Jeppe Thulin Østerberg, PhD-student, firstname.lastname@example.org and Michael Broberg Palmgren, professor, dr. scient. email@example.com, Institute for Plant and Environmental Sciences, University of Copenhagen
CRISPR/Cas9 is the new rising star among methods in molecular biology. The technique is a nuclease that is pre-programmed to identify a specific DNA sequence and cleave it. This ability to cut DNA in a selected location enables the molecular biologist to make use of the DNA repair mechanisms native to all cellular life. CRISPR gene editing hit plant biology three years ago, and since then the tool has been promising for application in plant research including basic science and targeted plant breeding in agriculture.
DNA repair mechanisms
When genomic DNA is broken in a living cell, mechanisms will activate to repair the break. These mechanisms are broadly labeled non-homologous end joining (NHEJ) and homology directed repair (HDR). Non-matching ends of broken DNA strands are joined, and the breaks are repaired by copying from any similar DNA that might be nearby – most often the complementary strand on the second copy of the same chromosome. In common for these repair mechanisms is that they can be used for editing the cleaved sequence.
In the case of NHEJ, the joining of non-homologous strands is error prone, and often leads to insertions, deletions, or substitutions of DNA in the programmed cut site. These are fairly random, although they most often tend to be insertions or deletions of 1 to 20 base pairs. HDR is used to deliver a partially complementary DNA strand to the cut site, enabling the cellular repair mechanisms to repair the Cas9 induced cut, by copying off the delivered DNA strand. The sequence around the programmed cut site is altered by altering the delivered DNA strand i.e. genome editing.
CRISPR revolutionizes programmable nucleases
Programmable nucleases have been examined since the 1980s, and include systems such as the zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). ZFNs and TALENs are able to target and cleave double stranded DNA. However, they are more demanding to program, as the DNA recognizing entity consists of subsets of nucleotide-recognizing peptide sequences. Each of such newly programmed nucleases requires the de novo construction of a specific peptide.
The CRISPR system was a revelation when it was published in 2012 (1,2). Programming of CRISPR/Cas9 requires only the fusion to a RNA strand complementary to the required cleavage site. The Cas (CRISPR associated) protein remains the same. This reduces the cost of programming for the endonuclease, going from a series of cloning events to construct a new protein, to simply design and order an oligonucleotide and clone it into a predesigned plasmid.
The CRISPR/Cas9 programmable nuclease system is derived from the prokaryotic adaptive type II immune system. It uses the ability of subtypes of the Cas protein family to function as RNA guided endonucleases, when merged with a specific type of guide RNA. In the bacterial immune system, the Cas systems are preprogrammed with virus DNA recognizing RNA from previous viral infections, and render immunity against previously experienced virus by recognizing and cleaving viral DNA as soon as it enters the cell. In 2013 the first applications of targeted mutagenesis in model and crop plants using the CRISPR/Cas system in plant genome editing were published (e.g. 3). Already in this first wave of publications, plants were transformed with the CRISPR/Cas systems using both NHEJ and HDR.
Plant CRISPR in practice
Practical usage of the CRISPR/Cas system in plant genome editing has identified two hurdles. First, NHEJ is much more feasible than HDR since it is easier to use CRISPR as a hammer on a gene by destroying it than to do text editing by introducing single point mutations. Furthermore, introducing recessive mutations that confer loss-of-function by deleting the gene is easier than introducing dominant mutations leading to gain-of-function genes with new abilities. Second, when introducing the CRISPR/Cas9 machinery into plants by transformation, the resulting plant often contains a mixture of transformed and non-transformed plant cells. When mosaic plants develop it is not certain that the germ cells contain the construct and that the introduced changes are inheritable.
Use of knockout plants for basic research
Homologous recombination into plant genomes has historically had low success rates. Accordingly, knockout plants, where specific genes have been turned off, are investigated by using plants from large gene banks of randomly generated mutant plants. Such libraries of plant mutants where the affected gene has been identified serve as a biological resource center for researchers studying the function of specific plant genes. However, aside from well-established model plants such as Arabidopsis thaliana (Figure 1), where mutant libraries are well established, the access to mutant libraries of other plant species is limited.
The ability of CRISPR-Cas9 gene editing to knockout genes directly, has the potential to replace the construction of new plant mutant libraries in basic plant research. An example is the use of CRISPR-Cas9 in the examination of symbiotic nitrogen fixation related genes in Lotus japonicus, a group of plants where only a few mutant lines exist (4).
Another inherent issue in plant research using knockout mutants is the complex ploidy level of several main crop plants. Bread wheat (Triticum aestivum) is hexaploid and has several copies of the same genes. Investigation of the role of specific genes using gene inactivation often requires knockout of all gene copies to see an effect. The task of generating plants with knockouts of all copies of a gene is an arduous task. In contrast, the design of programmable nucleases enables to target DNA sequences that are common for all copies of the same gene in order to acquire a full knockout plant.
Use of CRISPR in agriculture
Although the field of modern plant breeding encompasses a large array of methods to achieve crops of interest, it is in part based on the examination of randomly mutagenized mutant plants and the focused breeding of desired qualities that emerge in these mutants. Random mutations have for a long period of time been uncharted and solely selected based on the resulting traits. Genetic analysis has led to understanding of the underlying genetic traits that cause mutations we deem desirable in crops, and which we in part determine as domestication traits. It also stems from the knowledge accumulating from research of knockout plants, especially in the major model crop plants such as rice, maize and wheat. This information enables plant breeders to target genes for knockout by genetic screening of mutant libraries.
Use of the CRISPR/Cas9 gene editing holds the potential to speed up plant breeding efforts significantly. The use of this information has already been used to abolish mildew infection on hexaploid wheat, by knock out of the host recognition factors that enable the mildew fungus to recognize wheat as its host. Furthermore, targeting genes responsible for production or transport of toxic plant compounds can be used to create less toxic versions of crops like quinoa and cassava that today require lengthy processing before they are ready for human consumption.
As the CRISPR technology matures in plant biology, it holds potential to perform feats of genetic engineering that will likely enable breeders to create desirable traits from ancient crop types, such as fertilization requirements or disease resistance (5). CRISPR gene editing is still young in the plant field, but the coming decade will likely yield a plethora of applications that might change both the way crop plants are made and which crops we grow.
1. Gasiunas G, Barrangou R, Horvath P, Siksnys V Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America. 2012;109(39): E2579–86.
2. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096): 816–21.
3. Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S: Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 2013;31: 691-693.
4. Wang L, Wang L, Tan Q, Fan Q, Zhu H, Hong Z, Zhang Z, Duanmu D. Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9. Front Plant Sci. 2016;7:1333. 5. Palmgren MG, Edenbrandt AK, Vedel SE, Andersen MM, Landes X, Østerberg JT, Falhof J, Olsen LI, Christensen SB, Sandøe P, Gamborg C, Kappel K, Thorsen BJ, Pagh P. Are we ready for back-to-nature crop breeding? Trends Plant Sci. 2015;20:155-64.
The article was published in BioZoom no. 4 2016.