Download Site Directed Nucleases (SDN) for targeted

January 2015
Site Directed Nucleases (SDN) for targeted genome modification
Crop improvement, including the development of new traits, necessitates the continuous production of
new biological diversity, either through crosses of existing plants (new combinations of existing genetic
variation) or through the generation of new genetic variation through mutagenesis. With the
development of targeted genome editing technologies it is becoming feasible to advance biological
diversity in crops in a predictable manner. The basis of current targeted genome editing applications is
the capacity to induce a DNA double strand break (DSB) at a selected location in the genome where the
modification is intended. Directed repair of the DSB allows for targeted genome editing. Such
applications can be applied to generate mutations (targeted mutations or precise native gene editing) as
well as precise insertion of genes (cisgenes, intragenes, or transgenes).
Different approaches can be used to achieve targeted DNA breaks, including Meganucleases (MN), Zinc
Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered
Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (CRISPR/Cas) (Gaj et al.,
2013, Podevin et al., 2013). Collectively, these are often discussed under the acronym site directed
nucleases (SDNs), pointing out to the general principle of the technology to use a DNA cutting enzyme
(nuclease) for the generation of the targeted (or site directed) DNA break.
Variants of SDN applications are often categorized as SDN-1, SDN-2 and SDN-3 depending on the
outcome of the DNA double strand break repair.
SDN-1: When the SDN is used in the absence of a DNA repair template (see SDN-2/-3), the outcome is a
targeted, non-specific genetic deletion mutation. In this case, the position of the DNA DSB is precisely
selected, but the DNA repair by the host cell is random and results in small nucleotide deletions,
additions or substitutions. Alternatively, if two DSBs are induced on either side of a targeted DNA
sequence, SDN-1 can result in removal of larger DNA regions (e.g. promoter or whole gene). In either
case, screening and selection of the targeted change allows for identification of the desired genomic
mutational outcome.
SDN-2: Is used to generate gene editing mutations. In this case, a SDN is used to generate a targeted DSB
and a DNA repair template (a short DNA sequence identical to the targeted DSB DNA sequence except
for one or a few nucleotide changes) is used to repair the DSB. The outcome is a targeted and
predetermined point mutation in the desired gene of interest.
SDN-3: When the SDN is used along with a DNA repair template that contains new DNA sequence (e.g.
gene), the outcome of the technology would be the integration of that DNA sequence into the plant
genome. The most likely application illustrating the use of SDN-3 would be the insertion of cisgenic,
intragenic, or transgenic expression cassettes at a selected genome location.
SDNs may be delivered to the cells in various ways. SDNs can be delivered transiently (without transgene
integration) or could be stably integrated in an intermediate plant that serves as a delivery vehicle for
the nuclease activity to a recipient plant. After the cross, the SDN is expressed and then segregated away
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from the recipient plant line resulting in a final product containing only the targeted modification.
Methods to deliver the nuclease via protein are also being contemplated.
The advantage of SDN-1 and SDN-2 applications compared to classical mutagenesis is the precision with
which the desired variation can be created and the ability to efficiently screen for the desired product.
Once the desired product is identified, crossing and segregation is used to select for plants that only
contain the desired changes and serve as donor material for elite plant variety development though
traditional breeding. Because SDN-1 and SDN-2 applications result in precise targeted mutations, the
resulting plants are comparable to plants generated by other mutagenesis techniques. Due to the long
history and role in production of improved crop varieties, mutagenesis is globally considered to
contribute to the production of safe, reliable and sustainable crops. Based on these facts, it can be
concluded that products produced by SDN-1 and SDN-2 applications should be viewed and treated by
regulatory authorities in the same manner as traditional mutational products.
The SDN-3 application allows precise choice of the genomic location to insert a cisgene, intragene, or a
transgene. SDN-3 is highly beneficial for stacking several traits together so that they are inherited as a
single genetic locus; this helps avoid lengthy trait introgression processes and enables efficient
incorporation of traits into elite lines for the development of new varieties. Another benefit of the
precise gene insertion via SDN-3 is the ability to select a favorable genomic location for optimal gene
introduction and expression.
Plants generated through SDN-3 offer reduced regulatory oversight compared to traditional transgenic
products on the market today. The idea being that once the background has been assessed in terms of
characterization of the insertion site, it would not need to be reassessed again for subsequent
applications and instead the assessment would just focus on the inserted gene.
Gaj, T., Gersbach, C.A., Barbas III, C.F., 2013. Zfn, talen, and crispr/cas-based methods for genome
engineering. Trends in Biotechnology 31, 397-405.
Podevin, N., Davies, H.V., Hartung, F., Nogue´, F., Casacuberta, J.M., 2013. Site-directed nucleases: A
paradigm shift in predictable, knowledge-based plant breeding. Trends in Biotechnology 31, 375-383.
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