In planta test system for targeted cellular mutagenesis by injection of oligonucleotides to apical meristem of maize seedlings

Genome-editing tools from Oligonucleotide-Directed Mutagenesis (ODM) to CRISPR system use synthetic oligonucleotides for targeted exchange of nucleotides. Presently, majority of genome-editing protocols are dependent on the in vitro cell or tissue culture systems with somaclonal variation, and limitations in plant regeneration. Therefore, here, we report an alternative in planta cellular test system for optimization of the ODM, based on the injection of oligonucleotide solution into the apical meristematic region of haploid maize seedlings. Using 5′-fluorescein-labeled oligonucleotides, we detected accumulation of synthetic DNA molecules in cells of the shoot apical meristem and of the vascular bundles of leaf primordia. For silencing or knocking down of the phytoene desaturase gene in somatic cells, 41-mer long single-stranded oligonucleotides with TAG stop codon were injected into maize seedlings. We detected out-growing M1 plantlets that developed leaves with white stripes or pale-green color. Confocal microscopy of white stripes showed that in addition to the chlorophyll fluorescence-deficient tissue region, chlorophyll containing cells are present in white stripes. The Ion Torrent sequencing of DNA samples from the white stripes indicated 0.13–1.50% read frequency for the TAG stop codon in the phytoene desaturase gene. Appearance of chlorotic abnormalities supports the mutagenic nature of oligonucleotide molecules after injection into the shoot apical meristem region of maize seedling. The described protocol provides basis for early seedling stage characterization of functionality of a mutagenic oligonucleotide with different chemistry and testing efficiency of various treatment combinations at plant level.

Clock genes and environmental cues coordinate Anopheles pheromone synthesis, swarming, and mating

Anopheles mating is initiated by the swarming of males at dusk followed by females flying into the swarm. Here, we show that mosquito swarming and mating are coordinately guided by clock genes, light, and temperature. Transcriptome analysis shows up-regulation of the clock genes period (per) and timeless (tim) in the head of field-caught swarming Anopheles coluzzii males. Knockdown of per and tim expression affects Anopheles gambiae s.s. and Anopheles stephensi male mating in the laboratory, and it reduces male An. coluzzii swarming and mating under semifield conditions. Light and temperature affect mosquito mating, possibly by modulating per and/or tim expression. Moreover, the desaturase gene desat1 is up-regulated and rhythmically expressed in the heads of swarming males and regulates the production of cuticular hydrocarbons, including heptacosane, which stimulates mating activity.

Efficient Genome Editing in Populus Using CRISPR/Cas12a

The ability to create targeted mutations using clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 in support of forest tree biotechnology is currently limited. CRISPR/Cas12a is a novel CRISPR effector protein that not only broadens the CRISPR/Cas targeting range but also enables the generation of large-fragment deletions. In this study, a CRISPR/Cas12a system was evaluated for the induction of targeted mutations in the woody tree poplar (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) using the CRISPR/Cas12a genome-targeting system, and the results indicated that the AsCas12a system is the most efficient. We further optimized the co-cultivation temperature after Agrobacterium-mediated transformation from 22 to 28°C to increase the Cas12a nuclease editing efficiency in poplar. AsCas12a showed the highest mutation efficiency, at 70%, and the majority of editing sites were composed of large-fragment deletions. By using this simple and high-efficiency CRISPR/Cas12a system, multiple targets can be modified to obtain multigene simultaneous knockout mutants in tree species, which will provide powerful tools with which to facilitate genetic studies of forest trees.