Biology subjects

John Aach

Publications and source records attributed to John Aach.

4 recordsLinked to original sources

bioRxiv2016

Assembly of Radically Recoded E. coli Genome Segments

The large potential of radically recoded organisms (RROs) in medicine and industry depends on improved technologies for efficient assembly and testing of recoded genomes for biosafety and functionality. Here we describe a next generation platform for conjugative assembly genome engineering, termed CAGE 2.0, that enables the scarless integration of large synthetically recoded E. coli segments at isogenic and adjacent genomic loci. A stable tdk dual selective marker is employed to facilitate cyclical assembly and removal of attachment sites used for targeted segment delivery by sitespecific recombination. Bypassing the need for vector transformation harnesses the multi Mb capacity of CAGE, while minimizing artifacts associated with RecA-mediated homologous recombination. Our method expands the genome engineering toolkit for radical modification across many organisms and recombinase-mediated cassette exchange (RMCE).

Synthetic Biology↗

bioRxiv2016

Genome Editing With Targeted Deaminases

Precise genetic modifications are essential for biomedical research and gene therapy. Yet, traditional homology-directed genome editing is limited by the requirements for DNA cleavage, donor DNA template and the endogenous DNA break-repair machinery. Here we present programmable cytidine deaminases that enable site-specific cytidine to thymidine (C-to-T) genomic edits without the need for DNA cleavage. Our targeted deaminases are efficient and specific in Escherichia coli, converting a genomic C-to-T with 13% efficiency and 95% accuracy. Edited cells do not harbor unintended genomic abnormalities. These novel enzymes also function in human cells, leading to a site-specific C-to-T transition in 2.5% of cells with reduced toxicity compared with zinc-finger nucleases. Targeted deaminases therefore represent a platform for safer and effective genome editing in prokaryotes and eukaryotes, especially in systems where DSBs are toxic, such as human stem cells and repetitive elements targeting.

Bioengineering↗

bioRxiv2014

Highly-efficient Cas9-mediated transcriptional programming

The RNA-guided bacterial nuclease Cas9 can be reengineered as a programmable transcription factor by a series of changes to the Cas9 protein in addition to the fusion of a transcriptional activation domain (AD)1-5. However, the modest levels of gene activation achieved by current Cas9 activators have limited their potential applications. Here we describe the development of an improved transcriptional regulator through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to Cas9. We demonstrate its utility in activating expression of endogenous coding and non-coding genes, targeting several genes simultaneously and stimulating neuronal differentiation of induced pluripotent stem cells (iPSCs).

Synthetic Biology↗

bioRxiv2014

CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes

CRISPR/Cas9 systems enable many molecular activities to be efficiently directed in vivo to user-specifiable DNA sequences of interest, including generation of dsDNA cuts and nicks, transcriptional activation and repression, and fluorescence. CRISPR targeting relies on base pairing of short RNA transcripts with their target DNA sequences that must also be adjacent to fixed DNA motifs. However, rules for Cas9 targeting specificity are incompletely known. With increasing numbers of Cas9 systems being developed and deployed in more and more organisms, there is now strong need for a flexible and rational method for finding Cas9 sites with low off-targeting potential. We address this through the CasFinder system, which we demonstrate by generating human and mouse exome-wide catalogs of specific sites for three varieties of Cas9 - S. pyogenes, S. thermophilus (ST1), and N. meningitidis - that each target 56-74% of all exons. We also generate reduced sets of up to 3 targets per gene for use in high-throughput Cas9-based gene knockout screens that target 75-80% of all genes.

Genomics↗