Silvana Konermann
Massachusetts Institute of Technology
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Publication
Featured researches published by Silvana Konermann.
Nature Protocols | 2017
Julia Joung; Silvana Konermann; Jonathan S. Gootenberg; Omar O. Abudayyeh; Randall Jeffrey Platt; Mark D. Brigham; Neville E. Sanjana; Feng Zhang
Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9–15 weeks, followed by 4–5 weeks of validation.
Nature | 2017
Julia Joung; Jesse M. Engreitz; Silvana Konermann; Omar O. Abudayyeh; Vanessa Verdine; François Aguet; Jonathan S. Gootenberg; Neville E. Sanjana; Jason Wright; Charles P. Fulco; Yuen-Yi Tseng; Charles H. Yoon; Jesse S. Boehm; Eric S. Lander; Feng Zhang
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.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.
Nature Biotechnology | 2016
James E. Dahlman; Omar O. Abudayyeh; Julia Joung; Jonathan S. Gootenberg; Feng Zhang; Silvana Konermann
In the version of this article initially published, when discussing the data in Figure 2b, on p. 1160, we wrote, “...targeting the same HBG1/2 promoter and found they had 32 and 55 perturbed transcripts....” This should have been “31 and 55 perturbed transcripts” as in the sentence in the figure legend discussing the same data. The error has been corrected in the HTML and PDF versions of the article.
Nature Protocols | 2018
Julia Joung; Silvana Konermann; Jonathan S. Gootenberg; Omar O. Abudayyeh; Randall Jeffrey Platt; Mark D. Brigham; Neville E. Sanjana; Feng Zhang
In the published version of this paper, Step 64 of the Procedure reads, “Refer to Steps 37–39 for NGS analysis of the sgRNA distribution.” This step should refer the reader to Steps 35–39. This text has not been corrected in the original paper.
bioRxiv | 2016
Julia Joung; Silvana Konermann; Jonathan S. Gootenberg; Omar O. Abudayyeh; Randall Jeffrey Platt; Mark D. Brigham; Neville E. Sanjana; Feng Zhang
Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial immune system CRISPR (clustered regularly interspaced short palindromic repeats) has been adapted for genome-scale screening by combining Cas9 with guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom-or ready-made guide RNA libraries are constructed and packaged into lentivirus for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified from the screen, we further describe strategies for confirming the screening phenotype as well as genetic perturbation through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 6-10 weeks followed by 3-4 weeks of validation.
Archive | 2013
Feng Zhang; Mark D. Brigham; Le Cong; Silvana Konermann; Neville E. Sanjana
Archive | 2014
Feng Zhang; Mark D. Brigham; Le Cong; Silvana Konermann
Archive | 2015
Silvana Konermann; Alexandro Trevino; Mark D. Brigham; Fei Ran; Patrick Hsu; Chie-yu Lin; Osamu Nureki; Hiroshi Nishimasu; Ryuichiro Ishitani; Feng Zhang
Cell | 2018
Silvana Konermann; Peter Lotfy; Nicholas J. Brideau; Jennifer Oki; Maxim N. Shokhirev; Patrick Hsu
Archive | 2015
Feng Zhang; Silvana Konermann; James E. Dahlman; Omar O. Abudayyeh