Lecture 16 The future of plant biotechnology: genome editing and concluding perspectives Neal Stewart & Agnieska Piatek

Discussion questions What is the main dichotomy between innovation and caution (or risk, or the perception of risk)? What is genome editing? What are the current tools for genome editing? What are zinc-finger nucleases, TALENs, and CRISPR? How might they alter the future of plant biotechnology? How do feelings and trust influence plant biotechnology?

Problems in plant biotechnology: might be addressed with new technologies Agrobacterium- and especially biolistics-mediated transformation are imprecise: many transgenic events must be produced. Transgenic plants are regulated because they are transgenic- is there another way? Hint: genome editing. What is synthetic biology? Will it help solve problems or complicate them?

Figure 17.3 An abstraction hierarchy that supports the engineering of genetic systems. The parts [such as promoters, ribosome binding sites (RBS) in prokaryotes, coding regions and terminators] are DNA sequence-based and sometimes context-dependent, but can be engineered rationally to produce different devices such as inputs, logic gates and outputs, which permit assembly into artificial systems for further practical, desirable applications.

Figure 17.8

Genome editing Altering the sequence of DNA “in situ” Targeted mutagenesis Knock-outs Point mutations Gene insertions or “trait landing pads” Ideally leaving no transgene footprint Is genome engineering plant breeding, genetic engineering or both?

Genome editing tools in plants Meganucleases: 1990s Oligonucleotide-directed mutagenesis: late 1990s Zinc finger nucleases (ZFNs): mid 2000s Transcription activator like effector nucleases (TALENs): early 2010s Clustered regularly spaced short palindromic repeats (CRISPR): 2013

Zinc finger nucleases www.bmb.psu.edu, www.wpclipart.com, www.faculty.ucr.edu

Figure 17.5 Figure 17.5 Engineered Zinc-finger nucleases (ZFNs; a) and transcription activator-like effector nucleases (TALENs; b) for targeted genome modification (Reprinted from Liu et al., 2013b). Each nuclease contains a custom-designed DNA binding domain and the non-specific DNA-cleavage domain of the FokI endonuclease which has to dimerize for DNA cleavage within the spacer regions between the two binding sites. The spacer regions between the monomers of ZFNs and TALENs are 5-7 bp and 6-40 bp in length, respectively.

ZFNs in gene therapy Nature 435:577

Transcription activator-like effectors (TALEs) Transcription factors Secreted by Xanthomonas bacteria via type III secretion system Bind promoter sequences in the host plant Recognize plant DNA sequences Plant cell TF TF TF Gene Gene Promoter Nucleus www.plantwise.org

Structure and DNA binding code of TALEs Central repeat domain AD NLS L T P E Q V V A I A S H D G G K Q A L E T V Q R L L P V L C Q A H G 1 34 12 13 TS Repeats (1.5 to 33.5) Repeat variable diresidue (RVD) NG = T HD = C NI = A NN = G or A DNA binding code 12 13

TCCCAAATTTGATCG How to engineer TALEs? = C Engineered central repeat domain N’ TS C’ AD NLS HD NI NG NK DNA Target TCCCAAATTTGATCG HD Prerequisite of T nucleotide preceeding the DNA target sequence = C NI = A NG = T NK = G

Why engineer TALEs? For targeted genome mutagenesis and editing: TALEN B TALEN A 5’ 3’ 5’ 3’ Target sequence A Target sequence B 5’ 3’ FokI dimer Outcome: Deletion Chromosomal deletion Point mutation Insertion AACGT TTGCA

Why engineer TALEs? V X = or For targeted genome regulation TFs TALE-TF Modulator V X mRNA transcripts 5’ 3’ 3’ 5’ Promoter Target sequence Gene Modulator = or Repressor Activator

RNA based genome engineering platform PAM

CRISPR – Bacterial Immunity Clustered Regularly Interspaced Short Palindromic Repeats Acquired adaptive immunity in bacteria against viruses First described in 1987 Streptococcus pyogenes CRISPR array tracrRNA Cas9 Cas1 Cas2 Csn2 spacers direct repeats

Mechanism of CRISPR-mediated immunity in bacteria 3. Interference Virus DNA Plasmid DNA 1. Acquisition leader 1 2 3 4 n Cas CRISPR array Cas locus crRNA Pre-crRNA 2. Expression Cas proteins

CRISPR genome editing

CRISPR variants DNA surgeon.With just a guide RNA and a protein called Cas9, researchers first showed that the CRISPR system can home in on and cut specific DNA, knocking out a gene or enabling part of it to be replaced by substitute DNA. More recently, Cas9 modifications have made possible the repression (lower left) or activation (lower right) of specific genes. Published by AAAS E Pennisi Science 2013;341:833-836

Last questions of the semester Is food too hot (emotionally) to be addressed by biotechnology? Where on earth? What is the scientist’s role here? What about non-food plant biotechnology such as bioenergy? What about genome editing?

“Ordinary tomatoes do not contain genes, while genetically modified ones do” 1996 - 1998 People in different countries have varied knowledge about the facts of genetics and biotechnology. Slide courtesy of Tom Hoban

American consumers’ trust in biotechnology information sources Slide courtesy of Tom Hoban

Source of information trusted most to tell the truth about biotechnology (includes all European countries) Slide courtesy of Tom Hoban

Way forward Technological innovations will continue Until disaster strikes (think Fukushima) And then innovations will resume But they will be (more) regulated So there is a balance between innovationpotential risks and regulationensure safety

Trends Plant biotech plant synthetic biology Designed components More precise gene integration and regulation Building a crop from scratch? Transgenicsgenome editing Regulations? What about a tiered approach? Increasing gap between public’s knowledge of science and technology and science and technology advances But…patents expire and economics shift…times change

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