Short hairpin RNA (shRNA) technology enables stable and regulated gene repression. For establishing experimentally versatile RNAi tools and minimizing toxicities, synthetic shRNAs can be embedded into endogenous microRNA contexts. However, due to our incomplete understanding of microRNA biogenesis, such "shRNAmirs" often fail to trigger potent knockdown, especially when expressed from a single genomic copy. Following recent advances in design of synthetic shRNAmir stems, here we take a systematic approach to optimize the experimental miR-30 backbone. Among several favorable features, we identify a conserved element 3' of the basal stem as critically required for optimal shRNAmir processing and implement it in an optimized backbone termed "miR-E", which strongly increases mature shRNA levels and knockdown efficacy. Existing miR-30 reagents can be easily converted to miR-E, and its combination with up-to-date design rules establishes a validated and accessible platform for generating effective single-copy shRNA libraries that will facilitate the functional annotation of the genome.

The recent development of CRISPR-Cas systems as easily accessible and programmable tools for genome editing and regulation is spurring a revolution in biology. Paired with the rapid expansion of personalized and reference genomic sequence information, technologies based on CRISPR-Cas are enabling nearly unlimited genetic manipulation even in previously difficult contexts, including human cells. Although much attention has focused on the potential of CRISPR-Cas to cure Mendelian diseases, the technology also holds promise to transform the development of therapies to treat complex heritable and somatic disorders. Here we discuss how CRISPR-Cas can impact the next generation of drugs through accelerating the identification and validation of high-value targets, uncovering high confidence biomarkers and developing differentiated breakthrough therapies. We focus on the promises, pitfalls and hurdles of this revolutionary gene editing technology, and also discuss key aspects of different CRISPR-Cas screening platforms and offer our perspectives on the best practices in genome engineering.

Summary MEK inhibitors are clinically active in BRAFV600E melanomas, but only marginally so in KRAS-mutant tumors. Here we found that MEK inhibitors suppress ERK signaling more potently in BRAFV600E- than in KRAS-mutant tumors. To understand this, we performed an RNAi screen in a KRAS-mutant model and found that CRAF knockdown enhanced MEK inhibition. MEK activated by CRAF was less susceptible to MEK inhibitors than when activated by BRAFV600E. MEK inhibitors induced RAF-MEK complexes in KRAS mutant models, and disrupting such complexes enhanced inhibition of CRAF-dependent ERK signaling. Newer MEK inhibitors not only target MEK catalytic activity, but also impair its reactivation by CRAF: either by disrupting RAF-MEK complexes or by interacting with Ser 222 to prevent RAF-mediated phosphorylation in the complex.

Summary Short hairpin RNAs (shRNAs) provide powerful experimental tools by enabling stable and regulated gene silencing through programming of endogenous microRNA pathways. Since requirements for efficient shRNA biogenesis and target suppression are largely unknown, many predicted shRNAs fail to efficiently suppress their target. To overcome this barrier, we developed a “Sensor assay” that enables the biological identification of effective shRNAs at large scale. By constructing and evaluating 20,000 RNAi reporters covering every possible target site in 9 mammalian transcripts, we show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. Our unbiased analyses reveal that potent shRNAs share various predicted and previously unknown features associated with specific microRNA processing steps, and suggest a new model for competitive strand selection. Together, our study establishes a powerful tool for large-scale identification of highly potent shRNAs and provides new insights into sequence requirements of effective RNAi.

Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo1. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown. We demonstrated that this system improves the study of essential genes and was sufficiently robust to eradicate aggressive cancer in mice by suppressing a single gene. Further, we applied this system for in vivo negative-selection screening with pooled shRNAs and propose a streamlined, inexpensive workflow that will facilitate the use of RNA interference (RNAi) for the identification and evaluation of essential therapeutic targets.

Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility for synthetic genome engineering, agricultural genomics, and biomedical applications. Although bacteria harboring CRISPR-Cas9 or CRISPR-Cas3 adaptive immune systems sometimes acquire mobile genetic elements encoding anti-CRISPR proteins that inhibit Cas9, Cas3, or the DNA-binding Cascade complex, no such inhibitors have been found for CRISPR-Cas12a. Here we use a comprehensive bioinformatic and experimental screening approach to identify three different inhibitors that block or diminish CRISPR-Cas12a-mediated genome editing in human cells. We also find a widespread connection between CRISPR self-targeting and inhibitor prevalence in prokaryotic genomes, suggesting a straightforward path to the discovery of many more anti-CRISPRs from the microbial world.

The CRISPR-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disrupting the enzyme’s binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the Estrogen Receptor α Ligand Binding Domain. This protein displayed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNA s (shRNA s), offers something not easily achieved with traditional genetic approaches—inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNA s to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNA s to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.