineering: Genetic engineering in bacteria using homologous recombination. In: Current Protocols in Molecular Biology. Hoboken, N.J.: John Wiley & Sons, Inc., pp. Chapter 1 Unit 16 p. 11-24.
  12. 1 2 Li, X; Thomason, LC; Sawitzke, JA; Costantino, N; Court, DL (2013). "Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli". Nucleic Acids Research. 41 (22): e204. doi:10.1093/nar/gkt1075. PMC   3905872 . PMID   24203710.
  13. ↑ A Novel Selection Marker for Efficient DNA Cloning and Recombineering in E. coli
  14. ↑ Costantino N., Court D. L. (2003). "Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants". Proceedings of the National Academy of Sciences of the United States of America. 100 (26): 15748–15753. Bibcode:2003PNAS..10015748C. doi: 10.1073/pnas.2434959100 . PMC   307639 . PMID   14673109.
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  18. ↑ van Kessel J. C., Hatfull G. F. (2008). "Mycobacterial recombineering". Mycobacteria Protocols. Methods in Molecular Biology. 435. pp. 203–215. doi:10.1007/978-1-4939-2450-9_10. ISBN   978-1-4939-2449-3. PMID   25779316.
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  20. ↑ van Kessel J. C., Marinelli L. J., Hatfull G. F. (2008). "Recombineering mycobacteria and their phages". Nature Reviews Microbiology. 6 (11): 851–857. doi:10.1038/nrmicro2014. PMC   3503148 . PMID   18923412.CS1 maint: multiple names: authors list (link)
  21. ↑ DiCarlo James E., Conley Andrew J., Penttilä Merja, Jäntti Jussi, Wang Harris H., Church George M. (2013). "Yeast Oligo-Mediated Genome Engineering (YOGE)". ACS Synthetic Biology. 2 (12): 741–749. doi:10.1021/sb400117c. PMC   4048964 . PMID   24160921.CS1 maint: multiple names: authors list (link)
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  25. ↑ Li, X; Jun, Y; Erickstad, M; Brown, S; Parks, A; Court, D; Jun, S (2016). "tCRISPRi: tunable and reversible, one-step control of gene expression". Scientific Reports. 6: 39096. Bibcode:2016NatSR...639076L. doi:10.1038/srep39076. PMC   5171832 . PMID   27996021.
  26. ↑ Brumos J, Zhao C, Gong Y, Soriano D, Patel AP, Perez-Amador MA, Stepanova AN, Alonso JM (2019). "An improved recombineering toolset for plants". The Plant Cell. 32 (1): 100–122. doi:10.1105/tpc.19.00431. PMC   6961616 . PMID   31666295.CS1 maint: multiple names: authors list (link)
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