ARGOS8 variants generated by CRISPR‐Cas9 improve maize grain yield under field drought stress conditions

Abstract

SummaryMaize ARGOS8 is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing ARGOS8 have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of ARGOS8 native expression variation in drought‐tolerant breeding, a diverse set of over 400 maize inbreds was examined for ARGOS8 mRNA expression, but the expression levels in all lines were less than that created in the original ARGOS8 transgenic events. We then employed a CRISPR‐Cas‐enabled advanced breeding technology to generate novel variants of ARGOS8. The native maize GOS2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5′‐untranslated region of the native ARGOS8 gene or was used to replace the native promoter of ARGOS8. Precise genomic DNA modification at the ARGOS8 locus was verified by PCR and sequencing. The ARGOS8 variants had elevated levels of ARGOS8 transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the GOS2 promoter. A field study showed that compared to the WT, the ARGOS8 variants increased grain yield by five bushels per acre under flowering stress conditions and had no yield loss under well‐watered conditions. These results demonstrate the utility of the CRISPR‐Cas9 system in generating novel allelic variation for breeding drought‐tolerant crops.

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…So far, reports about CRISPR-assisted knock-in experiments in plants describe circular donor templates ranging from 0.47 to 5.2 kb in total length, including homology sequences of 46 bp to 1.6 kb on each side of the sequence of interest [2,6,8,10,43,67,68] ( Table 1). As for free DNA fragments, they varied from 72 to 476 bp including homology arms, of even length or not, of 46 to 146 bp [6,11,67,69].…”

“…Rare are the situations (involving the same protocol, target and plant species) allowing to compare the impact of the nature and length of the homologies on knock-in efficiency. In maize, despite the fact that the targets were different, by bombarding immature embryos with a circular DNA donor template, knock-in seemed more efficient with homology sequences of 1 kb framing a donor cassette of 3.1 kb (2.5-4.1%), than with 0.4 kb homologies surrounding a 1.8 kb donor cassette (1%) [2,6]. However, no absolute rules regarding the link between CRISPR-induced gene knock-in efficiency and the length of the homology arms can be drawn from the available data in plants.…”

“…Regarding the donor DNA, long donor templates (with long sequences of interest or long homology arms) can be delivered to the cells on the same or on another plasmid as the CRISPR-Cas system [2,6,8,27,41,69], or as linear dsDNA fragments, provided as free molecules [67] or released in planta from a plasmid by inserting them between CRISPR target sites [3,10,67]. Shorter templates (with short homology arms or small replacement sequences) can be introduced into the cells as ssDNA oligonucleotides (sense or antisense) [6,11,67,75].…”

“…Genomic modifications can be obtained either by repairing the induced double-strand breaks (DSB) by error-prone end joining, such as alternative non-homologous end joining (NHEJ-alt) that will generate mutations (gene knock-out), or by end joining or homologous recombination of an exogenous donor DNA (gene knock-in) leading to allele replacement or targeted transgene insertion [1][2][3]. The system can also be used to trigger targeted chromosomal rearrangements potentially useful to increase mapping resolution of specific traits [4] or to regulate gene expression by using the catalytically inactivated Cas9 (dCas9) fused to various effector domains [5].…”

“…Indeed, whereas the knock-out of endogenous plant loci is generally easy to achieve (with mutagenesis efficiencies varying from 2% [6] to 100% [7] of mutated individuals), CRISPR-induced gene knock-in of a donor DNA by homology-driven repair (HDR) is much more difficult to perform in higher plants, with efficiencies rarely reaching a few percent [2,6,[8][9][10][11]. It explains why among the CRISPR-induced traits already reported in the literature, such as diseases tolerances [12][13][14], adaptation to drought [2], modified ripening profiles [15], male sterility and factors impacting yield [9,16,17], most of them are derived from gene knock-out, and still very few from gene knock-in. Yet, many potential applications of the CRISPR-Cas9 system to plant breeding require the insertion of alleles of interest into elite lines and rely on the development of gene knock-in protocols with good efficiency.…”