Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes

Abstract

Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR–Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9–gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species.

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

“…The CRISPR-Cas system can be stably or transiently transfected into the plant cells as nucleic acids (plasmids or DNA fragments) [6,73] or pre-assembled ribonucleoprotein complexes (RNPs) [74][75][76].…”

“…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].…”

“…Very often, to increase the proportion of Cas9 protein effectively addressed to the nucleus, one or two nuclear localization signals (such as A. tumefaciens bipartite VirD2 T-DNA border endonuclease carboxyl terminal NLS, or the monopartite Simian virus 40 NLS) are incorporated into the expression cassettes at the amino and/or carboxyl termini of the Cas9 open reading frame [12,73,75,88].…”

“…Using Cas9 RNA or protein presents the advantage of not having to work on optimizing expression by defining sequences codon-optimized for the host plant and selecting appropriate promoters. Preassembled complexes of purified Cas9 protein and guide RNA (RNPs) were transfected into protoplasts of Arabidopsis thaliana, Nicotiana attenuata, Petunia x hybrida, grapevine, apple, lettuce and rice [74,76,95] and bombarded into immature embryos of bread and durum wheat [73,96], and maize [75]. In all cases, RNPs showed good cleavage efficiency (4-46%), with (when tested) no detectable mosaicism, suggesting a very early action of the nuclease before the first cell division [74,76].…”

“…Nevertheless, the rapid degradation of the RNAs is considered beneficial as it reduces the time during when the genome is exposed to nuclease activity, which limits the risk of generating off-target mutations [74]. So far, the only experiment reporting gene knock-in using a RNP complex was performed in maize by bombardment of immature embryos [75]. By using a ssDNA donor template of 127 bp bearing 7 substitutions, replacement of the target gene could be obtained with a knockin frequency of 2-2.5% [75], which is much better than the exact same experiment performed using a CRISPR-Cas9 system provided to the cell on two separate plasmids (0.3-0.4%) [6].…”