Hairy CRISPR: Genome Editing in Plants Using Hairy Root Transformation

CRISPR/Cas-mediated genome editing is a powerful tool of plant functional genomics. Hairy root transformation is a rapid and convenient approach for obtaining transgenic roots. When combined, these techniques represent a fast and effective means of studying gene function. In this review, we outline the current state of the art reached by the combination of these approaches over seven years. Additionally, we discuss the origins of different Agrobacterium rhizogenes strains that are widely used for hairy root transformation; the components of CRISPR/Cas vectors, such as the promoters that drive Cas or gRNA expression, the types of Cas nuclease, and selectable and screenable markers; and the application of CRISPR/Cas genome editing in hairy roots. The modification of the already known vector pKSE401 with the addition of the rice translational enhancer OsMac3 and the gene encoding the fluorescent protein DsRed1 is also described.

Translation initiation from sequence variants of the bacteriophage T7 g10RBS in Escherichia coli and Agrobacterium fabrum

Background The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine–Dalgarno (SD) sequence augmented by an upstream translational “enhancer” (Enh) element, supporting protein production at many times the level seen with simple synthetic SD-containing sequences. The objective of this study was to dissect the g10RBS to identify simpler derivatives that exhibit much of the original translation efficiency. Methods and results Twenty derivatives of g10RBS were tested using multiple promoter/reporter gene contexts. We have identified one derivative (which we call “CON_G”) that maintains 100% activity in E. coli and is 33% shorter. Further minimization of CON_G results in variants that lose only modest amounts of activity. Certain nucleotide substitutions in the spacer region between the SD sequence and initiation codon show strong decreases in translation. When testing these 20 derivatives in the alphaproteobacterium Agrobacterium fabrum, most supported strong reporter protein expression that was not dependent on the Enh. Conclusions The g10RBS derivatives tested in this study display a range of observed activity, including a minimized version (CON_G) that retains 100% activity in E. coli while being 33% shorter. This high activity is evident in two different promoter/reporter sequence contexts. The array of RBS sequences presented here may be useful to researchers in need of fine-tuned expression of recombinant proteins of interest.

Cellular translational enhancer elements that recruit eukaryotic initiation factor 3

Translation initiation is a highly regulated process which broadly affects eukaryotic gene expression. Eukaryotic initiation factor 3 (eIF3) is a central player in canonical and alternative pathways for ribosome recruitment. Here we have investigated how direct binding of eIF3 contributes to the large and regulated differences in protein output conferred by different 5′- untranslated regions (5′-UTRs) of cellular mRNAs. Using an unbiased high-throughput approach to determine the affinity of budding yeast eIF3 for native 5′-UTRs from 4,252 genes, we demonstrate that eIF3 binds specifically to a subset of 5′-UTRs that contain a short unstructured binding motif, AMAYAA. eIF3 binding mRNAs have higher ribosome density in growing cells and are preferentially translated under certain stress conditions, supporting the functional relevance of this interaction. Our results reveal a new class of translational enhancer and suggest a mechanism by which changes in core initiation factor activity enact mRNA-specific translation programs.