Spatial Sequencing: A Perspective.

Fluorescent spatial sequencing brings next-generation sequencing into a new realm capable of identifying nucleic acids in the cell's natural environment. For the first time, scientists are able to multiplex the assignment of specific locations to hundreds of transcriptional targets and lay the foundation for understanding how genetic changes control the fate of each cell within the tissue microenvironment. In this perspective, we discuss the capabilities of fluorescent spatial sequencing in the context of other spatial imaging technologies and describe how these new technologies offer a data-rich, multiomic solution to many research applications.

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.

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.

Highly multiplexed subcellular RNA sequencing in situ.

Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay.

Rapid gene cloning using terminator primers and modular vectors.

We seek to create useful biological diversity by exploiting the modular nature of genetic information. In this report we describe experiments that focus on the modular nature of plasmid cloning vectors. Bacterial plasmids are modular entities composed of origins of replication, selectable markers and other components.