Publications by authors named "Neville E Sanjana"

44 Publications

Pluripotent stem cell-derived models of neurological diseases reveal early transcriptional heterogeneity.

Genome Biol 2021 Mar 4;22(1):73. Epub 2021 Mar 4.

Department of Genetics, The Alexander Silberman Institute of Life Sciences, Edmond J. Safra Campus, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel.

Background: Many neurodegenerative diseases develop only later in life, when cells in the nervous system lose their structure or function. In many forms of neurodegenerative diseases, this late-onset phenomenon remains largely unexplained.

Results: Analyzing single-cell RNA sequencing from Alzheimer's disease (AD) and Huntington's disease (HD) patients, we find increased transcriptional heterogeneity in disease-state neurons. We hypothesize that transcriptional heterogeneity precedes neurodegenerative disease pathologies. To test this idea experimentally, we use juvenile forms (72Q; 180Q) of HD iPSCs, differentiate them into committed neuronal progenitors, and obtain single-cell expression profiles. We show a global increase in gene expression variability in HD. Autophagy genes become more stable, while energy and actin-related genes become more variable in the mutant cells. Knocking down several differentially variable genes results in increased aggregate formation, a pathology associated with HD. We further validate the increased transcriptional heterogeneity in CHD8+/- cells, a model for autism spectrum disorder.

Conclusions: Overall, our results suggest that although neurodegenerative diseases develop over time, transcriptional regulation imbalance is present already at very early developmental stages. Therefore, an intervention aimed at this early phenotype may be of high diagnostic value.
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http://dx.doi.org/10.1186/s13059-021-02301-6DOI Listing
March 2021

The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types.

Elife 2021 02 11;10. Epub 2021 Feb 11.

New York Genome Center, New York, United States.

A novel variant of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has recently emerged and rapidly surpassed others in prevalence. This mutation is in linkage disequilibrium with an ORF1b protein variant (P314L), making it difficult to discern the functional significance of the Spike D614G mutation from population genetics alone. Here, we perform site-directed mutagenesis on wild-type human-codon-optimized Spike to introduce the D614G variant. Using multiple human cell lines, including human lung epithelial cells, we found that the lentiviral particles pseudotyped with Spike D614G are more effective at transducing cells than ones pseudotyped with wild-type Spike. The increased transduction with Spike D614G ranged from 1.3- to 2.4-fold in Caco-2 and Calu-3 cells expressing endogenous ACE2 and from 1.5- to 7.7-fold in A549 and Huh7.5 overexpressing ACE2. Furthermore, -complementation of SARS-CoV-2 virus with Spike D614G showed an increased infectivity in human cells. Although there is minimal difference in ACE2 receptor binding between the D614 and G614 Spike variants, the G614 variant is more resistant to proteolytic cleavage, suggesting a possible mechanism for the increased transduction.
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http://dx.doi.org/10.7554/eLife.65365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891930PMC
February 2021

Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

Cell 2021 01 24;184(1):92-105.e16. Epub 2020 Oct 24.

New York Genome Center, New York, NY, USA; Department of Biology, New York University, New York, NY, USA. Electronic address:

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.
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http://dx.doi.org/10.1016/j.cell.2020.10.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584921PMC
January 2021

The D614G mutation in SARS-CoV-2 Spike increases transduction of multiple human cell types.

bioRxiv 2020 Jun 15. Epub 2020 Jun 15.

