Precision editing of the CHO genome via CRISPR-based prime editing: Progress and challenges

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Genome Engineering to Improve Acetate and Cellulosic Hydrolysate Tolerance in E. coli for Improved Cellulosic Biofuel Production

Engineering organisms for improved performance using lignocellulose feedstocks is an important step toward a sustainable fuel and chemical industry. Cellulosic feedstocks contain carbon and energy in the form of cellulosic and hemicellulosic sugars. Pretreatment processes that hydrolyze lignocellulose into its component sugars often also result in the accumulation of growth inhibitory compounds, such as acetate and furfural among others. Engineering tolerance to these inhibitors is a necessary step for the efficient production of biofuels and biochemicals. For this end we use multiple genome-wide and targeted tools to alter the genetic makeup of E. coli so we can obtain the desired trait of growth on lignocellulosic hydrolysate and tolerance to inhibitory concentrations of acetate. Each of these tools used introduces mutations within a population. These populations are placed in a selection environment where the fittest survive. The change in population genotypes is then analyzed. We applied a recently reported strategy for engineering tolerance towards the goal of increasing Escherichia coli growth in elevated acetate concentrations (Lynch, Warnecke et al. 2007). We performed selections upon an E. coli genome library using a moderate selection pressure. These studies identified a range of high-fitness genes that are normally involved in membrane and extracellular processes, are key regulated steps in pathways, and are involved in pathways that yield specific amino acids and nucleotides. Supplementation of the products and metabolically-related metabolites of these pathways increased growth rate in acetate. Directed evolution has been used successfully to increase tolerance to a variety of inhibitors on a variety of microorganisms. However, the number of unique and non-neutral mutations searched has been limited. With recent advances in DNA synthesis and recombination technologies, new advanced tools can be used. We report a two step strategy that can search a very large number of mutations that are more likely to improve the tolerance of the organism. First, the trackable multiplex recombineering (TRMR) tool searches a genome-wide library for single mutations which have a mutation which either turns up or down gene expression. Based on microarray analysis, a small number of targets are selected for recursive multiplex recombineering. We constructed and searched a library of mutations in the ribosomal binding site of targeted genes, including clones which have multiple mutations. We conducted this strategy in two inhibitory environments (acetate and lignocellulosic hydrolysate). For both cases, we successfully found single mutants from the first step, but in the second step, we found no tolerant mutants for acetate and multiple tolerant single mutants for the hydrolysate. A model was applied to predict the outcome of these selections with varying epistatic effects. This strategy is capable of searching a very large mutational space, but without prior knowledge of epistatic interaction, successful multiple mutants are not guaranteed

Dynamic pH profiles drive higher cell specific and volumetric productivity

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Combined effect of ammonia stress and cell line age on CHO cell derived VRC01 monoclonal antibody glycosylation

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The PEG13-DMR and brain-specific enhancers dictate imprinted expression within the 8q24 intellectual disability risk locus

Background: Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9. Results: Utilizing allele-specific RT-PCR, bisulphite sequencing, chromatin immunoprecipitation and chromosome conformation capture (3C) we show the reciprocal expression of the novel, paternally expressed, PEG13 non-coding RNA and maternally expressed KCNK9 genes in brain, and the biallelic expression of flanking transcripts in a range of tissues. We identify a tandem-repeat region overlapping the PEG13 transcript that is methylated on the maternal allele, which binds CTCF-cohesin in chromatin immunoprecipitation experiments and possesses enhancer-blocker activity. Using 3C, we identify mutually exclusive approximately 58 and 500 kb chromatin loops in adult frontal cortex between a novel brain-specific enhancer, marked by H3K4me1 and H3K27ac, with the KCNK9 and PEG13 promoters which we propose regulates brain-specific expression. Conclusions: We have characterised the molecular mechanism responsible for reciprocal allelic expression of the PEG13 and KCNK9 transcripts. Therefore, our observations may have important implications for identifying the cause of intellectual disabilities associated with the 8q24 locu

Measurement of innate immune response biomarkers in peritoneal dialysis effluent using a rapid diagnostic point-of-care device as a diagnostic indicator of peritonitis

Peritonitis is the commonest complication of peritoneal dialysis and a major reason for treatment failure. Current diagnosis is based on clinical symptoms, cloudy effluent and a dialysate white cell count (over 100 cells/μl). A rapid point-of-care diagnostic test would accelerate diagnosis and potentially improve outcomes from infection. Here, in a clinical audit project, we used PERiPLEX®, a point-of-care device which detects when levels of matrix metalloproteinase-8 and interleukin-6 are elevated above a threshold within minutes in dialysis effluent, to assess whether it could confirm or exclude peritonitis in 107 patients undergoing peritoneal dialysis. Mean patient age was 64.6 years with a median duration of peritoneal dialysis of 3.5 months (interquartile range 6.4 – 31.5 months). Presence of peritonitis was confirmed by clinical criteria. There were 49 positive tests of which 41 patients had peritonitis, three had other causes of intra-peritoneal inflammation, three had severe urosepsis and two patients required no treatment. Fifty eight tests were negative with one patient having a false negative result. The positive predictive value of the test was 83.7% (95% confidence interval 72.8 – 90.8) and the negative predictive value was 98.3% (89.1 – 99.8). Sensitivity and specificity were 97.6% (87.4 – 99.9) and 87.7% (77.2 – 94.5) respectively. Thus, PERiPLEX® could be used as a rapid point-of-care test that can aid the diagnosis or exclusion of peritonitis with a high negative predictive value