Genetic engineering methods

Steve Strauss Steps in genetic engineering Genetic engineering vastly increases the genetic diversity available for breeding

Difficulties of inter-species crosses

F. Nogue – INRA France Overview of genetic engineering

Traditional x plant breeding

Variety Variety A B

Genetic x engineering any gene source What are GMOs? • GMO = genetically modified organism – Same as GE or GEO = genetic engineering = creation of recombinant DNA modified organisms • It’s the method: Native or “foreign” genes, modified traits or new traits • Genes isolated in chemical form, changed in a test tube, and re-inserted asexually – Vs. making crosses or random mutations in conventional breeding • Powerful breeding tool – relatively simple traits can be designed – but without constraints from native gene pools – That’s why its called genetic engineering, though we are modifying, not building, a new organism The acronyms, evolving in meaning • GE (genetic engineering) = GM (genetic modification) = asexual modification and/or insertion of DNA

GMO = genetically modified organism GEO = genetically engineered organism

The terms “biotechnology” or “modern biotechnology” often equated with GE or GM methods

Transgenic = GE, or transfer of genes between distant species Cisgenic, intragenic for transfer or modification of genes from closely related species How are GMO crops produced

Step 1 Getting whole plants back from cultured cells = organismal cloning Differentiation of new plant organs (shoots, roots, embryos) from single cells First step is de- differentiation into “callus” after treatment with the plant hormone auxin

Leaf-discs Shoots produced first, then roots, using specific plant hormones for each step Somatic embryogenesis – shoot-root axis differentiated as a unit

Immature cotyledon Somatic embryos

Repetitive embryogenesis = cloning Step 2 Getting DNA into plant cells

Main methods • Agrobacterium tumefaciens • Biolistics [gene gun] Agrobacterium is a natural plant genetic engineer Agrobacterium engineering

Gene of interest

T-DNA

Ti Plasmid Engineered plant cell

Agrobacterium tumefaciens Historically, each insertion was unique in terms of where it landed in plant genome Cocultivation of Agrobacterium with plant tissues Agrobacterium in contact with wounded plant tissues during cocultivation Gene gun bombardment of plant tissues in Petri dish DNA coated metal particles after “gene-gun” insertion into tissues Transgenic cassava via biolistics - GUS reporter gene Step 3 Selection of transgenic cells Only a few cells get engineered

Challenge: Recover plants from that one cell so new plant is not chimeric (i.e., not genetically variable within the organism) Antibiotics in plant tissue culture limit growth to engineered cells Other kinds of genes can also be used to favor transgenic cells (e.g., sugar uptake, herbicide resistance, hormone sensitivity) Antibiotic selection of transgenic tissues Then plants are propagated normally (seeds, cuttings) and tested for health and new qualities

Propagation of poplars Growth in the field in tissue culture Genetic engineering is defined as…

A. Transfer of genes from one species to another B. Modifying genes to produce new traits C. Asexual modification of inherited DNA D. A method with risks and benefits distinct from that of conventional breeding E. Creation of novel-appearing life forms Why bother with in vitro culture when doing GE, when we can simply treat whole plants with Agrobacterium (like happens in Nature)?

A. Resulting plants would not be sterile B. Its just routine scientific technique C. The resulting plants would not be genetically homogenous D. Plants (but not animals) have cell walls that stop Agrobacterium gene transfer E. Antibiotic selection is much less efficient vs. that in Petri dishes Poor control over transgene insertion • Location in genome ~random – Chromosomal environment important to level and pattern of expression, not just promoters – Expression varies a great deal among gene insertion events • Also varying among events – Number of copies varies from one to dozens – Orientation of gene varies – Genes can be turned off, or be unstable Large consequences of poor transgene/transformation control for biotechnology • Gene transfer and tissue culture process is itself mutagenic – Insertion into/near genes – Increased random mutation in genome from stress of hormones/new developmental path • Thus, extensive selection after gene insertion for desirable events prior to commercial use – Hundreds screened, over many generations • Event = unit of regulation worldwide Interpreting significance of GE’s unintended effects on genome • How does it compare to conventional breeding? – Lots of unintended genetic change in making hybrids, inbreeding, random mutagenesis • Lots of genetic variation in gene expression and gene content in nature – Gene presence and absence highly variable • No urgency to regulate traditional breeding comparable to GE in spite of this Time to end event-specific regulation? No greater unintended impacts from GE vs. breeding Coming: Gene editing technology for diverse traits

CRISPRs

TALENs

Adapted from Pennisi, Science, 2013) CRISPRs: Predictable, stable, certain change of DNA sequence ~50% biallelic mutation rate for genes in poplar

Wild type`

Non-mutants

Insertion

Mutants

Data from Estafania AG-2 AG-1 Elorriaga, PhD target site Small deletions target site student, OSU Large deletion Three types of Site Directed Nuclease (SDN) “genome editing” Genetic engineering has a similar function to introgression – “gene purification”

Recombinant DNA (or GM) allows a single gene to be introduced into a genome. This method can be faster and more precise than conventional breeding Elite tomato Poor tomato but disease resistant

Elite, disease resistant tomato Source of gene Once a gene is (disease-resistant introduced into the plant) plant genome it functions like any Gene of interest other gene

Isolate gene of interest using molecular biology Recombine into methods recipient plant DNA Why are GE methods used sometimes and molecular breeding others?

Molecular breeding

1. Desired trait must 2. Genetic resources 3. Plant should be be present in must be available to propagated population or a wild breeders (compatible sexually, and relative that can relatives) efficiently hybridize

Photo credits: Gramene.org Why are GM methods used sometimes and molecular breeding others?

Molecular breeding

1. Desired trait must be 2. Genetic resources 3. Plant should be present in population must be available propagated sexually GM

3. Plant can be 1. Gene can come from 2. Genetic resources not propagated vegetatively; any source or created required anew sex not needed

Photo credits: Gramene.org ETH Life International Summary • Genetic engineering requires knowledge of gene-trait connection, and both insertion and regeneration methods – Biological and physical vectors for gene insertion • Terms used to define genetic engineering evolving, confusing – Trans – vs. cis/intra-genic • Much variation among insertions with “old” methods • Genome editing targets gene changes and insertions with high efficiency