Disadvantages of randomly induced mutations for reverse genetics

• Mutations induced by insertion, chemical, and physical mutagens are randomly distributed along the

• Large number of mutants (more than number of ) are needed to be created so that all genes are covered

• Difficulty or time/labor consuming in identifying the mutation sites as well as in isolating mutants for every Site specific mutation

• Target mutagenesis is a method that is used to make specific and intentional changes to the DNA sequence of a target gene • Posttranscriptional gene silencing - Antisense RNA - RNA interference (RNAi) • Homologous recombination (HR) • Gene or genome editing - zinc-finger nucleases (ZFNs) - Transcription activator-like effector nucleases (TALENs) - CRISPR-Cas9 Antisense RNA

• Antisense RNA (asRNA), also referred to as antisense transcript, is transcribed from the lagging strand of a gene and is complementary to a specific mRNA or sense transcript with which it hybridizes, and thereby block its translation into protein • asRNA occur naturally and the primary function is to regulate their own gene expression • Notably, more than 30% of annotated transcripts in humans have antisense transcription

Pelechano and Steinmetz, 2013 Antisense RNA

• Antisense transcripts can be classified into short (<200 nucleotides) and long (>200 nucleotides) non-coding RNAs (ncRNAs) • Antisense RNAs can be produced synthetically and have found wide spread use as research tools for gene silencing • https://en.wikipedia.org/wiki/ Antisense_RNA Using antisense RNA to study gene function --inhibition of carotenoid biosynthesis in transgenic tomatoes • Find a candidate gene • During tomato fruit ripening the expression levels of many genes, encoding specific enzyme responsible for the physical changes in the fruit increase

• Several gene cDNA that correspond to ripenning-enhanced mRNAs identified, however function of the encoded protein is unknown

• pTOM5 may be involved in carotenoid biosynthesis

• In this study, antisense RNA to show that the pTOM5 gene product is part of this biosynthetic pathway Inhibition of carotenoid biosynthesis in transgenic tomatoes • A chimeric pTOM5 antisens gene was constructed and then transferred into tomato plants an antisense gene mutation by using Agrobacterium

• Transformed plants have corolla with less intense yellow pigmentation Inhibition of carotenoid biosynthesis in transgenic tomatoes

• The yellow fruit have drastically reduced levels of carotenoids, particular lycopene, compared to normal ripening fruit, indicating that the protein encoded by pTOM5 mRNA is involved in either the synthesis or regulation of synthesis of carotenoids in the fruit Development and commercialization of reduced lignin

• Alfalfa (Medicago sativa) is the most cultivated forage legume in the world • Genetically engineered (GE) glyphosate-resistant alfalfa is deregulated in 2010 • Since 2015, an alfalfa variety with reduced lignin (RL) has been available as a stacked trait with glyphosate resistance • Approximately 15% of the alfalfa currently grown in the U.S. is GE, and this is expected to grow to up to 50% within the next 10 years

Barros et al., 2018 Development and commercialization of reduced lignin

• Lignin reduction was achieved by downregulating the gene encoding caffeoyl-CoA 3-O- methyltransferase (CCoAOMT), and development of the commercial product, branded as HarvXtra

Barros et al., 2018 Development and commercialization of reduced lignin a) With a 5–10 days harvest delay, RL alfalfa harvested on a 35–40 days harvest interval showed 20% gain in forage mass and no significant reduction in RFQ compared to reference cultivars harvested on a 30-day harvest interval b) Reducing the cutting frequency improved the persistence of the stand

Barros et al., 2018 microRNA

• A short (20-30 nt) noncoding RNA • Negatively regulate gene expression by pairing to mRNAs of protein-coding genes to direct their post-transcriptional repression First identified microRNA --lin-4 in C. elegans

• lin-4 and lin-14, induced mutants, show abnormal larva development from L1 to L2 • lin-14 is a protein coding gene

Lin-4 Lin-14

(12h) (7h) (8h) (14h)

(10h)

Lee et al., 1993 First identified microRNA --lin-4 in C. elegans

• lin-4 does not encode a protein, but encodes a pair of small RNA • A 21 nt of lin-4 RNA complementary to a repeated sequence element in the 3︎’ UTR of lin-14 mRNA • lin-4 regulates lin-14 translation via an antisense RNA-RNA interaction, reduce lin-14 protein level without change in level of lin-14 mRNA

