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

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.¬

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

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

Programmable RNA recognition and cleavage by CRISPR/Cas9.

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags

Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts

Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts

Identification of a Minimal Peptide Tag for <i>in Vivo</i> and <i>in Vitro</i> Loading of Encapsulin

The encapsulation of enzymes and other proteins within a proteinaceous shell has been observed in many bacteria and archaea, but the function and utility of many such compartments are enigmatic. Efforts to study these functions have been complicated by the size and complexity of traditional protein compartments. One potential system for investigating the effect of compartmentalization is encapsulin, a large and newly discovered class of protein shells that are typically composed of two proteins: a protomer that assembles into the icosahedral shell and a cargo protein packaged inside. Encapsulins are some of the simplest known protein shell systems and readily self-assemble <i>in vivo</i>. Systematic characterization of the effects of compartmentalization requires the ability to load a wide range of cargo proteins. Here, we demonstrate that foreign cargo can be loaded into the encapsulin from <i>Thermotoga maritima</i> both <i>in vivo</i> and <i>in vitro</i> by fusion of the cargo protein with a short C-terminal peptide present in the native cargo. To facilitate biochemical characterization, we also develop a simple and rapid purification protocol and demonstrate the thermal and pH stability of the shell. Efforts to study the biophysical effects of protein encapsulation have been problematic in complex compartments, but the simplicity of assembling and loading encapsulin makes it an ideal system for future experiments exploring the effects of encapsulation on proteins