Enhanced Cas12a editing in mammalian cells and zebrafish.

Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells.

Publisher Correction: Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing.

The original version of this Article contained errors in the author affiliations. Mehmet Fatih Bolukbasi was incorrectly associated with Bluebird Bio., Cambridge, MA, USA and Ankit Gupta was incorrectly associated with Exonics Therapeutics, Watertown, MA, USA.

SIR proteins create compact heterochromatin fibers.

Heterochromatin is a silenced chromatin region essential for maintaining genomic stability and driving developmental processes. The complicated structure and dynamics of heterochromatin have rendered it difficult to characterize. In budding yeast, heterochromatin assembly requires the SIR proteins-Sir3, believed to be the primary structural component of SIR heterochromatin, and the Sir2-4 complex, responsible for the targeted recruitment of SIR proteins and the deacetylation of lysine 16 of histone H4.

Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing.

The development of robust, versatile and accurate toolsets is critical to facilitate therapeutic genome editing applications. Here we establish RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, as versatile genome engineering systems. In both of these formats, Cas9-Cas9 fusions display an expanded targeting repertoire and achieve highly specific genome editing.

Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing.

Efficient genome editing with Cas9-sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications. Translational efforts to develop other RNA therapeutics have shown that judicious chemical modification of RNAs can improve therapeutic efficacy by reducing susceptibility to nuclease degradation. Guided by the structure of the Cas9-sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and we develop an optimal set of chemical modifications for in vivo applications.

Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo.

The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals.

Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery.

The simplicity of site-specific genome targeting by type II clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas9 nucleases, along with their robust activity profile, has changed the landscape of genome editing. These favorable properties have made the CRISPR-Cas9 system the technology of choice for sequence-specific modifications in vertebrate systems. For many applications, whether the focus is on basic science investigations or therapeutic efficacy, activity and precision are important considerations when one is choosing a nuclease platform, target site and delivery method.

DNA-binding-domain fusions enhance the targeting range and precision of Cas9.

The CRISPR-Cas9 system is commonly used in biomedical research; however, the precision of Cas9 is suboptimal for applications that involve editing a large population of cells (for example, gene therapy). Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but they often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site in the human genome over a large cohort of treated cells.

miR-1289 and "Zipcode"-like Sequence Enrich mRNAs in Microvesicles.

Despite intensive studies, the molecular mechanisms by which the genetic materials are uploaded into microvesicles (MVs) are still unknown. This is the first study describing a zipcode-like 25 nucleotide (nt) sequence in the 3'-untranslated region (3'UTR) of mRNAs, with variants of this sequence present in many mRNAs enriched in MVs, as compared to their glioblastoma cells of origin. When this sequence was incorporated into the 3'UTR of a reporter message and expressed in a different cell type, it led to enrichment of the reporter mRNA in MVs.