Lee et al., 1993 First identified microRNA --lin-4 in C. elegans

• Let-7, another gene in the C. elegans, encodes a 22 bp regulatory RNA found in 2000, which promotes the transition from L4 to adult • Hundreds of microRNA genes and families were identified in diverse organisms

lin-4 lin-14

(12h) (7h) (8h) (14h)

(10h) let-7 lin-41

Lee et al., 1993; Reinhart et al., 2000 microRNA regulates gene expression

• MicroRNAs (miRNAs) assemble with Argonaute proteins into miRNA-induced silencing complexes (miRISCs) to direct post- transcriptional silencing of complementary mRNA targets • miRNAs silence gene expression by repressing translation and accelerating target mRNA degradation • microRNA regulates gene expression microRNA recognize sites

• Conserved pairing to the seed region (7-8 nt) of microRNA

Bartel, 2004, 2009 microRNA identified in diverse species

• In human, more than 5,300 genes involved in diverse biological functions/processes are targeted/regulated by those small RNAs – Cell fate (tissue development) – Cancer • In plants, genes regulated by microRNA are involved in development in plants and abiotic stress

Bartel, 2009 Example 1: MicroRNA control of PHABULOSA in leaf development

Ile Gly Met Lys Pro Gly Pro Asp ------AUU GGG AUG AAG CCU GGU CCG GAU------CCC UAC UUC GGA CCA GGC miR166

Gly (GGU) Asp (GAU) Gly (GGA)

Mallory et al., 2004 Example 2: MicroRNA control of PHABULOSA in leaf development

• Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA- guided cleavage of these mRNAs in vitro (performed or taking place in a test tube, culture dish, or elsewhere outside a living organism).

• Confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb-d mutants. Artificial microRNA (amiRNA) for a specific gene silencing to study gene function

• http://wmd3.weigelworld.org/cgi-bin/webapp.cgi • This tool was initially implemented for Arabidopsis thaliana, but has now been extended to >90 additional species

Schwab et al., The Plant Cell 2006 Example 1: Gene silencing of FT using amiRNA

• amiR-ft-1 targets FT, a gene related to promotion of flowering • The fragment was placed behind a promoter • Transgenic plants were generated by Agrobacterium mediated transformation Wild type -10 amiR--1

Schwab et al., The Plant Cell 2006 MicroRNA and gene therapy

• First FDA approved RNAi therapy Hereditary Transthyretin (TTR) amyloidosis (ATTR amyloidosis)

• Hereditary transthyretin amyloidosis is an autosomal dominant, multisystemic, progressive, life-threatening disease caused from deposition of insoluble ATTR amyloid fibrils in various organs and tissues • Hereditary transthyretin amyloidosis is inexorably progressive, with survival of 2 to 15 years after the onset of neuropathy • TTR is predominantly produced by hepatocytes and circulates as a homotetrameric complex that functions as a transporter for thyroxine and vitamin A • However, TTR monomers can undergo a major conformational transformation to aggregate in a highly ordered and abnormal amyloid fibril form (ATTR); Deposition of wild-type (wt) ATTR typically occurs in older patients, giving rise to wt-ATTR amyloidosis, formerly known as senile systemic amyloidosis

Gofieau et al., 1996; Giaever et al., 2014 Hereditary Transthyretin (TTR) amyloidosis (ATTR amyloidosis)

• Mutations in the TTR gene give rise to variants that destabilize the tetramer such that TTR can more readily undergo the conformational change to amyloid, and these genetic changes underlie the various hereditary ATTR amyloidosis (h- ATTR amyloidosis) clinical syndromes. The liver is the primary source of circulating tetrameric transthyretin protein • The liver is the primary source of circulating tetrameric transthyretin protein • Current treatment options for hereditary transthyretin amyloidosis are limited and include orthotopic liver transplantation and transthyretin tetramer stabilizers (tafamidis or diflunisal). However, many patients who are treated with these approaches continue to have disease progression • Patisiran, a hepatically directed investigational RNAi therapeutic agent, harnesses this process to reduce the production of mutant and wild-type transthyretin by targeting the 3′ untranslated region of transthyretin mRNA Homologous Recombination (HR) induce targeted gene mutation Example 1: deletion project

