Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns.
Highlights
The development of technologies related to genetic improvement, such as transgenesis and more recently, genome editing, have changed the way humans have grown food for thousands of years.Today, the most promising tool to DNA manipulation is Clustered regularly interspaced short palindromic repeats (CRISPR) (Clustered Regularly Interspaced ShortPalindromic Repeats), a gene-editing technology that has been adapted from the bacterial immune system against viral infections [1]
While aa longer longer exposure exposure time to the ribonucleoproteins complex (RNP) complex led to a higher mutation mutation index indexfor forall allgRNAs gRNAstested, tested,the theincrease increaseininmutation mutation rate was consistent among rate was notnot consistent among all all guide RNA molecule (gRNA)
Our experiments demonstrated the suitability of the polyethylene glycol (PEG)-delivered CRISPR-Cas9 RNPs system for gene-editing screening in maize
Summary
The development of technologies related to genetic improvement, such as transgenesis and more recently, genome editing, have changed the way humans have grown food for thousands of years. CRISPR-Cas technology can be used as RNP complexes without the introgression and expression of a transgenic cassette in the host genome [8] Such an approach would avoid a number of generations of backcrossing, expression vectors, and other invasive methods of cell penetration (e.g., biolistics) that can lead to gene disruption, including large deletions, partial trisomy, genome shattering events, and plant mosaicism [9]. To develop a standard protocol for different maize varieties, we designed gRNAs and primers complementary to coding regions in exon three that are conserved in the species, in order to evaluate the efficiency and spectrum of DNA changes generated by CRISPR-Cas technology in maize, and add relevant information to the safety of gene-edited organisms. This efficient and relatively easy assay method for the selection of gRNA suitable for editing of genes of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively
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