Exploring the connection between genomic organization and cellular fitness
Taylor Ward
Within the nucleus, chromosomes fold into complex, 3-dimensional structures that play a vital role in gene regulation. These structures include chromosome territories defined by physical organization, as well as both long and short range interactions between and within chromosomes. These interactions are an essential component of gene regulation, with effects on gene expression being exerted from over hundreds of kilobases (kb) away. Current methods for studying these chromosome interactions and their connection of cellular fitness are limited to detecting endogenous chromosomal contacts, and include conformation capture (3C) technologies or live-cell imaging by microscopy. The biological importance of chromosomal organization has been established, but the causal relationship between cellular fitness and chromosomal interactions is still unclear. To investigate this causal relationship, I have been working on a novel assay that manipulates chromosomal interactions using a dCas9-DNA binding domain fusion. Nuclease-deficient dCas9 offers the flexibility to target the fusion protein to anywhere in a genome by the design of the sgRNA. The DNA binding domain portion can be targeted to a single, fixed genomic locus by integrating the corresponding recognition sequence into the genome. An sgRNA target site and the fixed genomic locus are brought into proximity by the dCas9-DNA binding domain fusion to generate an artificial tether. These tethers can be created in a massively parallel assay wherein each cell in the population receives a different sgRNA and generates a unique artificial tether (Figure 1A). I am interested in using this assay to interrogate the impact of chromosomal conformation on cellular fitness. I hypothesize that there are certain chromosomal conformations that are allowed and others that are not allowed, and these can be distinguished by differences in cellular fitness. By comparing the fitness of strains carrying the same sgRNA library members but different fixed locus sites, I expect to find position-dependent effects (Figure 1B). For example, if a tether between an sgRNA target site and a genomic position within the interior of the genome is allowed, while the same target site tethered to the nuclear periphery results in a severe decrease in fitness, this result might suggest a strong effect exerted by the positional dependence of gene regulation. While work so far on developing this tethering assay has been done in baker’s yeast, future directions include developing this approach for human tissue culture applications, opening the door to investigate the impact of differences in chromosomal organization between different cell types in multicellular organisms.
Figure 1. sgRNA library members result in unique tethers with varying effects on fitness.
(A) Schematic depicting four different tethers in cells with dCas9 binding sites indicated by colors. (B) Histograms describe expected results for changing frequencies of sgRNAs depending on their tethering effect. These changes in frequencies can be used to calculate fitness scores.
(A) Schematic depicting four different tethers in cells with dCas9 binding sites indicated by colors. (B) Histograms describe expected results for changing frequencies of sgRNAs depending on their tethering effect. These changes in frequencies can be used to calculate fitness scores.
Continuous directed evolution
Bryan Andrews and Ben Brandsen
Continuous directed evolution, in which mutations are continuously introduced into a gene of interest that undergoes selection for function, offers a powerful means to rapidly explore wide sequence spaces, which would otherwise require time-intensive long-term evolution approaches or labor-intensive mutational scans. We are developing a general method for continuous evolution that relies on error-prone transcription followed by RNA-templated repair of dsDNA breaks, a mechanism of DNA repair that has been observed in bacteria, yeast, and mammalian cells.
To develop this method in Escherichia coli and Saccharomyces cerevisiae, we generated a broken kanamycin resistance gene that has the potential to be repaired, leading to resistance. In this design, a T7 RNA polymerase promoter is introduced downstream of the gene of interest, leading to antisense transcription of the gene (Figure 1). We express a T7 RNA polymerase with increased error rate to generate the antisense transcript. RNA-templated repair incorporates mutations from the antisense transcript into the gene. Rare mutations that repair the kanamycin resistance gene can be detected by growth in kanamycin.
We are evaluating RNA-templated repair in several different strain backgrounds. We aim to identify specific gene knockout(s) that improve the efficiency of this process. We will use this continuous directed evolution method to evaluate known evolutionary trajectories in order to confirm that it recapitulates previous evolutionary paths determined by other methods. We also plan to use this method for affinity maturation of protein-ligand and protein-protein interactions.
To develop this method in Escherichia coli and Saccharomyces cerevisiae, we generated a broken kanamycin resistance gene that has the potential to be repaired, leading to resistance. In this design, a T7 RNA polymerase promoter is introduced downstream of the gene of interest, leading to antisense transcription of the gene (Figure 1). We express a T7 RNA polymerase with increased error rate to generate the antisense transcript. RNA-templated repair incorporates mutations from the antisense transcript into the gene. Rare mutations that repair the kanamycin resistance gene can be detected by growth in kanamycin.
We are evaluating RNA-templated repair in several different strain backgrounds. We aim to identify specific gene knockout(s) that improve the efficiency of this process. We will use this continuous directed evolution method to evaluate known evolutionary trajectories in order to confirm that it recapitulates previous evolutionary paths determined by other methods. We also plan to use this method for affinity maturation of protein-ligand and protein-protein interactions.
Figure 1. Strategy for continuous directed evolution. The T7 RNA polymerase promoter is introduced downstream of the gene of interest, and an error-prone T7 RNA polymerase generates antisense transcript of the gene. After a dsDNA break, the antisense RNA is used as a template for repair, transferring mutations from the RNA to the DNA.
Rapid identification and quantitative assessment of plant enhancers in full-genome libraries
Mike Dorrity and Josh Cuperus
Plants respond to environmental stimuli by tightly controlled changes in gene expression, requiring a dynamic regulatory network. Despite this need, a key mode of dynamic regulation, long-range activation or repression, appears to be absent from chromatin interaction datasets (3C, Hi-C) in Arabidopsis thaliana. The apparent lack of distal physical interactions among chromosomes suggests that Arabidopsis may not use typical enhancers, though they are frequent among other higher eukaryotes. We describe the use of an alternative method to identify and functionally characterize enhancers, STARR-seq (from Arnold et al Science 2013 399, 1074). This method relies on simultaneously measuring the activity of a library of gene constructs, each containing a fragment of genomic DNA inserted into a transcribed sequence. If a fragment does not possess an enhancer element, the construct is expressed only at low level due to the minimal promoter driving transcription. If a fragment is able to confer distal activation, the gene is expressed at higher level and the functional enhancer sequence, codified in the transcript, increases in abundance.
We have optimized the method for transient expression in plants by testing a digest of a plasmid carrying the known viral 35S enhancer in the STARR-seq experimental context. Individual fragments of the enhancer showed enrichment (top 5 shown) when present. We foresee this method being useful for addressing biological questions of gene regulation as well as for application to emerging challenges in crop design.
We have begun using Arabidopsis genomic DNA in the STARR-seq assay. One such example is presented below, where we see enrichment of intergenic regions. Some of these enrichment peaks overlap with known chromatin-accessible regions. Overall, peaks in our STARR-seq library have enrichment of regions that are classified in genomic categories similar to those of hypersensitive sites, with the notable exception of transposons. This outcome is expected, given that transposons are heavily methylated in planta but are not methylated in STARR-seq.