Recently, a novel isolate of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has emerged and rapidly surpassed others in prevalence, including the original SARS-CoV-2 isolate from Wuhan, China. This Spike variant is a defining feature of the most prevalent clade (A2a) of SARS-CoV-2 genomes worldwide. Using phylogenomic data, several groups have proposed that the D614G variant may confer increased transmissibility leading to positive selection, while others have claimed that currently available evidence does not support positive selection. Furthermore, in the A2a clade, this mutation is in linkage disequilibrium with a ORF1b protein variant (P314L), making it difficult to discern the functional significance of the Spike D614G mutation from population genetics alone. Here, we perform site-directed mutagenesis on a human codon-optimized spike protein to introduce the D614G variant and produce SARS-CoV-2-pseudotyped lentiviral particles (S-Virus) with this variant and with D614 Spike. We show that in multiple cell lines, including human lung epithelial cells, that S-Virus carrying the D614G mutation is up to 8-fold more effective at transducing cells than wild-type S-Virus. This provides functional evidence that the D614G mutation in the Spike protein increases transduction of human cells. Further we show that the G614 variant is more resistant to cleavage in vitro and in human cells, which may suggest a possible mechanism for the increased transduction. Given that several vaccines in development and in clinical trials are based on the initial (D614) Spike sequence, this result has important implications for the efficacy of these vaccines in protecting against this recent and highly-prevalent SARS-CoV-2 isolate.
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http://dx.doi.org/10.1101/2020.06.14.151357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310625PMC
June 2020

Massively parallel Cas13 screens reveal principles for guide RNA design.

Nat Biotechnol 2020 06 16;38(6):722-727. Epub 2020 Mar 16.

New York Genome Center, New York, NY, USA.

Type VI CRISPR enzymes are RNA-targeting proteins with nuclease activity that enable specific and robust target gene knockdown without altering the genome. To define rules for the design of Cas13d guide RNAs (gRNAs), we conducted massively parallel screens targeting messenger RNAs (mRNAs) of a green fluorescent protein transgene, and CD46, CD55 and CD71 cell-surface proteins in human cells. In total, we measured the activity of 24,460 gRNAs with and without mismatches relative to the target sequences. Knockdown efficacy is driven by gRNA-specific features and target site context. Single mismatches generally reduce knockdown to a modest degree, but spacer nucleotides 15-21 are largely intolerant of target site mismatches. We developed a computational model to identify optimal gRNAs and confirm their generalizability, testing 3,979 guides targeting mRNAs of 48 endogenous genes. We show that Cas13 can be used in forward transcriptomic pooled screens and, using our model, predict optimized Cas13 gRNAs for all protein-coding transcripts in the human genome.
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http://dx.doi.org/10.1038/s41587-020-0456-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294996PMC
June 2020

High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation.

Cell Rep 2020 03;30(9):2859-2868.e5

New York Genome Center, New York, NY, USA; Department of Biology, New York University, New York, NY, USA. Electronic address:

A key limitation of the widely used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG), but their activity has been measured at only a small number of endogenous sites. Here, we devise a high-throughput Cas9 pooled competition screen to compare the performance of Cas9 variants at thousands of genomic loci for gene knockout, transcriptional activation, and inhibition. We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cleavage. Of the PAM-flexible variants, we find that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combine xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants.
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http://dx.doi.org/10.1016/j.celrep.2020.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558435PMC
March 2020

Generation of a knock-in MAP2-tdTomato reporter human embryonic stem cell line with inducible expression of NEUROG2/1 (NYGCe001-A).

Stem Cell Res 2019 12 1;41:101643. Epub 2019 Nov 1.

New York Genome Center, New York, NY, USA; Department of Biology, New York University, New York, NY, USA. Electronic address:

Overexpression of NEUROG2 and NEUROG1 (NEUROG2/1) in human embryonic stem cells (hESCs) rapidly produces functional networks of excitatory and inhibitory neurons. To facilitate the use of this efficient inducible human neuron model in neuroscience research, we generated hESCs with doxycycline-inducible NEUROG2/1 via lentivirus and a tdTomato fluorescent reporter knock-in at the MAP2 locus using the CRISPR nuclease Cas9. Upon doxycycline-driven induction of NEUROG2/1, these hESCs differentiate within days into cells that are uniformly MAP2 immunoreactive and tdTomato fluorescent.
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http://dx.doi.org/10.1016/j.scr.2019.101643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919562PMC
December 2019

Immunomagnetic cell sorting.

