Precision genome engineering and agriculture: opportunities and regulatory challenges

Abstract

Plant agriculture is poised at a technological inflection point. Recent advances in genome engineering make it possible to precisely alter DNA sequences in living cells, providing unprecedented control over a plant's genetic material. Potential future crops derived through genome engineering include those that better withstand pests, that have enhanced nutritional value, and that are able to grow on marginal lands. In many instances, crops with such traits will be created by altering only a few nucleotides among the billions that comprise plant genomes. As such, and with the appropriate regulatory structures in place, crops created through genome engineering might prove to be more acceptable to the public than plants that carry foreign DNA in their genomes. Public perception and the performance of the engineered crop varieties will determine the extent to which this powerful technology contributes towards securing the world's food supply.

Figure 1. Schematics of the four classes of sequence-specific nucleases.

(A) The meganuclease, I-SceI, is shown bound to its DNA target. The catalytic domain, which also determines DNA sequence specificity, is shown in red. (B) A ZFN dimer is illustrated bound to DNA. ZFN targets are bound by two zinc-finger DNA binding domains (dark blue) separated by a 5–7-bp spacer sequence. FokI cleavage occurs within the spacer. Each zinc finger typically recognizes 3 bp. (C) Depicted is a TALEN dimer bound to DNA. The DNA binding domains are in dark blue. The two TALEN target sites are typically separated by a 15–20-bp spacer sequence. Like ZFNs, the TAL effector repeat arrays are fused to FokI. Each TAL effector motif recognizes one base. (D) The CRISPR/Cas9 system recognizes DNA through base pairing between DNA sequences at the target site and a CRISPR-based guide RNA (gRNA). Cas9 has two nuclease domains (shown by red arrowheads) that each cleave one strand of double-stranded DNA.

Figure 2. Targeted genome engineering by non-homologous end-joining or homologous recombination using sequence-specific nucleases.

(A) NHEJ-mediated repair can result in small deletions or insertions at the target sites that can disrupt gene function (knock-outs, left). DNA fragments can be inserted via NHEJ-mediated ligation to create targeted insertions (knock-ins, right). (B) When two cuts are made by SSNs, NHEJ-mediated repair can result in either deletions or inversions of large genomic regions (left) or targeted gene deletions or chromosomal translocations (right). (C) HR-mediated repair, involving a homologous DNA template, leads to gene replacement or gene insertion.