• Life with 6000 genes – Yeast, single cell, first genome sequence of an eukaryote – Genome size of 12 Mb and 5,885 protein-coding genes • Saccharomyces Genome Deletion Project using homologous recombination (HR) – A complete, systematic deletion collection to identify essential genes and understand gene function • http://www-sequence.stanford.edu/group/ yeast_deletion_project/deletions3.html

Gofieau et al., 1996; Giaever et al., 2014 Example 1: Yeast deletion project

• More than 21,000 mutant strains that carry precise deletions of 6,000 open reading frames • The yeast deletion collection represents the first, systematically constructed deletion collection available for any organism • 18.7% (1105) of the genes proved essential for growth on rich glucose medium • The mutant strains have been used in world-wide laboratories in >1000 genome-wide screens

Giaever et al., Science 1999; Nature 2002; Genetics 2014 Example 1: Yeast deletion project

• Screens include phenotypic annotations of 3,489 genes

Giaever et al., Genetics 2014 Example 2. Gene interaction network in yeast

• Genetic interactions occur when mutations in two or more genes combine to generate an unexpected phenotype • An extreme negative or synthetic lethal genetic interaction occurs when two mutations, neither lethal individually, combine to cause cell death • Constructing more than 23 million double mutants, tested most of the ~6000 genes in the yeast for all possible pairwise genetic interactions, • A positive genetic interaction describes a double mutant that exhibits a fitness that is greater than expected based on the combination of the two corresponding single mutants • Conversely, a negative or synthetic lethal/ sick genetic interaction is identified when a double mutant displays a fitness defect that is more extreme than expected • identifying nearly 1 million interactions, including ~550,000 negative and ~350,000 positive interactions, spanning ~90% of all yeast genes

Costanzo et al., Science 2016 Homologous recombination (HR) does not work well in plants

• High frequency of DNA integration at non-homologous sites by illegitimate recombination • Typically 105 to 107 illegitimate recombination events for every homologous recombination event • Makes it impractical to screen large numbers of transformed plants to identify the rare individuals that have undergone homologous recombination

Wright et al., The Plant Journal 2005 Gene/genome editing

• A little more than 20 years ago, the concept of genome editing (at that point termed gene targeting) came to a new era with the discovery that nucleases could be engineered to create site-specific DNA DSBs that could stimulate homologous recombination more than 1000-fold • The DNA break is the foundation for genome editing and can lead to either gene disruption by error-prone non-homologous end joining (NHEJ) that creates INDELs or precise gene addition or repair by HR using an exogenously supplied repair template with homology to the targeted site • While disruptive NHEJ is largely uncontrollable in terms of genotypic outcome, HR is a precise process with the ability to alter the genome with nucleotide resolution.

Bak et al., Trends in genetics 2018 DNA double strand break and repair

• Non-homologous end joining (NHEJ) • Homology-directed repair (HDR)

Urnov et al. Nature review genetics 2010 Gene/genome editing

• However, HR is also theoretically more difficult to achieve because it mainly operates during the S and G2 phases of the cell cycle when a sister chromatid is available for repair and it is in competition with NHEJ since introduced INDELs or substitutions can prevent further cleavage at the site. • HR relies on efficient and nontoxic delivery of DNA repair templates that need to not only enter cells but also efficiently reach the nucleus • Genome editing is a process that exploits the natural cellular pathways that repair DNA breaks • A key advance was the discovery of engineered nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like (TAL) effector nucleases (TALENs), that can generate site-specific DSBs.

