Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d

Forward Error Correction for DNA Data Storage

AbstractWe report on a strong capacity boost in storing digital data in synthetic DNA. In principle, synthetic DNA is an ideal media to archive digital data for very long times because the achievable data density and longevity outperforms today's digital data storage media by far. On the other hand, neither the synthesis, nor the amplification and the sequencing of DNA strands can be performed error-free today and in the foreseeable future. In order to make synthetic DNA available as digital data storage media, specifically tailored forward error correction schemes have to be applied.For the purpose of realizing a DNA data storage, we have developed an efficient and robust forwarderror-correcting scheme adapted to the DNA channel. We based the design of the needed DNA channel model on data from a proof-of-concept conducted 2012 by a team from the Harvard Medical School [1]. Our forward error correction scheme is able to cope with all error types of today's DNA synthesis, amplification and sequencing processes, e.g. insertion, deletion, and swap errors.In a successful experiment, we were able to store and retrieve error-free 22 MByte of digital data in synthetic DNA recently. The found residual error probability is already in the same order as it is in hard disk drives and can be easily improved further. This proves the feasibility to use synthetic DNA as longterm digital data storage media

Precise Cas9 targeting enables genomic mutation prevention

Here, we present a generalized method of guide RNA “tuning” that enables Cas9 to discriminate between two target sites that differ by a single-nucleotide polymorphism. We employ our methodology to generate an in vivo mutation prevention system in which Cas9 actively restricts the occurrence of undesired gain-of-function mutations within a population of engineered organisms. We further demonstrate that the system is scalable to a multitude of targets and that the general tuning and prevention concepts are portable across engineered Cas9 variants and Cas9 orthologs. Finally, we show that the mutation prevention system maintains robust activity even when placed within the complex environment of the mouse gastrointestinal tract.National Human Genome Research Institute (U.S.) ( Grant P50 HG005550)Wyss Institute for Biologically Inspired EngineeringUnited States. Defense Threat Reduction Agency (Grant HDTRA1-15-1-0051)Paul G. Allen Frontiers Grou

Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d