The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

The new frontier of genome engineering with CRISPR-Cas9

Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

http://stacks.cdc.gov/view/cdc/29957/cdc_29957_DS1.pdf

CRISPR-Cas systems for editing, regulating and targeting genomes

Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

A specific protein, bound to DNA, can activate transcription of a wide array of genes in many eukaryotes. Further analysis suggests a general outline for how eukaryotic transcriptional activators function and are controlled.

How eukaryotic transcriptional activators work

50 Years of Image Analysis

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided doublestranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.

/pdf/programmable-repression-and-activation-of-bacterial-gene-1aqlr6197j.pdf

Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

https://www.cell.com/cell/pdf/0092-8674(86)90833-0.pdf

Cooperative binding of λ repressors to sites separated by integral turns of the DNA helix

Repressor structure and the mechanism of positive control

Many transcriptional activators in prokaryotes are known to bind near a promoter and contact RNA polymerase, but it is not clear whether a protein-protein contact between an activator and RNA polymerase is enough to activate gene transcription Here we show that contact between a DNA-bound protein and a heterologous protein domain fused to RNA polymerase can elicit transcriptional activation; moreover, the strength of this engineered protein-protein interaction determines the amount of gene activation Our results indicate that an arbitrary interaction between a DNA-bound protein and RNA polymerase can activate transcription We also find that when the DNA-bound 'activator' makes contact with two different components of the polymerase, the effect of these two interactions on transcription is synergistic

Activation of prokaryotic transcription through arbitrary protein–protein contacts

The present invention makes available an interaction trap system (hereinafter "ITS") which is derived using recombinantly engineered prokaryotic cells.

Ann Hochschild

Papers

Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

Cooperative binding of λ repressors to sites separated by integral turns of the DNA helix

Repressor structure and the mechanism of positive control

Activation of prokaryotic transcription through arbitrary protein–protein contacts

Interaction trap assay, reagents and uses thereof