Bak et al., Trends in genetics 2018 Genome editing

• Key requirements for highly specific nucleases – Site-specific recognition of target sequence – Easy to be engineered for user-defined sequences so that any gene can be edited Zinc-Finger domain

• Zinc-Finger domain, a DNA binding domain of a transcription factor • Tandem repeats of such engineered zinc fingers can be used to target desired genomic DNA sequences Zinc-Finger Nucleases (ZFNs)

• Engineered Zinc-finger domains fused to a DNA cleavage domain of a restriction enzyme • One finger recognize 3bp DNA sequence, up to 6 fingers each side • Two adjacent binding events occur in the correct orientation and with appropriate spacing for dimer formation

Urnov et al. Nature review genetics 2010 Disadvantage of Zinc-Finger Nucleases

• One Zinc-Finger recognizes 3 bp, not flexible or easy to be designed for broad target sequences Transcription Activator-Like Effectors (TALE)

• TALEs, a transcriptional activator produced by a plant pathogen • Specifically bind and regulate plant genes • Increases plant susceptibility to pathogen, or trigger plant defense

Bogdanove et al., Current Opinion in Plant Biology 2010 Transcription Activator-Like Effectors (TALE)

• DNA binding domain contains 30 tandem repeats of a 33- to 35- amino-acid-sequence motif • The amino acid sequence of each repeat is invariant, with the exception of two adjacent amino acids (the repeat variable diresidue, RVD) • RVD recognize different DNA base pairs, One-to-one match between the RVDs and the nucleotides in the target DNA sequence

Christian et al., Genetics 2010 Transcription Activator-Like Effectors Nucleases (TALEN)

• Modularity of the repeats enables rapid construction of TALE nucleases (TALENs) with novel specificities to target double-strand breaks at specific locations • Modified TALEs by adopting the molecular architecture used for ZFNs

Christian et al., Genetics 2010 Example: Improving cold storage and processing traits in potato through TALEN editing

• Cold storage of potato is commonly used to reduce sprouting and extend postharvest shelf life, but accumulate reducing sugars (e.g., glucose and fructose)

• The reducing sugars interacted with free amino acid cause brown, bitter-tasting products and elevated levels of acrylamide (a potential carcinogen) Example: Improving cold storage and processing traits in potato through TALEN editing

• Vacuolar invertase gene (Vlnv) encodes a protein that breaks down sucrose to glucose and fructose Example: Improving cold storage and processing traits in potato through TALEN editing

Clasen et al., Plant Biotechnology 2015 Discovery of CRISPR

• Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) was first identified in E. Coli genome in 1987 • Until 2005, it was found that these spacers were homologous to viral and plasmid sequences, suggesting a role in defense mechanism

Horvath and Barrangou, Science 2010 CRISPR/Cas9 in immunity system

• A Cas9 complex cleave the viral DNA and integrate the novel spacer into the CRISPR locus • The CRISPR repeat- spacer is transcribed to crRNA • crRNA bind to a Cas complex and guide Cas to degrade the invading viral DNA

Horvath and Barrangou, Science 2010 CRISPR/Cas9 can be programed for a target DNA sequence

• The Cas9 is guided by two-RNA structure, tracrRNA and targeting crRNA • The target sequence consists of a 20-bp DNA sequence complementary to the gRNA, followed by trinucleotide sequence (5'-NGG-3') called the protospacer adjacent motif (PAM) • The Cas nuclease digests both strands of the genomic DNA 3-4 nucleotides 5' of the PAM sequence

Jinek et al., Science 2012 Engineering quantitative trait variation for crop improvement by genome editing

• Numerous quantitative trait loci (QTL) and genome-wide association studies (GWAS) in both plants and animals have revealed many of the genetic changes driving evolution, domestication, and breeding occurred in cis-regulatory regions • Compared to mutations in coding sequences that alter protein structure, cis- regulatory variants are frequently less pleiotropic and often cause subtle phenotypic change by modifying the timing, pattern, or level of gene expression • Expanding cis-regulatory variation holds promise not only for crop improvement, but also for elucidating principles underlying the control of quantitative traits • In plants, this technology has primarily been used to engineer mutations in coding sequences, with the goal of creating null alleles for functional studies • In this study, the authors generated dozens of novel cis-regulatory alleles for three genes that regulate fruit size, inflorescence architecture, and plant growth habit in tomato

Rodríguez-Leal et al., Cell 2017 Engineering quantitative trait variation for crop improvement by genome editing

• The major feature of tomato domestication was a dramatic increase in fruit size, caused in large part by an increase in the number of locules (seed compartments)

• The ancestor of tomato (S. pimpinellifolium, S.pim) produces small bilocular fruits, and the fasciated (fas) and locule number (lc) QTL were major contributors to increased locule number, and thus fruit size, in domesticated tomato (Solanum lycopersicum, S.lyc) (Figure 1B)

