Occurrence and Nature of Off-Target Modifications by CRISPR-Cas Genome Editing in Plants

Abstract

CRISPR-Cas-based genome editing allows for precise and targeted genetic modification of plants. Nevertheless, unintended off-target edits can arise that might confer risks when present in gene-edited food crops. Through an extensive literature review we gathered information on CRISPR-Cas off-target edits in plants. Most observed off-target changes were small insertions or deletions (1-22 bp) or nucleotide substitutions, and large deletions (>100 bp) were rare. One study detected the insertion of vector-derived DNA sequences, which is important considering the risk assessment of gene-edited plants. Off-target sites had few mismatches (1-3 nt) with the target sequence and were mainly located in protein-coding regions, often in target gene homologues. Off-targets edits were predominantly detected via biased analysis of predicted off-target sites instead of unbiased genome-wide analysis. CRISPR-Cas-edited plants showed lower off-target mutation frequencies than conventionally bred plants. This Review can aid discussions on the relevance of evaluating off-target modifications for risk assessment of CRISPR-Cas-edited plants.

Figure 1

Schematic overview of the types of SDN modifications. The asterisks (*) signify nucleotides (in color) that are not identical to the native host sequence (in gray) around the double-stranded break introduced by the SDN. Such nonidentical nucleotides are introduced either through substitution or through insertion of nucleotides during the process of DNA break repair. SDN-1 applications can generate 1 base pair (bp) up to a small number of base insertions/deletions (indels) without providing a donor DNA template, through nonhomologous end-joining (NHEJ). Occasionally larger deletions may occur as a result of alternative repair mechanisms such as microhomology-mediated end-joining (MMEJ). SDN-2 applications can generate precise and small genetic modifications at the target site, ranging from point mutations to small indels, by means of a donor DNA template for homology-directed repair (HDR). SDN-3 applications can insert entire DNA cassettes into a target site, by providing a large donor DNA template of the desired gene, which leads to insertion by HDR or NHEJ and a transgenic plant if the donor originates from an unrelated species.

Figure 2

Schematic overview of the mutation frequencies of different plant breeding techniques, based on our literature study.