An siRNA-guided ARGONAUTE protein directs RNA polymerase V to initiate DNA methylation

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

Modification of improved-genome editing via oviductal nucleic acids delivery (i-GONAD)-mediated knock-in in rats

Background Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency. Results The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 μM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different. Conclusions We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.

Staying and Returning Dynamics of Young Children's Attention

In this paper, we decompose sustained attending behavior into components of continuous attention maintenance and attentional transitions and study how each of these components develops in young children. Our results in two experiments suggest that changes in children's ability to return attention to a target locus after distraction (“Returning”) play a crucial role in the development of sustained attention between the ages of 3.5-6 years, perhaps to a greater extent than changes in the ability to continuously maintain attention on the target (“Staying”). We further distinguish Returning from the behavior of transitioning attention away from task (“Leaving”) and provide evidence that Leaving is more strongly influenced by bottom-up factors, while Returning is invariant to these same bottom-up factors, suggesting a potentially greater contribution of top-down factors in Returning. Overall, these results (a) suggest the importance of understanding the cognitive process of transitioning attention for understanding sustained attention and its development, (b) provide an empirical paradigm within which to study this process, and (c) begin to characterize basic features of this process, namely its development and its relative dependence on top-down and bottom-up influences on attention.

An siRNA-guided Argonaute protein directs RNA Polymerase V to initiate DNA methylation

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA Polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without preexisting DNA methylation. We find that Pol V follows and is dependent upon the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of preexisting DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits

Gene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9’s homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to spCas9, miCas9 enhances double-stranded DNA mediated large size gene knock-in rates, systematically reduces off-target insertion and deletion events, maintains or increases single-stranded oligodeoxynucleotides mediated precise gene editing rates, and effectively reduces on-target insertion and deletion rates in knock-in applications. Furthermore, we demonstrate that this fusion motif can work as a “plug and play” module, compatible and synergistic with other Cas9 variants. MiCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.

203 Genome Modifications by Sleeping Beauty Transposition and CRISPR/Cas9 to Improve Cow Milk Composition for Human Consumption

Genome manipulation of cattle represents a powerful tool to increase the nutritional value and reduce allergenicity of cow milk for human consumption. This could be accomplished by improving the amount of polyunsaturated fatty acids (ω-3 and ω-6) and simultaneously abolishing β-lactoglobulin (BLG), a potent allergen for predisposed humans. The aim of this study was to introduce the sequence for a desaturase construct (mFAT-2, from C. elegans), which is able to catalyse the synthesis of ω-3 and ω-6 fatty acids, into the bovine genome by Sleeping Beauty (SB) transposition, and simultaneously knocking out the bovine β-lactoglubulin gene using CRISPR/Cas9 system. The sgRNA (AAGTGCCTCCTGCTTGCCC) targeted to BLG exon 1 was synthesised as an oligo linker and cloned into the px459-Cas9. The mutation activity of the designed sgRNA at the target locus was determined by T7 endonuclease assay I (T7EI) mismatch detection assay. Briefly, bovine fetal fibroblasts (BFF) were seeded at 0.5 × 105 cells per well of a 24-well plate in triplicate, when the cells reached 80% confluence (12–24 h), cultures were transfected with 1 μg of px459-Cas9::BLG plasmid co-expressing Cas9 and sgRNA using polyethylenimine reagent (PEI; 3 ng μL−1). After 3 days of puromycin selection, genomic DNA from transfected cells were extracted and the sequence of interest was PCR-amplified and digested by T7EI restriction enzyme. Digestion products showed a mutation efficiency at the target locus of 29%. Subsequently, we chemically cotransfected 0.5 × 105 BFF with 0.5 μg of knockout vector (px459-Cas9::BLG) and 0.5 μg of SB plasmids (carrying mFAT-2 cDNA for mammary gland-specific expression) using 3 ng μL−1 PEI in triplicate. At 48 h post-transfection, cell cultures were subjected to 3 days of puromycin and 21 days of neomycin selection. PCR analysis of antibiotic resistant colonies revealed the presence of mFAT-2 transgene in almost 70% of the analysed cells lines. Genotyping of BLG exon 1 was performed by direct sequencing of PCR amplicons using primers flanking the target site. Despite the appreciable gene mutation activity of the sgRNA sequence previously determined by T7EI assay (29%), none of the cell lines analysed showed modification in the BLG target locus. We speculate that the SB vector might have disrupted the activity of targeting vector. We are currently performing additional experiments to accomplish gene addition (mFAT) and gene knockout (BGL) in one step using these highly efficient and precise transgenic tools. Genetically modified cells will be used as nuclear donor to produce transgenic cows by somatic cells nuclear transfer. The financial support of CONICET, UNRC and FONCYT is gratefully acknowledged.

Phenotypic plasticity promotes recombination and gene clustering in periodic environments

While theory offers clear predictions for when recombination will evolve in changing environments, it is unclear what natural scenarios can generate the necessary conditions. The Red Queen hypothesis provides one such scenario in natural populations, but it requires interaction with antagonistic species such as host-parasite systems. We present a novel scenario for the evolution of recombination in finite populations: the genomic storage effect due to phenotypic plasticity. Using an analytic approximation and Monte Carlo simulations we demonstrate that balanced polymorphism and recombination evolve between a target locus that codes for a seasonally selected trait and a plasticity modifier locus that modulates the effects of target-locus alleles. Unlike in prior models, evolution of recombination by this plasticity effect does not require antagonistic inter-specific interactions or a steady influx of mutation, and it occurs even when a single target locus expresses a trait under selection. Furthermore, we show that selection will suppress the recombination rate among multiple polymorphic target loci, even in the absence of epistasis among them, which produces a cluster of linked loci under selection. These results provide a novel biological scenario for the evolution of recombination and supergenes.

Phenotypic plasticity promotes recombination and gene clustering in periodic environments

While theory offers clear predictions for when recombination will evolve in changing environments, it is unclear what natural scenarios can generate the necessary conditions. The Red Queen hypothesis provides one such scenario in natural populations, but it requires interaction with antagonistic species such as host-parasite systems. We present a novel scenario for the evolution of recombination in finite populations: the genomic storage effect due to phenotypic plasticity. Using an analytic approximation and Monte Carlo simulations we demonstrate that balanced polymorphism and recombination evolve between a target locus that codes for a seasonally selected trait and a plasticity modifier locus that modulates the effects of target-locus alleles. Unlike in prior models, evolution of recombination by this plasticity effect does not require antagonistic inter-specific interactions or a steady influx of mutation, and it occurs even when a single target locus expresses a trait under selection. Furthermore, we show that selection will suppress the recombination rate among multiple polymorphic target loci, even in the absence of epistasis among them, which produces a cluster of linked loci under selection. These results provide a novel biological scenario for the evolution of recombination and supergenes.