Rodríguez-Leal et al., Cell 2017 Engineering quantitative trait variation for crop improvement by genome editing

• 10% of fruits from S.pim plants carrying a CRISPR/Cas9-induced 4-bp deletion in the CArG element developed three locules (S.pim- lcCR), (Figure 1F)

• Validated in a domesticated tomato variety (S.lyc. cv. M82) whose fruits develop two (60%) or three (40%) locules (Figure 1G). We found that 70% of fruits from S.lyc-lcCR plants carrying a 5-bp deletion in the repressor motif developed three or more locules

Rodríguez-Leal et al., Cell 2017 CRISPR/Cas9 Mutagenesis of the SlCLV3 Promoter Generates Novel cis-Regulatory Alleles

A. Model showing how an allelic series of SlCLV3 transcriptional alleles could provide a range of quantitative effects on floral organ number B. Schematic of SlCLV3 promoter targeted by eight gRNAs (numbered blue arrowheads). C. PCR showing multiple deletion alleles in four T0 plants. Amplicons were obtained using primers spanning the entire target region.

D. Weak and strong effects on flower morphology and fruit size were observed among T0 lines. Number of floral organs and locules are indicated

Rodríguez-Leal et al., Cell 2017 CRISPR/Cas9 Mutagenesis of the SlCLV3 Promoter Generates Novel cis-Regulatory Alleles

• Obtaining homozygous mutants from CRISPR/Cas9 in first generation transgenics is rare • Sequenced the of homozygous T2 progeny and revealed a second allele in T0-1 with a complex rearrangement (designated SlCLV3CR-pro1-2) and a large 7.3- Kbp deletion allele in T0-2 that spanned the SlCLV3 coding sequence (SlCLV3CR- pro2-2)

• Increase in floral organs for SlCLV3CR-pro2-2 homozygotes matched slclv3CR mutants, confirming SlCLV3CR-pro2-2 is a null allele • Plants homozygous for the original T0-1 and T0-2 alleles (SlCLV3CR-pro1-1 and SlCLV3CR-pro2-1, respectively) showed slightly weaker effects than slclv3CR plants, indicating hypomorphic alleles

• SlCLV3CR-pro1-2 homozygotes resembled WT, explaining the weak phenotype of the original biallelic T0-1 plant • These results demonstrate that CRISPR/Cas9 transgenes carrying diverse gRNAs targeting various regions of a promoter can effectively create novel cis-regulatory mutations and alleles with phenotypic effects

Rodríguez-Leal et al., Cell 2017 CRISPR/Cas9 cis-Regulatory Mutagenesis Can Fine-Tune Diverse Traits

Rodríguez-Leal et al., Cell 2017 High-throughput functional genetics using CRISPR/Cas9

Shalem et al., 2015 Shalem et al., 2015 Example: High-throughput functional genetics using CRISPR/Cas9

• Melanoma is the most dangerous type of skin cancer. Globally, in 2012, it occurred in 232,000 people and resulted in 55,000 deaths • ~60% of melanomas carry an activating mutation in the gene encoding the serine–threonine protein kinase B- RAF (BRAF) • 90% of reported BRAF mutations result in a substitution of glutamic acid for valine at amino acid 600 (the V600E mutation)

Flaherty et al., 2010 Example: High-throughput functional genetics using CRISPR/Cas9

• Vemurafenib (known as PLX4032) – A small molecule, is an inhibitor of BRAF with the V600E mutation – Causes programmed cell death in melanoma cell – Vemurafenib received FDA approval for the treatment of late-stage melanoma on August 17, 2011

• Most side effects appeared to be proportional to the dose

Flaherty et al., 2010 Example: High-throughput functional genetics using CRISPR/Cas9

• Goal: What genes are related to the medicine (PLX) responses? • A genome-scale CRISPR/Cas9 knockout (GeCKO) library targeting 18,080 genes, 5 targets per gene • Transduced the human melanoma cell line A375 • Screen for genes whose loss is involved in resistance to vemurafenib

Shalem et al., 2014 Science Example: High-throughput functional genetics using CRISPR-cas9

• Loss of function of NF2 cause resistance to PLX – All 5 targets of the gene showed resistance, not false positive error

Shalem et al., 2014 Science Gene-edited CRISPR mushroom escapes US regulation

• “The mushroom can be cultivated and sold without passing through the agency’s regulatory process — making it the first CRISPR-edited organism to receive a green light from the US government.”

