Genetic “Engineering” for Crop Improvement Xiwen Cai North Dakota State University, Fargo, ND

Outline Definition of genetic engineering Genetic engineering at the whole plant level Genetic engineering at the cell level Genetic engineering at the chromosome level Genetic engineering at the DNA level

Engineering Engineering is the application of scientific, economic, social, and practical knowledge in order to invent, design, build, maintain, research, and improve structures, machines, devices, systems, materials, and processes. wikipedia.org

“Genetic Engineering” Genetic engineering, also called genetic modification, is the direct manipulation of an organism's genome using biotechnology. New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. An organism that is generated through genetic engineering is considered to be a genetically modified organism (GMO). wikipedia.org

Genetic Engineering Genetic engineering, also called genetic modification, is the manipulation of an organism's genome at whole plant, cell, chromosome, and DNA/RNA levels using conventional and modern genetic, cytogenetic, and genomic technologies. The end products of genetic engineering are genetically modified organisms (GMOs) no matter how they are modified genetically.

Are “Genetically Modified” Crops And Foods New? We have been genetically modifying plants and animals for a very long time or since the dawn of civilization! Almost all crop plants have been extensively modified genetically compared to their wild relatives.

Genetic Engineering At Whole Plant Level Conventional breeding – homologous meiotic recombination-based - Select parents and make crosses - Homologous meiotic recombination - Select recombinants of interest - Evaluate traits of recombinants - Release varieties

Genetic Engineering At Whole Plant Level Mutation breeding (perennials and others with narrow genetic variation…) - Induce mutations - Select mutants of interest - Evaluate traits of mutants - Release varieties

Genetic Engineering At Cell Level Cell/tissue culture - Structural variation in chromosomes and DNA Nuclear substitution - Nucleus-cytoplasm interaction - Male sterility and heterosis in crop production

Genetic Engineering At Chromosome Level Induce homoeologous meiotic recombination

Goatgrass-Derived Rust Resistance Gene Genetics 187:1011-1021 (2011)

Genetic Engineering At Chromosome Level Induce chromosome mutations - Irradiation-induced Chen et al. 2008. Science in China Series C: Life Sciences:51: 346-352

Genetic Engineering At Chromosome Level Induce chromosome mutations - Gametocidal chromosome-induced Endo and Gill, J. Hered 87:295-307 (1996)

Genetic Engineering At Chromosome Level Induce chromosome mutations - Misdivision-induced Friebe et al. Cytogenet Genome Res 109:293–297 (2005)

Genetic Engineering At Chromosome Level Manipulate chromosomal behavior in meiosis and mitosis - Meiotic restitution

Genetic Engineering At Chromosome Level Manipulate chromosomal behavior in meiosis and mitosis - Wide hybridization-induced chromosome elimination e.g. Wheat x Maize hybridization system for wheat haploid production – doubled haploid mapping population

Genetic Engineering At Chromosome Level Manipulate ploidy level - haploidy, diploidy, and polyploidy - Chemical-induced chromosome doubling - Meiotic restitution-induced chromosome doubling - Wide hybridization-resulted chromosome elimination

Genetic Engineering At DNA Level Transformation

Genetic Engineering At DNA Level Viral infection, microinjection, and bombardment with DNA-coated tungsten particles Griffiths et al. 1999

Genetic Engineering At DNA Level Genome editing Artificially engineered nuclease ("molecular scissor”)-mediated genome modifications, including insertion, substitution, deletion, and inversion, etc. Create site-specific double-stranded breaks (DSBs) by engineered nucleases Repair DSBs and bring in DNA sequence changes by homologous recombination (HR) and nonhomologous end-joining (NHEJ)

Genetic Engineering At DNA Level Genome editing - Create site-specific DSBs Engineered nucleases: Meganucleases – Recognize long DNA sequence (14-40 bp) and many engineered versions: less toxicity and high costs Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) – Restriction endonuclease consisting of a specific DNA-binding domain at the N-terminal and a non-specific DNA cleavage domain at the C-terminal – Protein-guided ZFNs are generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences. TALENs are generated by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. Transcription activator-like effectors (TALEs) can be quickly engineered to bind practically any desired DNA sequence. wikipedia.org

