We have previously reported the genetic correction of Huntington’s disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR.

Genomic robustness is the extent to which an organism has evolved to withstand the effects of deleterious mutations. We explored the extent of genomic robustness in budding yeast by genome wide dosage suppressor analysis of 53 conditional lethal mutations in cell division cycle and RNA synthesis related genes, revealing 660 suppressor interactions of which 642 are novel. This collection has several distinctive features, including high co-occurrence of mutant-suppressor pairs within protein modules, highly correlated functions between the pairs and higher diversity of functions among the co-suppressors than previously observed. Dosage suppression of essential genes encoding RNA polymerase subunits and chromosome cohesion complex suggests a surprising degree of functional plasticity of macromolecular complexes, and the existence of numerous degenerate pathways for circumventing the effects of potentially lethal mutations. These results imply that organisms and cancer are likely able to exploit the genomic robustness properties, due the persistence of cryptic gene and pathway functions, to generate variation and adapt to selective pressures.

 Abstract-Huntington's disease is a hereditary neurodegenerative disease in which a few cell types in the sub-cortical brains undergo selective degeneration leading to neurological and cognitive decline and death. The disease is caused by expanded CAG repeat within the first exon of the Huntington gene (HTT). However, the mechanisms of molecular pathogenesis of this disease are not well understood. There is currently no therapy or a cure. We used a model of the disease in the simple organism Saccharomyces cerevisiae that previous workers in the field had successfully utilized to determine putative molecular mechanisms related to the disease. Using this model, we identified 41 novel genes that appear to suppress the toxic effects of the mutant genes. We generated a network of physical and genetic interactions among the human homologs of these novel suppressor genes, and analysis of this network allowed us to propose for the first time that mRNA degradation mechanisms might be affected in the diseased cells.Index Terms-huntington's disease, systems biology, disease model, genetic modifier, suppressor network, disease gene network

Chromosome stability models are usually qualitative models derived from molecular-genetic mechanisms for DNA repair, DNA synthesis, and cell division. While qualitative models are informative, they are also challenging to reformulate as precise quantitative models. In this report we explore how (A) laboratory experiments, (B) quantitative simulation, and (C) seriation algorithms can inform models of chromosome stability. Laboratory experiments were used to identify 19 genes that when over-expressed cause chromosome instability in the yeast Saccharomyces cerevisiae. To better understand the molecular mechanisms by which these genes act, we explored their genetic interactions with 18 deletion mutations known to cause chromosome instability. Quantitative simulations based on a mathematical model of the cell cycle were used to predict the consequences of several genetic interactions. These simulations lead us to suspect that the chromosome instability genes cause cell-cycle perturbations. Cell-cycle involvement was confirmed using a seriation algorithm, which was used to analyze the genetic interaction matrix to reveal an underlying cyclical pattern. The seriation algorithm searched over 1014 possible arrangements of rows and columns to find one optimal arrangement, which correctly reflects events during cell cycle phases. To conclude, we illustrate how the molecular mechanisms behind these cell cycle events are consistent with established molecular interaction maps.

Mutational robustness is the extent to which an organism has evolved to withstand the effects of deleterious mutations. We explored the extent of mutational robustness in the budding yeast by genome wide dosage suppressor analysis of 53 conditional lethal mutations in cell division cycle and RNA synthesis related genes, revealing 660 suppressor interactions of which 642 are novel. This collection has several distinctive features, including high co-occurrence of mutant-suppressor pairs within protein modules, highly correlated functions between the pairs, and higher diversity of functions among the co-suppressors than previously observed. Dosage suppression of essential genes encoding RNA polymerase subunits and chromosome cohesion complex suggest a surprising degree of functional plasticity of macromolecular complexes and the existence of degenerate pathways for circumventing potentially lethal mutations. The utility of dosage-suppressor networks is illustrated by the discovery of a novel connection between chromosome cohesion-condensation pathways involving homologous recombination, and Huntington's disease.Biological interaction networks are robust to perturbation 1,2,3,4,5 because of several features, including power-law network topology, redundancy, modularity, and their dynamic properties 2,6,7,8,9,10,11,12,13,14,15,16,17 . Although modularity is a common feature of interaction networks 12,18,19,20,21,22 , the contribution of modularity to genetic robustness is difficult to determine. Recent studies have revealed dynamic interaction among apparently unrelated gene modules in response to genotoxic stress, suggesting the existence of highly reconfigurable networks of gene and protein modules as well as of unexpectedly plastic macromolecular complexes 19 . Rewiring of signaling and/or metabolic networks have been observed in cancer cells that evolved chemotherapy resistance 23,24 . It has been speculated that genetic and epigenetic changes could accomplish network rewiring 25,26 . Complex genomic changes reminiscent of functional rewiring are associated with rapid evolutionary adaptation in response to mutation in an essential gene 27 .The suppression of essential gene mutations has been classically employed to investigate gene function, and suppressors provide clues to mechanisms of evolution 28 .More recently, genome wide dosage suppressor discovery has contributed novel insights into biological processes 29,30 . By seeking dosage suppressors of 53 mutant genes, mostly encoding cell cycle/DNA replication related genes or RNA polymerase/RNA modification related genes, we have uncovered 660 pairs of "dosage-suppressor" interactions, which supplement 1,626 dosage-suppressor pairs reported previously in the literature 31 and 254 interactions that were discovered in a recent high throughput experiment 29 . The set of interaction pairs discovered in this study overlaps significantly with known protein-protein and genetic interaction pairs, suggesting related molecular 4 mechanisms that connect the suppressed mutant...