Seamless editing of the chloroplast genome in plants

Abstract

BackgroundGene editing technologies enable the precise insertion of favourable mutations and performance enhancing trait genes into chromosomes whilst excluding all excess DNA from modified genomes. The technology gives rise to a new class of biotech crops which is likely to have widespread applications in agriculture. Despite progress in the nucleus, the seamless insertions of point mutations and non-selectable foreign genes into the organelle genomes of crops have not been described. The chloroplast genome is an attractive target to improve photosynthesis and crop performance. Current chloroplast genome engineering technologies for introducing point mutations into native chloroplast genes leave DNA scars, such as the target sites for recombination enzymes. Seamless editing methods to modify chloroplast genes need to address reversal of site-directed point mutations by template mediated repair with the vast excess of wild type chloroplast genomes that are present early in the transformation process.ResultsUsing tobacco, we developed an efficient two-step method to edit a chloroplast gene by replacing the wild type sequence with a transient intermediate. This was resolved to the final edited gene by recombination between imperfect direct repeats. Six out of 11 transplastomic plants isolated contained the desired intermediate and at the second step this was resolved to the edited chloroplast gene in five of six plants tested. Maintenance of a single base deletion mutation in an imperfect direct repeat of the native chloroplast rbcL gene showed the limited influence of biased repair back to the wild type sequence. The deletion caused a frameshift, which replaced the five C-terminal amino acids of the Rubisco large subunit with 16 alternative residues resulting in a ~30-fold reduction in its accumulation. We monitored the process in vivo by engineering an overlapping gusA gene downstream of the edited rbcL gene. Translational coupling between the overlapping rbcL and gusA genes resulted in relatively high GUS accumulation (~0.5 % of leaf protein).ConclusionsEditing chloroplast genomes using transient imperfect direct repeats provides an efficient method for introducing point mutations into chloroplast genes. Moreover, we describe the first synthetic operon allowing expression of a downstream overlapping gene by translational coupling in chloroplasts. Overlapping genes provide a new mechanism for co-ordinating the translation of foreign proteins in chloroplasts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0857-6) contains supplementary material, which is available to authorized users.

Mini‐synplastomes for plastid genetic engineering

“…Conventional tobacco chloroplast engineering typically uses homologous arms of ~1 kb (much longer for lettuce) (Daniell et al ., 2019b; Kumari et al ., 2019; Kwon et al ., 2016, 2018; Meeker et al ., 1988; Park et al ., 2020; Ruhlman et al ., 2010) although the use of smaller direct repeats of up to 174 bp in length has been used for direct‐repeat mediated excision of marker genes (Iamtham and Day, 2000). In terms of efficiency, even with the goal of introduction of a single SNP into the native plastome, homology arms of ~ 600 bp are recommended (Martin Avila et al ., 2016). Similarly, as indicated in the generation of NICE1, imperfect direct repeats as small as 16 bp can facilitate effective recombination in plastids (Staub and Maliga, 1994).…”

Transgene insertion into the plastid genome alters expression of adjacent native chloroplast genes at the transcriptional and translational levels

“…In bacteria, translation of adjacent reading frames in polycistronic mRNAs is often coupled in that translation of the upstream coding sequence activates the translation of downstream reading frame(s) (Jackson et al ., 2007; Yamamoto et al ., 2016). Translational coupling of adjacent reading frames was also observed in plastid translation (Adachi et al ., 2012; Fu et al ., 2021; Martin Avila et al ., 2016; Yukawa and Sugiura, 2008; Zoschke et al ., 2013) and has been exploited in chloroplast biotechnology (Martin Avila et al ., 2016). Hence, in addition to the transcriptional overexpression of genes downstream of aadA , the strongly translated aadA reading frame may also activate the translation of downstream reading frames that are located on the same mRNA by translational coupling or by increasing the local ribosome density.…”

“…Third, transgenes should be inserted at maximum distance to downstream genes to dampen read‐through effects on gene expression. Fourth, methods for post‐transformation excision of marker genes are available (Lutz et al ., 2006; Martin Avila et al ., 2016) and can be used to omit transgene expression effects on neighbouring genes, at least in the final stage of generating transplastomic lines. Fifth, recent progress with marker‐free manipulation of the plastid genome and ectopic chloroplast transgene expression provide additional options to avoid neighbouring‐gene effects altogether (Maliga, 2022).…”

The Power of CRISPR-Cas9-Induced Genome Editing to Speed Up Plant Breeding

“…Importantly, the CRISPR-Cas9-mediated gene drive technology (called as mutagenic chain reaction (MCR) [109]) can be used to produce stable homozygous (biallelic) mutant lines by using HDR-driven propagation of the CRISPR-Cas9-cassette to the companion chromosome. Moreover, such concept can also be applied for editing organelle genomes (e.g., chloroplast) in order to overcome the high copy number of genomes and reversion of mutations [110]. …”