Genome editing

From Wikipedia, the free encyclopedia
Jump to: navigation, search

Genome editing, or genome editing with engineered nucleases (GEEN) is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or "molecular scissors." The nucleases create specific double-stranded break (DSBs) at desired locations in the genome, and harness the cell’s endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) and nonhomologous end-joining (NHEJ). There are currently four families of engineered nucleases being used: Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas system, and engineered meganuclease re-engineered homing endonucleases.[1][2][3]


It was chosen by Nature Methods as the 2011 Method of the Year.[5]


A common approach in the modern biological research is to manipulate the genetic sequence (genotype) of an organism (or a single cell) and observe the impact of this change on the organism (phenotype). Such approach is called reverse genetics and its significance for the modern biology lies in its relative simplicity. In contrast, in forward genetics a new phenotype is first observed and then its genetic basis is studied. This course is more complex, since phenotypic changes are often a result of multiple genetic interactions.

Among the key aspects of reverse genetic analysis is the ability to modify the genetic code. This can be achieved by:

Both approaches have several drawbacks:

  • They are less successful in other organisms.[citation needed]
  • They also require stringent selection steps and thus addition of selection specific sequences, along with those incorporated into the DNA.
  • They can be quite inefficient - e.g. in mouse embryonic stem cells treated with donor DNA, in only 1 of a million the DNA got incorporated at the desired position.[6]


Hence, genomic editing with engineered nucleases, a rapidly growing technology is a promising new approach. It overcomes these shortcomings and uses relatively simple concepts.

Double stranded breaks and their repair[edit]



NHEJ is error prone such that it was shown to cause mutations at the repair site in approximately 50% of DSB in mycobacteria [7] and also its low fidelity has been linked to mutational accumulation in leukemias.[8] Thus if one is able to create a DSB at a desired gene in multiple samples, it is very likely that mutations will be generated at that site in some of the treatments because of errors created by the NHEJ infidelity.



Site-specific double stranded breaks[edit]


Current engineered nucleases[edit]

The current groups of engineered nucleases used for GEEN. Matching colors signify DNA recognition patterns

Meganucleases, found commonly in microbial species, have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific.[10][11] This can be exploited to make site-specific DSB in genome editing; however, the challenge is that not enough meganucleases are known, or may ever be known, to cover all possible target sequences. To overcome this challenge, mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences.[11] Others have been able to fuse various meganucleases and create hybrid enzymes that recognize a new sequence.[12] Yet others have attempted to alter the DNA interacting aminoacids of the meganuclease to design sequence specific meganucelases in a method named rationally designed meganuclease (US Patent 8,021,867 B2).

Meganuclease have the benefit of causing less toxicity in cells compared to methods such as ZFNs likely because of more stringent DNA sequence recognition;[11] however, the construction of sequence specific enzymes for all possible sequences is costly and time consuming as one is not benefiting from combinatorial possibilities that methods such as ZFNs and TALENs utilize. So there are both advantages and disadvantages.



Endogenous genes targeted.jpg


Listed below are some specific tasks this method can carry out:

Overview of GEEN workflow and editing possibilities

Targeted gene addition in plants[edit]

Genome editing using ZFN provides a new strategy for genetic manipulation in plants and is likely to assist engineering desired plant traits by modifying endogenous genes. For instance, site-specific gene addition in major crop species can be used for 'trait stacking' whereby several desired traits are physically linked to ensure their co-segregation during the breeding processes.[14] Progress in such cases have been recently reported in Arabidopsis thaliana [16] [17][18] and Zea mays. In Arabidopsis thaliana, using ZFN-assisted gene targeting, two herbicide-resistant genes (tobacco acetolactate synthase SuRA and SuRB) were introduced to SuR loci with as high as 2% transformed cells with mutations.[19] In Zea mays, disruption of the target locus was achieved by ZFN-induced DSBs and the resulting NHEJ. ZFN was also used to drive herbicide-tolerance gene expression cassette (PAT) into the targeted endogenous locus IPK1 in this case.[20] Such genome modification observed in the regenerated plants has been shown to be inheritable and was transmitted to the next generation.[20]

Several optimizations need to be made in order to improve editing plant genomes using ZFN-mediated targeting.[21] These include the reliable design and subsequent test of the nucleases, the absence of toxicity of the nucleases, the appropriate choice of the plant tissue for targeting, the routes of introduction or induction of enzyme activity, the lack of off-target mutagenesis, and a reliable detection of mutated cases.[21]

Gene therapy[edit]




Prospect and Current Limitations[edit]

In the future, research into genome editing with engineered nucleases needs to focus on improving safety and specificity of the nucleases. For example, improving the ability to detect off-target events can improve our ability to learn about ways of preventing them. In addition, zinc-fingers used in ZFNs are seldom completely specific, and some may cause toxicity. However, the toxicity has been reported to be reduced by modifications done on the cleavage domain of the ZFN.[22]

In addition, the study by Dana Carroll looking at modifying the genome with engineered nucleases shows the requirement of better understanding of the basic recombination and repair machinery of DNA. In the future, a possible method to identify secondary targets would be to capture broken ends from cells expressing the ZFNs and to sequence the flanking DNA using high-throughput sequencing.[22]


Lastly, one must also realize that although GEEN has higher efficiency than many other methods in reverse genetics, it is still not highly efficient as in many cases less than half of the treated populations obtain the desired changes.[19] For example, when one is planning to use the cell's NHEJ to create a mutation, the cell's HDR systems will also be at work correcting the DSB with lower mutational rates.

See also[edit]

Further reading[edit]


  1. ^ Esvelt, KM.; Wang, HH. (2013). "Genome-scale engineering for systems and synthetic biology.". Mol Syst Biol 9 (1): 641. doi:10.1038/msb.2012.66. PMC 3564264. PMID 23340847. 
  2. ^ Tan, WS.; Carlson, DF.; Walton, MW.; Fahrenkrug, SC.; Hackett, PB. (2012). "Precision editing of large animal genomes.". Adv Genet 80: 37–97. doi:10.1016/B978-0-12-404742-6.00002-8. PMC 3683964. PMID 23084873. 
  3. ^ Puchta, H.; Fauser, F. (2013). "Gene targeting in plants: 25 years later.". Int. J. Dev. Biol 57: 629–637. doi:10.1387/ijdb.130194hp. 
  4. ^ Fire, A. et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans" Nature 391 (6669), 806-811 (1998).
  8. ^ Feyruz Virgilia, R., DNA double strand breaks (DSB) and non-homologous end joining (NHEJ) pathways in human leukemia. Cancer Letters 193 (1), 1-9 (2003).
  26. ^ Witzany, G. The agents of natural genome editing. J Mol Cell Biol 3: 181-189 (2011). PMID: 21459884 doi:10.1093/jmcb/mjr005