A tethering assay to analyze chromosome structure
Taylor Wang
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. The biological importance of chromosomal organization has been established, but the causal relationship between cellular fitness and chromosomal interactions is still unclear. This causal relationship represents unknown territory that can be explored by methods that can artificially manipulate chromosomes to create new interactions and study their effects on fitness.
We use a novel assay that manipulates chromosomal interactions by taking advantage of Cas9 targeting flexibility from the clustered regularly interspersed short palindromic repeats (CRISPR) system for genome editing. Guide RNAs (gRNAs) provide the basis for flexibility by targeting Cas9 binding in the genome through sequence complementarity. I adapted this flexibility for my needs by using a nuclease deficient Cas9 (dCas9) that binds without making incisions. This dCas9 is expressed as a fusion to the sequence specific DNA-binding domain (DBD) of LexA. The LexA-dCas9 fusion creates tethers by bringing an anchor locus—containing the lexA operator sequence—into close proximity of the dCas9 binding site (Figure 1A). These tethers can be created in multiplex wherein each cell in a population receives a different gRNA library member and generates a unique tether. A fitness selection for tethering will result in changing gRNA frequencies which can be read out by Illumina sequencing (Figure 1B).
We use a novel assay that manipulates chromosomal interactions by taking advantage of Cas9 targeting flexibility from the clustered regularly interspersed short palindromic repeats (CRISPR) system for genome editing. Guide RNAs (gRNAs) provide the basis for flexibility by targeting Cas9 binding in the genome through sequence complementarity. I adapted this flexibility for my needs by using a nuclease deficient Cas9 (dCas9) that binds without making incisions. This dCas9 is expressed as a fusion to the sequence specific DNA-binding domain (DBD) of LexA. The LexA-dCas9 fusion creates tethers by bringing an anchor locus—containing the lexA operator sequence—into close proximity of the dCas9 binding site (Figure 1A). These tethers can be created in multiplex wherein each cell in a population receives a different gRNA library member and generates a unique tether. A fitness selection for tethering will result in changing gRNA frequencies which can be read out by Illumina sequencing (Figure 1B).
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.
In pilot experiments, we have been testing the efficacy of the tethering technology by tethering a plasmid-to- genome—instead of genome-to-genome—and using mCherry fluorescence as a reporter for the presence or absence of a tether. This plasmid-to-genome tethering experiment relied on three components: 1] exogenous constitutively expressed Gal4 DBD-VP16; 2] the LexA-dCas9 tethering protein; and 3] another exogenous plasmid that is bound by the dCas9 of the tethering protein and that also contains Gal4 binding sites (Figure 2). With this design we can test our hypothesis of trans-activator diffusion from one tethered locus to the other. If one of these loci is the reporter locus, this diffusion should allow for transcriptional activation of mCherry.
Moving forward, we also intend to adapt this system for genome-to-genome tethering at a single site that will act like pTW1 by binding the Gal4 DBD-VP16 activator, i.e. the Gal4 UAS on the pTW1 plasmid will instead be moved to a genomic locus. gRNA design will target the dCas9 to this genomic locus instead of the plasmid, and tethering of this locus to the reporter should result in activation if genome-to-genome tethering is possible. These experiments would open the door to the feasibility of multiplex library tethering experiments to investigate the effect of perturbing genomic organization on cellular fitness.
A method for continuous directed evolution
Ben Brandsen
Continuous directed evolution, in which mutations are continuously introduced into a gene of interest and selected for function, offers a powerful means to rapidly explore wide sequence space, which would otherwise require time-intensive long-term evolution approaches or labor-intensive mutational scans. I am developing a method for continuous evolution in S. cerevisiae that relies on T7 RNA polymerase mediated mutagenesis that uses a fusion of T7 RNA polymerase and the cytidine deaminase AID. A similar strategy has been demonstrated in E. coli using T7 RNA polymerase and the cytidine deaminase rApobec1 (Moore, C. L. et al., J. Am. Chem. Soc. 2018).
I am testing this system by assessing the rate at which a new start codon is generated in a Kanamycin resistance gene lacking a start codon. After determining the rate of mutagenesis in yeast, I plan to use this system for many applications. I hope to evaluate known evolutionary trajectories to confirm that continuous evolution recapitulates previous evolutionary paths determined by other methods. I hope to use this method for affinity maturation of protein-ligand and protein-protein interactions. Finally, I hope to use this system for metabolic engineering by optimizing expression level and coding sequence of biosynthetic pathways exogenously expressed in yeast.
I am testing this system by assessing the rate at which a new start codon is generated in a Kanamycin resistance gene lacking a start codon. After determining the rate of mutagenesis in yeast, I plan to use this system for many applications. I hope to evaluate known evolutionary trajectories to confirm that continuous evolution recapitulates previous evolutionary paths determined by other methods. I hope to use this method for affinity maturation of protein-ligand and protein-protein interactions. Finally, I hope to use this system for metabolic engineering by optimizing expression level and coding sequence of biosynthetic pathways exogenously expressed in yeast.
Figure 1. Strategy for continuous directed evolution. A fusion of T7 RNA polymerase and cytidine deaminase AID is used to introduce mutations in regions of DNA downstream of a T7 promoter.
Empirical Estimation of Robustness from Deep Mutational Scans
Bryan Andrews (Collaboration with Peter Conlin and Ben Kerr)
Are genes more robust to errors than expected by chance?
Because of the way the genetic code is structured, different codons for the same amino acid have different ‘mutational neighborhoods.’ That is, if a single-base substitution occurs at an arginine codon, for instance, the set of possible new amino acids is very different if the original codon was an ‘cgg’ versus an ‘agg’, and the same is true for half of the amino acids: A, G, I, K, L, P, R, S, T & V.
Because of the way the genetic code is structured, different codons for the same amino acid have different ‘mutational neighborhoods.’ That is, if a single-base substitution occurs at an arginine codon, for instance, the set of possible new amino acids is very different if the original codon was an ‘cgg’ versus an ‘agg’, and the same is true for half of the amino acids: A, G, I, K, L, P, R, S, T & V.
As a result, the potential effects of a single-base substitution on a protein depends on what codons are used to encode that protein. In this project, in collaboration with Peter Conlin and Ben Kerr, we computationally reanalyzed published datasets in which proteins were subjected to Deep Mutational Scans, in order to test whether naturally occurring gene sequences are more robust to errors than would be expected by chance, a property we are calling ‘genetic robustness.’
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.
