Nucleic acid detection with CRISPR-Cas13a/C2c2

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.United States. Air Force Office of Scientific Research (Grant FA9550-14-1-0060)Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-14-1-0006)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institutes of Health (U.S.) (Grant 1R01-MH110049

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

Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood

Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes—for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR–Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.National Human Genome Research Institute (U.S.) (Grant R00-HG008171)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant F32-DK096822)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049

Nucleic acid detection with CRISPR-Cas13a/C2c2

Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.11137Nscopu

Genetically Encoding an Electrophilic Amino Acid for Protein Stapling and Covalent Binding to Native Receptors

[Image: see text] Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability

Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood

Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes—for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR–Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.National Human Genome Research Institute (U.S.) (Grant R00-HG008171)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant F32-DK096822)National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049