Evans, M. M. | bioRxiv Science

bioRxiv2020

A Safe Harbor-Targeted CRISPR/Cas9 Homology Independent Targeted Integration (HITI) System for Multi-Modality Reporter Gene-Based Cell Tracking

Imaging reporter genes can provide valuable, longitudinal information on the biodistribution, growth and survival of engineered cells in preclinical models and patients. A translational bottleneck to using reporter genes in patients is the necessity to engineer cells with randomly-integrating vectors. CRISPR/Cas9 targeted knock-in of reporter genes at a genomic safe harbor locus such as adeno-associated virus integration site 1 (AAVS1) may overcome these safety concerns. Here, we built Homology Independent Targeted Integration (HITI) CRISPR/Cas9 minicircle donors for precise AAVS1-targeted simultaneous knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency at the AAVS1 site using HITI vectors compared to homology-directed repair donor vectors. Characterization of select HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significantly increased Oatp1a1-mediated uptake of the clinically-approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. As few as 106 Oatp1a1-expressing cells in a 50 {micro}l subcutaneous injection could be detected in vivo with contrast-enhanced MRI. Contrast-enhanced MRI also improved the conspicuity of both sub-cutaneous and metastatic Oatp1a1-expressing tumours prior to them being palpable or even readily visible on pre-contrast images. Our work demonstrates the first CRISPR/Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multi-modal longitudinal in vivo imaging of cells. This work lays the foundation for safer, non-viral reporter gene tracking of multiple cell types.

bioengineering↗

bioRxiv2019

Highly expressed maize pollen genes display coordinated expression with neighboring transposable elements and contribute to pollen fitness

In flowering plants, the haploid male gametophyte (pollen) is essential for sperm delivery, double fertilization, and subsequent initiation of seed development. Pollen also undergoes dynamic epigenetic regulation of expression from transposable elements (TEs), but how this process interacts with gene regulation and function is not clearly understood. To identify components of these processes, we quantified transcript levels in four male reproductive stages of maize (tassel primordia, microspores, mature pollen, and isolated sperm cells) via RNA-seq. We found that, in contrast to Arabidopsis TE expression in pollen, TE transcripts in maize accumulate as early as the microspore stage and are also present in sperm cells. Intriguingly, coordinated expression was observed between the most highly expressed protein-coding genes and neighboring TEs, specifically in both mature pollen and sperm cells. To test the hypothesis that such elevated expression correlates with functional relevance, we measured the fitness cost (male-specific transmission defect) of GFP-tagged exon insertion mutations in over 50 genes highly expressed in pollen vegetative cell, sperm cell, or seedling (as a sporophytic control). Insertions in genes highly expressed only in seedling or primarily in sperm cells (with one exception) exhibited no difference from the expected 1:1 transmission ratio. In contrast, insertions in over 20% of vegetative cell genes were associated with significant reductions in fitness, showing a positive correlation of transcript level with non-Mendelian segregation. The gamete expressed2 (gex2) gene was the single sperm cell gene associated with reduced transmission when mutant (<35% for two independent insertions), and also triggered seed defects when crossed as a male, supporting a role for gex2 in double fertilization. Overall, our study demonstrates a developmentally programmed and coordinated transcriptional activation of TEs and genes, and further identifies maize pollen as a model in which transcriptomic data have predictive value for quantitative phenotypes.\n\nAuthor SummaryIn flowering plants, pollen is essential for delivering sperm cells to the egg and central cell for double fertilization, initiating the process of seed development. In plants with abundant pollen like maize, this process can be highly competitive. In an added layer of complexity, growing evidence indicates expression of transposable elements (TEs) is more dynamic in pollen than in other plant tissues. How these elements impact pollen function and gene regulation is not well understood. We used transcriptional profiling to generate a framework for both detailed analysis of TE expression and quantitative assessment of gene function during maize pollen development. TEs are expressed early and persist, many showing coordinate activation with highly-expressed neighboring genes in the pollen vegetative cell and sperm cells. Measuring fitness costs for a set of over 50 mutations indicates a correlation between elevated transcript level and gene function in the vegetative cell. Finally, we establish a role in fertilization for the gamete expressed2 (gex2) gene, identified based on its specific expression in sperm cells. These results highlight maize pollen as a powerful model for investigating the developmental interplay of TEs and genes, as well as for measuring fitness contributions of specific genes.

plant biology↗