Precision genetic modifications: a new era in molecular biology and crop improvement

Recently, the use of programmable DNA-binding proteins such as ZFP/ZFNs, TALE/TALENs and CRISPR/Cas has produced unprecedented advances in gene targeting and genome editing in prokaryotes and eukaryotes. These advances allow researchers to specifically alter genes, reprogram epigenetic marks, generate site-specific deletions and potentially cure diseases. Unlike previous methods, these precision genetic modification techniques (PGMs) are specific, efficient, easy to use and economical. Here we discuss the capabilities and pitfalls of PGMs and highlight the recent, exciting applications of PGMs in molecular biology and crop genetic engineering. Further improvement of the efficiency and precision of PGM techniques will enable researchers to precisely alter gene expression and biological/chemical pathways, probe gene function, modify epigenetic marks and improve crops by increasing yield, quality and tolerance to limiting biotic and abiotic stress conditions.     

Pseudouridine: the fifth RNA nucleotide with renewed interests

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Pseudouridine modification of U5 RNA in ribonucleoprotein particles assembled in vitro.

The formation of pseudouridine (psi) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of psi modification was detected when a combination of S100 and nuclear extracts was used for assembly. psi formation was inhibited when ATP and creatine phosphate or MgCl2 were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and psi formation showed that psi modification lags behind RNP assembly and that at very early time points, Sm-reactive U5 small nuclear RNPs are not modified. Two of three psi modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on psi formation was investigated. A mutation in the area of stem-loop I which contains the psi moieties or in the Sm binding sequence affected psi formation.     

Pseudouridine in RNA: what, where, how, and why

Pseudouridine (5-ribosyluracil) is a ubiquitous yet enigmatic constituent of structural RNAs (transfer, ribosomal, small nuclear, and small nucleolar). Although pseudouridine (psi) was the first modified nucleoside to be discovered in RNA, and is the most abundant, its biosynthesis and biological roles have remained poorly understood since its identification as a "fifth nucleoside" in RNA. Recently, a combination of biochemical, biophysical, and genetic approaches has helped to illuminate the structural consequences of psi in polyribonucleotides, the biochemical mechanism of U-->psi isomerization in RNA, and the role of modification enzymes (psi synthases) and box H/ACA snoRNAs, a class of eukaryotic small nucleolar RNAs, in the site-specific biosynthesis of psi. Through its unique ability to coordinate a structural water molecule via its free N1-H, psi exerts a subtle but significant "rigidifying" influence on the nearby sugar-phosphate backbone and also enhances base stacking. These effects may underlie the biological role of most (but perhaps not all) of the psi residues in RNA. Certain genetic mutants lacking specific psi residues in tRNA or rRNA exhibit difficulties in translation, display slow growth rates, and fail to compete effectively with wild-type strains in mixed culture. In particular, normal growth is severely compromised in an Escherichia coli mutant deficient in a pseudouridine synthase responsible for the formation of three closely spaced psi residues in the mRNA decoding region of the 23S rRNA. Such studies demonstrate that pseudouridylation of RNA confers an important selective advantage in a natural biological context.     

Grandfathering in a new era of parentage analysis

The advent of DNA fingerprinting and microsatellite techniques has revolutionized the way in which we investigate genetic pedigrees in the wild (Pemberton 2008). With large and often incomplete data sets consisting of hundreds to thousands of individuals over multiple generations becoming commonplace, new methods in parentage analysis are being developed to rise to the next generation of questions and challenges. In this issue, Christie et al. (2011) provide a simple yet elegant solution to the problem of identifying missing parents and assessing hybrid fitness in a mixed population of wild and hatchery steelhead trout (Oncorhynchus mykiss) where not all individuals can be sampled effectively. They develop a new method of grandparent analysis where parental genotypes can be reconstructed using data from candidate grandparent crosses and F2 offspring genotypes, allowing for new explorations of hybridization, migration and gene flow in wild populations.     

Transgenic trees for a new era

Summary Traditional breeding has been widely used in forestry. However, this technique is inefficient because trees have a long and complex life cycle that is not amenable to strict control by man. Fortunately, the development of genetic engineering is offering new ways of breeding and allowing the incorporation of new traits in plant species through the introduction of foreign genes (transgenes). The introduction of selected traits can be used to increase the productivity and commercial value of trees and other plants. For example, some species have been endowed with resistance to herbicide and pathogens such as insects and fungi. Also, it has been possible to introduce genes that modify development and wood quality, and induce sexual sterility. The development of transgenic trees has required the implementation of in vitro regeneration techniques such as organogenesis and somatic embryogenesis. Release of transgenic species into the agricultural market requires a standardized biosafety regulatory frame and effective communication between the scientific community and society to dissipate the suspicions associated with transgenic products.