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Aach, John

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Aach

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Aach, John
Author's Publications: search.filters.isAuthorOfPublication.8ef98d81-67a1-49ce-93f7-7be293f67327

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Now showing 1 - 10 of 23
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    Highly-efficient Cas9-mediated transcriptional programming
    (2015) Chavez, Alejandro; Scheiman, Jonathan; Vora, Suhani; Pruitt, Benjamin W.; Tuttle, M; Iyer, Eswar; Lin, Shuailiang; Kiani, Samira; Guzman, Christopher D.; Wiegand, Daniel; Ter-Ovanesyan, Dmitry; Braff, Jonathan L.; Davidsohn, Noah; Housden, Benjamin E; Perrimon, Norbert; Weiss, Ron; Aach, John; Collins, James; Church, George
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    On the design of clone-based haplotyping
    (BioMed Central, 2013) Lo, Christine; Liu, Rui; Lee, Jehyuk; Robasky, Kimberly; Byrne, Susan M; Lucchesi, Carolina; Aach, John; Church, George; Bafna, Vineet; Zhang, Kun
    Background: Haplotypes are important for assessing genealogy and disease susceptibility of individual genomes, but are difficult to obtain with routine sequencing approaches. Experimental haplotype reconstruction based on assembling fragments of individual chromosomes is promising, but with variable yields due to incompletely understood parameter choices. Results: We parameterize the clone-based haplotyping problem in order to provide theoretical and empirical assessments of the impact of different parameters on haplotype assembly. We confirm the intuition that long clones help link together heterozygous variants and thus improve haplotype length. Furthermore, given the length of the clones, we address how to choose the other parameters, including number of pools, clone coverage and sequencing coverage, so as to maximize haplotype length. We model the problem theoretically and show empirically the benefits of using larger clones with moderate number of pools and sequencing coverage. In particular, using 140 kb BAC clones, we construct haplotypes for a personal genome and assemble haplotypes with N50 values greater than 2.6 Mb. These assembled haplotypes are longer and at least as accurate as haplotypes of existing clone-based strategies, whether in vivo or in vitro. Conclusions: Our results provide practical guidelines for the development and design of clone-based methods to achieve long range, high-resolution and accurate haplotypes.
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    Optimization of scarless human stem cell genome editing
    (Oxford University Press, 2013) Yang, Luhan; Guell, Marc; Byrne, Susan M; Yang, Joyce; De Los Angeles, Alejandro; Mali, Prashant; Aach, John; Kim-Kiselak, Caroline; Briggs, Adrian; Rios, Xavier; Huang, Po-Yi; Daley, George; Church, George
    Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.
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    Barcoding cells using cell-surface programmable DNA-binding domains
    (2013) Mali, Prashant; Aach, John; Lee, Jehyuk; Levner, D; Nip, Lisa; Church, George
    We develop here a novel approach to barcode large numbers of cells through cell-surface expression of programmable zinc-finger DNA-binding domains (sZFs). We show sZFs enable double-stranded DNA to sequence-specifically label living cells, and also develop a sequential tagging approach to in situ image >3 cell types using just 3 fluorophores. Finally we demonstrate their broad versatility through ability to serve as surrogate reporters and facilitate selective cell capture and targeting.
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    Rational optimization of tolC as a powerful dual selectable marker for genome engineering
    (Oxford University Press, 2014) Gregg, Christopher; Lajoie, Marc; Napolitano, Michael G.; Mosberg, Joshua A.; Goodman, Daniel B.; Aach, John; Isaacs, Farren J.; Church, George
    Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by ∼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is ∼10-fold more efficient at achieving allelic diversity than previous best practices.
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    CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering
    (2013) Mali, Prashant; Aach, John; Stranges, P. Benjamin; Esvelt, Kevin Michael; Moosburner, Mark; Kosuri, Sriram; Yang, Luhan; Church, George
    Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes.1–7. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a novel transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TAL) effector proteins8, 9. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effector proteins can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.
