Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
The recent discovery and development of the CRISPR/Cas9 system for genome editing has transformed the molecular-biology landscape. The technology allows researchers to knock out or alter gene sequences in live organisms efficiently with a bacterial nuclease (Cas9) and a short guide RNA to direct the nuclease where to cut.
The CRISPR/Cas9 system originated in bacteria, where it serves as a form of adaptive immunity. But it works in a range of organisms, from primates to pathogens, enabling researchers to engineer genomes even in organisms that have less well-established genetic toolsets than mice and man—including plants [1, 2].
A 2014 review detailed that “[most] studies to date have been conducted in animal systems, but CRISPR/Cas9-mediated mutagenesis was recently demonstrated in Arabidopsis, tobacco, sorghum, rice and wheat, proving that this technique is applicable to both dicot and monocot plants” [1].
Here’s what you need to know when considering whether to use this powerful technology on plant systems.
Special delivery
CRISPR/Cas9 uses an RNA-guided nuclease to introduce DNA cleavage at a user-defined location. That event causes cellular machinery to try to fix the damage, either via nonhomologous end joining (NHEJ), a process that tends to introduce frame-shift mutations, and thus is used to create genetic knockouts, or homology-directed repair (HDR). In the latter case, researchers can guide the repair using a template DNA sequence, enabling them, for instance, to correct a mutation or insert a reporter gene. However, NHEJ tends to occur more frequently than HDR, meaning researchers often need to screen multiple clones to find one that has been correctly repaired.
The process works more or less identically in every organism studied, including plants. But plant researchers face problems other model systems do not. In particular, says Dan Voytas, director of the Center for Genome Engineering at the University of Minnesota and chief science officer at Calyxt, a company that develops genome-engineering technologies for agricultural applications, there’s the problem of nucleic acid delivery.
“You need to get [DNA] into cells or tissues from which you can recover a whole plant,” Voytas says.
Though some plants, like potato, tobacco and canola, are relatively simple to manipulate, others are notoriously difficult, Voytas explains. DNA delivery mechanisms that work for mammalian cells, such as electroporation and lipid transfection, rarely work on native plant cells, if for no other reason than their cell wall. Instead, plant researchers typically must rely on Agrobacterium infection, gene guns (“biolistics”) or “protoplasts” (plant cells from which the cell wall has been removed). More frustratingly, conditions that work for one species may not work for another. “The method for transforming rice is very different from transforming tomato or soybean,” Voytas says.
However the DNA is delivered, researchers then need to identify correctly modified cells and coax them back into a whole plant. And therein lies the other major difficulty, says Fuqiang Chen, principal R&D scientist at Sigma-Aldrich, a company that commercializes several genome-editing technologies: Plant regeneration—the equivalent of growing a genetically modified mouse from an embryo—can be painfully tedious.
“With mammalian cells you can do proof-of-concept experiments in two or three days. But plants are much more difficult,” Chen says. “Growth cycles are longer, transformation efficiency is low, and the protocols are not very friendly.”
In one recent study, Voytas and colleagues used an earlier genome-engineering strategy called TALENs to improve the tolerance of “Ranger Russet” potatoes for cold storage [3]. The team generated protoplasts, transformed with a plasmid encoding the TALEN proteins, and grew the transformants into whole plants. It took from 12 weeks to several months to produce organisms with shoots that could be harvested, at which point the plants had to be transferred to “rooting medium” for continued growth.
Another issue, Voytas says, is that plant researchers rarely use selectable markers, as agricultural clients are sensitive to consumers’ fear of genetically modified organisms (GMOs). Thus, these researchers often use transient expression and screen hundreds of organisms to find the desired transformants. In the potato study, for instance, Voytas’ team screened 600 shoots to find five that had only the desired mutations and no TALEN transgene integration into the host genome.
That said, Voytas’ study also highlights a significant advantage of genome-engineering technologies for plant researchers—the ability to target multiple alleles. Many plants are polyploid, meaning they carry multiple copies of a gene. Mammals are diploid (meaning a successful gene knockout requires hitting two copies of a gene), but plants can carry higher numbers, and genome-editing tools can hit them all. The potato gene Voytas targeted was tetraploid. Another recent study, which used CRISPR/Cas9 and TALENs to make mildew-resistant wheat, successfully knocked out six alleles in one organism by creating and crossing heterozygous strains [4].
Getting started
If you’re interested in using CRISPR/Cas9 in your own research, there’s no shortage of options. Reagents are available from Addgene, Agilent Technologies, Clontech, GE Dharmacon, Horizon Discovery, Origene, Sigma-Aldrich and Thermo Fisher Scientific, among others.
Sigma-Aldrich, for instance, offers a range of CRISPR/Cas9 plasmids, RNA-only formats, lentiviral particles and pooled CRISPR libraries, vectors and delivery systems. Agilent offers purified Cas9 protein and kits for guide-RNA transcription in vitro—tools useful mostly for in vitro experimentation. GE Dharmacon has the CRISPR RNA configurator program, which intuitively assists researchers in designing crRNAs by letting them simply enter the gene of interest.
Perhaps the broadest toolkit available today is that of Addgene, a nonprofit reagent repository. According to PLOS Biology, Addgene’s collection, as of June 2014, comprised “400 plasmids from 44 different labs” [5]. Joanne Kamens, the nonprofit’s executive director, says Addgene’s CRISPR catalog has since grown to represent about 50 labs and “a couple thousand” reagents, including both validated guide RNAs, empty vectors for guide-RNA cloning and Cas9-expression plasmids.
Most of those are not plant-specific, of course, but the collection does include 32 plant-specific plasmids from eight laboratories, Kamens says, including these [6]. Among the most popular are plasmids that express Cas9 and GFP via a plant-specific promoter and an empty backbone for expressing guide RNAs.
Addgene reagents are available to U.S. customers for $65 apiece (or less for larger orders), so the barrier to entry on CRISPR/Cas9 experimentation is low. But again, says Voytas, the delivery issue looms large. He advises researchers to spend time developing and optimizing media and protocols before dipping their toes in CRISPR technology. After all, the best plasmids in the world are useless if you cannot get them where they’re needed.
Says Voytas, “Genome engineering won’t go anywhere until you can get your DNA into the plant cell or tissue and then recover a plant from the [transformed] cell.”
References
[1] Lozano-Juste, J, Cutler, SR, “Plant genome engineering in full bloom,” Trends Plant Sci, 19:284-7, 2014. [PubMed ID: 24674878]
[2] Belhaj, K, et al., “Plant genome editing made easy: Targeted mutagenesis in model and crop plants using the CRISPR/Cas system,” Plant Methods, 9:39, 2013. [PubMed ID: 24112467]
[3] Clasen, BM, et al., “Improving cold storage and processing traits in potato through targeted gene knockout,” Plant Biotechnol J, 2015. doi:10.1111/pbi.12370. [PubMed ID: 25846201]
[4] Wang, Y, et al., “Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew,” Nat Biotechnol, 32:947-51, 2014. [PubMed ID: 25038773]
[5] Kamens, J, “Addgene: Making materials sharing ‘science as usual,’” PLOS Biol, 12:e1001991, 2014. [PubMed ID: 25387006]
[6] Xing, HL, et al., “A CRISPR/Cas9 toolkit for multiplex genome editing in plants,” BMC Plant Biology, 14:327, 2014. [PubMed ID: 25432517]