– Waltz, E., 2016. Gene-edited CRISPR mushroom escapes US regulation. Nature News, April 21 Gene editing is different from GMO

GMO Gene eding

Target gene CRISPR-Cas9

Substuon, Transformaon small inseron, Transformaon or deleon

Selfing or Selfing or backcross backcross Potential questions for final exam

• What are antisense RNA, MicroRNA, gene editing, Non- homologous end joining repair, homologous recombination repair? References

• Barros, J., Temple, S. and Dixon, R.A., 2019. Development and commercialization of reduced lignin alfalfa. Current Opinion in Biotechnology, 56, pp.48-54. • Bartel, D.P., 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. cell, 116(2), pp.281-297. • Bogdanove, A.J., Schornack, S. and Lahaye, T., 2010. TAL effectors: finding plant genes for disease and defense. Current opinion in plant biology, 13(4), pp.394-401. • Bortesi, Luisa, and Rainer Fischer. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology advances 33.1 (2015): 41-52. • Christian, Michelle, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186.2 (2010): 757-761. • Clasen, B.M., Stoddard, T.J., Luo, S., Demorest, Z.L., Li, J., Cedrone, F., Tibebu, R., Davison, S., Ray, E.E., Daulhac, A. and Coffman, A., 2016. Improving cold storage and processing traits in potato through targeted gene knockout. Plant biotechnology journal, 14(1), pp.169-176. • Costanzo, M., VanderSluis, B., Koch, E.N., Baryshnikova, A., Pons, C., Tan, G., Wang, W., Usaj, M., Hanchard, J., Lee, S.D. and Pelechano, V., 2016. A global genetic interaction network maps a wiring diagram of cellular function. Science, 353(6306), p.aaf1420. • Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., Dow, S., Lucau-Danila, A., Anderson, K., Andre, B. and Arkin, A.P., 2002. Functional profiling of the Saccharomyces cerevisiae genome. nature, 418(6896), p.387. • Giaever, G. and Nislow, C., 2014. The yeast deletion collection: a decade of functional genomics. Genetics, 197(2), pp.451-465. • Gregan, Juraj, et al. High-throughput knockout screen in fission yeast. Nature protocols 1.5 (2006): 2457-2464.

References

• Horvath, Philippe, and Rodolphe Barrangou. CRISPR/Cas, the immune system of bacteria and archaea. Science 327.5962 (2010): 167-170. • Jinek, Martin, et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337.6096 (2012): 816-821. • Pelechano, V. and Steinmetz, L.M., 2013. Gene regulation by antisense transcription. Nature Reviews Genetics, 14(12), p.880. • Rodríguez-Leal, D., Lemmon, Z.H., Man, J., Bartlett, M.E. and Lippman, Z.B., 2017. Engineering quantitative trait variation for crop improvement by genome editing. Cell, 171(2), pp.470-480. • Shalem, O., Sanjana, N.E., Hartenian, E., Shi, X., Scott, D.A., Mikkelsen, T.S., Heckl, D., Ebert, B.L., Root, D.E., Doench, J.G. and Zhang, F., 2014. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science, 343(6166), pp.84-87. • Shalem, O., Sanjana, N.E. and Zhang, F., 2015. High-throughput functional genomics using CRISPR–Cas9. Nature Reviews Genetics, 16(5), p.299. • Urnov, Fyodor D., et al. Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics 11.9 (2010): 636-646. • Waltz, E., 2016. Gene-edited CRISPR mushroom escapes US regulation. Nature News, 532, p.293. • Waterhouse, P.M., Graham, M.W. and Wang, M.B., 1998. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proceedings of the National Academy of Sciences, 95(23), pp.13959-13964. • Wright, D.A., Townsend, J.A., Winfrey, R.J., Irwin, P.A., Rajagopal, J., Lonosky, P.M., Hall, B.D., Jondle, M.D. and Voytas, D.F., 2005. High‐frequency homologous recombination in plants mediated by zinc‐finger nucleases. The Plant Journal, 44(4), pp.693-705.