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    Improved Cell-Free RNA and Protein Synthesis System
    (Public Library of Science, 2014) Li, Jun; Gu, Liangcai; Aach, John; Church, George
    Cell-free RNA and protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. Advances in reconstituted CFPS systems such as the Protein synthesis Using Recombinant Elements (PURE) system offer new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, protein microarrays, isotopic labeling, and incorporating unnatural amino acids. In this study, using firefly luciferase synthesis as a reporter system, we improved PURE system productivity up to 5 fold by adding or adjusting a variety of factors that affect transcription and translation, including Elongation factors (EF-Ts, EF-Tu, EF-G, and EF4), ribosome recycling factor (RRF), release factors (RF1, RF2, RF3), chaperones (GroEL/ES), BSA and tRNAs. The work provides a more efficient defined in vitro transcription and translation system and a deeper understanding of the factors that limit the whole system efficiency.
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    Genome-wide inactivation of porcine endogenous retroviruses (PERVs)
    (American Association for the Advancement of Science (AAAS), 2015) Yang, Luhan; Guell, Marc; Niu, D.; George, H.; Lesha, E.; Grishin, Dennis; Aach, John; Shrock, Ellen; Xu, W.; Poci, Jurgen; Cortazio, R.; Wilkinson, R. A.; Fishman, Jay; Church, George
    The shortage of organs for transplantation is a major barrier to the treatment of organ failure. While porcine organs are considered promising, their use has been checked by concerns about transmission of porcine endogenous retroviruses (PERVs) to humans. Here, we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all 62 copies of the PERV pol gene and demonstrated a > 1000-fold reduction in PERV transmission to human cells using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine- to-human xenotransplantation.
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    Optimizing complex phenotypes through model-guided multiplex genome engineering
    (BioMed Central, 2017) Kuznetsov, Gleb; Goodman, Daniel B.; Filsinger, Gabriel; Landon, Matthieu; Rohland, Nadin; Aach, John; Lajoie, Marc J.; Church, George
    We present a method for identifying genomic modifications that optimize a complex phenotype through multiplex genome engineering and predictive modeling. We apply our method to identify six single nucleotide mutations that recover 59% of the fitness defect exhibited by the 63-codon E. coli strain C321.∆A. By introducing targeted combinations of changes in multiplex we generate rich genotypic and phenotypic diversity and characterize clones using whole-genome sequencing and doubling time measurements. Regularized multivariate linear regression accurately quantifies individual allelic effects and overcomes bias from hitchhiking mutations and context-dependence of genome editing efficiency that would confound other strategies. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1217-z) contains supplementary material, which is available to authorized users.
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    Digital RNA allelotyping reveals tissue-specific and allele-specific gene expression in human
    (Nature Publishing Group, 2009) Zhang, Kun; Li, Jin Billy; Gao, Yuan; Egli, Dieter; Xie, Bin; Deng, Jie; Li, Zhe; Lee, Je-Hyuk; Aach, John; Leproust, Emily M; Eggan, Kevin; Church, George
    We developed a digital RNA allelotyping method for quantitatively interrogating allele-specific gene expression. This method involves ultra-deep sequencing of padlock captured SNPs from the transcriptome. We characterized four cell lines established from two human subjects in the Personal Genome Project. Approximately 11–22% of the heterozygous mRNA-associated SNPs show allele-specific expression in each cell line; and 4.3–8.5% are tissue-specific, suggesting the presence of tissue-specific cis-regulation. When applied to two pairs of sibling human embryonic stem cell lines, the sibling lines were more similar in allele-specific expression than were the genetically unrelated lines. We found that the variation of allelic ratios in gene expression among different cell lines is primarily explained by genetic variations, much more so than by specific tissue types or culturing conditions. Comparison of expressed SNPs on the sense and anti-sense transcripts suggested that allelic ratios are primarily determined by cis-regulatory mechanisms on the sense transcripts.