Genetic Engineering At DNA Level Genome editing - Create site-specific DSBs Engineered nucleases: wikipedia.org Protein-guided nucleases

Genetic Engineering At DNA Level Genome editing - Create site-specific DSBs Engineered nucleases: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system – RNA-guided

Genomic Structures of the CRISPR-Cas Systems in Streptococcus thermophilus Bacterium The cas genes are represented in gray, and the repeat-spacer array in black. The repeat and spacer (captured phage or plasmid nucleic acid) are detailed as black diamonds (T, terminal repeat) and white rectangles, respectively. Bottom line, consensus repeat sequence. L1 to L4, leader sequences. The predicted secondary structure of the CRISPR3 repeat is shown on the right. Science 327: 167-170 (2010)

CRISPR-Cas as an Immune System in Bacteria and Archaea Overview of the CRISPR/Cas mechanism of action. (A) Immunization process: After insertion of exogenous DNA from viruses or plasmids, a Cas complex recognizes foreign DNA and integrates a novel repeat-spacer unit at the leader end of the CRISPR locus. (B) Immunity process: The CRISPR repeat-spacer array is transcribed into a pre-crRNA that is processed into mature crRNAs, which are subsequently used as a guide by a Cas complex to interfere with the corresponding invading nucleic acid. Repeats are represented as diamonds, spacers as rectangles, and the CRISPR leader is labeled L. Science 327: 167-170 (2010)

Natural and Engineered CRISPR-Cas9 System (a) Naturally occurring CRISPR systems incorporate foreign DNA sequences into CRISPR arrays, which then produce crRNAs (CRISPR RNA) bearing “protospacer” regions that are complementary to the foreign DNA site. crRNAs hybridize to tracrRNAs (transactivating CRISPR RNA; also encoded by the CRISPR system) and this pair of RNAs can associate with the Cas9 nuclease. crRNA-tracrRNA:Cas9 complexes recognize and cleave foreign DNAs bearing the protospacer sequences. (b) The most widely used engineered CRISPR-Cas system utilizes a fusion between a crRNA and part of the tracrRNA sequence. This single gRNA complexes with Cas9 to mediate cleavage of target DNA sites that are complementary to the 5′ 20 nt of the gRNA and that lie next to a PAM (protospacer adjacent motifs) sequence. (c) Example sequences of a crRNA-tracrRNA hybrid and a gRNA. Nature Biotechnology 32: 347–355 (2014)

Genetic Engineering At DNA Level Genome editing - DSB repair DSB repair pathways: Non-homologous end joining (NHEJ) and homology directed repair (HDR) Nature Biotechnology 32: 347–355 (2014)

Applications of the CRISPR-Cas9 System in Genome Editing and Other Genome Studies (a,b) gRNA-directed Cas9 nuclease can induce indel mutations (a) or specific sequence replacement or insertion (b). (c) Pairs of gRNA-directed Cas9 nucleases can stimulate large deletions or genomic rearrangements (e.g., inversions or translocations). (d–f) gRNA-directed dCas9 can be fused to activation domains (d) to mediate upregulation of specific endogenous genes, heterologous effector domains (e) to alter histone modifications or DNA methylation, or fluorescent proteins (f) to enable imaging of specific genomic loci. TSS, transcription start site. Nature Biotech 32: 347–355 (2014)

Application of the CRISPR-Cas9 System in Plants (a) Assay scheme. (b) DNA gel with PCR bands obtained upon amplification using primers flanking the target site within the PDS gene of N. benthamiana. In lanes 1–3 the template genomic DNA was digested with MlyI, whereas in lane 4 nondigested genomic DNA was used. (c) Alignment of reads with Cas9-induced indels in PDS obtained from lane 1 of b. The wild-type sequence is shown at the top. The sequence targeted by the synthetic sgRNA is shown in red whereas the mutations are shown in blue. PAM, the protospacer-adjacent motif, was selected to follow the consensus sequence NGG. The changes in length and sequence are shown to the right. Nature Biotech 31: 691-693 (2013)

Acknowledgements Postdoctoral researchers, graduate students, visiting scientists, technicians, and undergraduate helpers in my lab My collaborators at NDSU, USDA-ARS, and other institutions in the US and other countries Funding agencies: USDA-NIFA-AFRI USWBSI National Science Foundation ND Wheat Commission

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