111 research outputs found
RNA targeting with CRISPR–Cas13
RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference1-3 can efficiently knockdown RNAs, but it is prone to off-target effects4, and visualizing RNAs typically relies on the introduction of exogenous tags5. Here we demonstrate that the class 2 type VI6,7 RNA-guided RNA-targeting CRISPR-Cas effector Cas13a8(previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049
Parallel genome-scale loss of function screens in 216 cancer cell lines for the identification of context-specific genetic dependencies
Using a genome-scale, lentivirally delivered shRNA library, we performed massively parallel pooled shRNA screens in 216 cancer cell lines to identify genes that are required for cell proliferation and/or viability. Cell line dependencies on 11,000 genes were interrogated by 5 shRNAs per gene. The proliferation effect of each shRNA in each cell line was assessed by transducing a population of 11M cells with one shRNA-virus per cell and determining the relative enrichment or depletion of each of the 54,000 shRNAs after 16 population doublings using Next Generation Sequencing. All the cell lines were screened using standardized conditions to best assess differential genetic dependencies across cell lines. When combined with genomic characterization of these cell lines, this dataset facilitates the linkage of genetic dependencies with specific cellular contexts (e.g., gene mutations or cell lineage). To enable such comparisons, we developed and provided a bioinformatics tool to identify linear and nonlinear correlations between these features
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Molecular mechanisms and applications of RNA targeting CRISPR endonucleases
Evolutionary pressure to protect against phage-induced lysis and rampant horizontal gene transfer has created a wide repertoire of defensive pathways in bacteria. CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated) systems are adaptive immune pathways that use RNA-guided nucleases to direct cleavage of invading nucleic acids. The programmable nature of these enzymes has enabled a revolution for DNA-targeting applications including gene editing, transcriptional control, and genomic imaging. In addition to DNA-targeting enzymes, specific subtypes of CRISPR-Cas systems recognize and degrade single stranded RNA (ssRNA) substrates. Repurposing these ssRNA-targeting enzymes into biotechnological tools is currently limited by a lack of mechanistic information. In this work, we address this issue by redirecting a well-studied DNA-targeting CRISPR nuclease, Cas9, to ssRNA targets and investigating the enzymatic mechanisms of a novel ssRNA-targeting CRISPR nuclease, Cas13a (formerly C2c2).Typically, Cas9 ignores ssRNA while searching for dsDNA targets due to ssRNA’s inherent single-stranded structure and lack of a protospacer adjacent motif (PAM). We redirected Cas9 to bind and recognize ssRNA targets through addition of a third component, a target-complementary DNA oligonucleotide or PAMmer, that provides a DNA:RNA hybrid PAM. Using primary microRNAs as a model system, we provide proof-of-concept evidence that Cas9:PAMmer complexes can mediate the isolation and subsequent mass spectrometry analysis of protein complexes bound to specific RNAs. The complexity of redirecting Cas9 to ssRNA substrates motivated us to investigate CRISPR proteins that natively target RNA. We focused on Cas13a, a predicted ribonuclease from Type VI CRISPR-Cas systems. We discovered that Cas13a possesses two distinct catalytic activities, one for site-specific cleavage of its CRISPR RNA (crRNA) and the second for nonspecific ssRNA degradation activated by target binding. These insights allowed us to establish a revised model for ssRNA-targeting by Type VI CRISPR-Cas systems. Through biochemical characterization of the entire Cas13a protein family, we revealed hidden diversity in substrate preferences and defined orthogonal enzyme subfamilies. These diverse Cas13a homologs can be harnessed in parallel for detection of distinct RNA species within complex mixtures for both bacterial immunity and diagnostic applications. Together, this work presents two novel biotechnological applications of CRISPR-Cas nucleases for RNA isolation and RNA detection.¬
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RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a.
CRISPR-Cas13a enzymes are RNA-guided, RNA-activated RNases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging, and RNA regulation. However, the relationship between target RNA binding and HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activation is poorly understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number and the position of mismatches between the guide and the target. We identify a central binding seed for which perfect base pairing is required for target binding and a separate nuclease switch for which imperfect base pairing results in tight binding, but not HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a RNA binding and cleavage behavior for RNA-targeting tool development
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RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes.
CRISPR adaptive immunity pathways protect prokaryotic cells against foreign nucleic acids using CRISPR RNA (crRNA)-guided nucleases. In type VI-A CRISPR-Cas systems, the signature protein Cas13a (formerly C2c2) contains two separate ribonuclease activities that catalyze crRNA maturation and ssRNA degradation. The Cas13a protein family occurs across different bacterial phyla and varies widely in both protein sequence and corresponding crRNA sequence conservation. Although grouped phylogenetically together, we show that the Cas13a enzyme family comprises two distinct functional groups that recognize orthogonal sets of crRNAs and possess different ssRNA cleavage specificities. These functional distinctions could not be bioinformatically predicted, suggesting more subtle co-evolution of Cas13a enzymes. Additionally, we find that Cas13a pre-crRNA processing is not essential for ssRNA cleavage, although it enhances ssRNA targeting for crRNAs encoded internally within the CRISPR array. We define two Cas13a protein subfamilies that can operate in parallel for RNA detection both in bacteria and for diagnostic applications
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