Bacteria herald a new era of gene editing.

The demonstration that nucleases guided by bacterial RNA can disrupt human genes represents a landmark in the rapidly developing field of genome engineering

Beyond the genome and into the clinic.

A report of BioMed Central's third annual Beyond the Genome conference, held at Harvard Medical School, Boston, September 27-29, 2012

Bind-n-Seq: high-throughput analysis of in vitro protein-DNA interactions using massively parallel sequencing.

Transcription factor-DNA interactions are some of the most important processes in biology because they directly control hereditary information. The targets of most transcription factor are unknown. In this report, we introduce Bind-n-Seq, a new high-throughput method for analyzing protein-DNA interactions in vitro, with several advantages over current methods. The procedure has three steps (i) binding proteins to randomized oligonucleotide DNA targets, (ii) sequencing the bound oligonucleotide with massively parallel technology and (iii) finding motifs among the sequences. De novo binding motifs determined by this method for the DNA-binding domains of two well-characterized zinc-finger proteins were similar to those described previously. Furthermore, calculations of the relative affinity of the proteins for specific DNA sequences correlated significantly with previous studies (R(2 )= 0.9). These results present Bind-n-Seq as a highly rapid and parallel method for determining in vitro binding sites and relative affinities

Methods for Scarless, Selection-Free Generation of Human Cells and Allele-Specific Functional Analysis of Disease-Associated SNPs and Variants of Uncertain Significance.

With the continued emergence of risk loci from Genome-Wide Association studies and variants of uncertain significance identified from patient sequencing, better methods are required to translate these human genetic findings into improvements in public health. Here we combine CRISPR/Cas9 gene editing with an innovative high-throughput genotyping pipeline utilizing KASP (Kompetitive Allele-Specific PCR) genotyping technology to create scarless isogenic cell models of cancer variants in ~1 month. We successfully modeled two novel variants previously identified by our lab in the PALB2 gene in HEK239 cells, resulting in isogenic cells representing all three genotypes for both variants. We also modeled a known functional risk SNP of colorectal cancer, rs6983267, in HCT-116 cells. Cells with extremely low levels of gene editing could still be identified and isolated using this approach. We also introduce a novel molecular assay, ChIPnQASO (Chromatin Immunoprecipitation and Quantitative Allele-Specific Occupation), which uses the same technology to reveal allele-specific function of these variants at the DNA-protein interaction level. We demonstrated preferential binding of the transcription factor TCF7L2 to the rs6983267 risk allele over the non-risk. Our pipeline provides a platform for functional variant discovery and validation that is accessible and broadly applicable for the progression of efforts towards precision medicine

Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.

Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations

Microstructure and hardness performance of AA6061 aluminium composite using friction stir processing

Rice husk ash (RHA) is an industrial waste that has become a potential reinforced material for aluminium matrix composite (AMCs) due to low cost and abundantly available resources. Friction stir processing (FSP) has been introduced as a method to modify surface properties of the metal and alloy including theirs composite as well. The present work reports the production and characterization of AA6061 and AA6061/5 vol% RHA using FSP using parameters rotation speed 1000 rpm and traversed speed 25 mm/min. The microstructure was studied using optical microscopy (OM). A homogenous dispersion of RHA particles was obtained in the composite. No agglomeration or segregation was observed. The produced composite exhibited a fine grain structure. An improvement in hardness profile was observed as AA6061/5 vol% RHA improves in hardness compared to FSPed of AA6061 without reinforcement

On the Performance of Copying Large Files Across a Contention-Based Network

Analytical and simulation models of interconnected local area networks, because of the large scale involved, are often constrained to represent only the most ideal of conditions for tractability sake. Consequently, many of the important causes of network delay are not accounted for. In this study, experimental evidence is presented to show how delay time in local area networks is significantly affected by hardware limitations in the connected workstations, software overhead, and network contention. The mechanism is a controlled experiment with two Vax workstations over an Ethernet. We investigate the network delays for large file transfers, taking into account the Vax workstation disk transfer limitations; generalized file transfer software such as NFS, FTP, and rcp; and the effect of contention on this simple network by the introduction of substantial workload from competing workstations. A comparison is made between the experimental data and a network modeling tool, and the limitations of the tool are explained. Insights from these experiments have increased our understanding of how more complex networks are likely to perform under heavy workloads.http://deepblue.lib.umich.edu/bitstream/2027.42/107873/1/citi-tr-89-3.pd