Nat Biomed Eng 2019 10;3(10):759-760

New York Genome Center, New York, NY, USA.

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http://dx.doi.org/10.1038/s41551-019-0459-3DOI Listing
October 2019

Identification of Cancer Drivers at CTCF Insulators in 1,962 Whole Genomes.

Cell Syst 2019 05 8;8(5):446-455.e8. Epub 2019 May 8.

Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10065, USA; Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065, USA; Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY 10021, USA; Caryl and Israel Englander Institute for Precision Medicine, New York Presbyterian Hospital, Weill Cornell Medicine, New York, NY 10065, USA. Electronic address:

Recent studies have shown that mutations at non-coding elements, such as promoters and enhancers, can act as cancer drivers. However, an important class of non-coding elements, namely CTCF insulators, has been overlooked in the previous driver analyses. We used insulator annotations from CTCF and cohesin ChIA-PET and analyzed somatic mutations in 1,962 whole genomes from 21 cancer types. Using the heterogeneous patterns of transcription-factor-motif disruption, functional impact, and recurrence of mutations, we developed a computational method that revealed 21 insulators showing signals of positive selection. In particular, mutations in an insulator in multiple cancer types, including 16% of melanoma samples, are associated with TGFB1 up-regulation. Using CRISPR-Cas9, we find that alterations at two of the most frequently mutated regions in this insulator increase cell growth by 40%-50%, supporting the role of this boundary element as a cancer driver. Thus, our study reveals several CTCF insulators as putative cancer drivers.
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http://dx.doi.org/10.1016/j.cels.2019.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917527PMC
May 2019

Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells.

Nat Methods 2019 05 22;16(5):409-412. Epub 2019 Apr 22.

Technology Innovation Laboratory, New York Genome Center, New York, NY, USA.

Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.
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http://dx.doi.org/10.1038/s41592-019-0392-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557128PMC
May 2019

Overexpression of NEUROG2 and NEUROG1 in human embryonic stem cells produces a network of excitatory and inhibitory neurons.

FASEB J 2019 04 30;33(4):5287-5299. Epub 2019 Jan 30.

Stanley Center for Psychiatric Research, Broad Institute, Cambridge, Massachusetts, USA.

Overexpression of mouse neurogenin ( Neurog) 2 alone or in combination with mouse Neurog2/1 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can rapidly produce high-yield excitatory neurons. Here, we report a detailed characterization of human neuronal networks induced by the expression of human NEUROG2 together with human NEUROG2/1 in hESCs using molecular, cellular, and electrophysiological measurements over 60 d after induction. Both excitatory synaptic transmission and network firing activity increased over time. Strikingly, inhibitory synaptic transmission and GABAergic cells were identified from NEUROG2/1 induced neurons (iNs). To illustrate the application of such iNs, we demonstrated that the heterozygous knock out of SCN2A, whose loss-of-function mutation is strongly implicated in autism risk, led to a dramatic reduction in network activity in the NEUROG2/1 iNs. Our findings not only extend our understanding of the NEUROG2/1-induced human neuronal network but also substantiate NEUROG2/1 iNs as an in vitro system for modeling neuronal and functional deficits on a human genetic background.-Lu, C., Shi, X., Allen, A., Baez-Nieto, D., Nikish, A., Sanjana, N. E., Pan, J. Q. Overexpression of NEUROG2 and NEUROG1 in human embryonic stem cells produces a network of excitatory and inhibitory neurons.
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http://dx.doi.org/10.1096/fj.201801110RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436650PMC
April 2019

Author Correction: Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.

Nat Protoc 2019 Jul;14(7):2259

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

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.
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http://dx.doi.org/10.1038/s41596-018-0063-0DOI Listing
July 2019

Effects of 3D culturing conditions on the transcriptomic profile of stem-cell-derived neurons.

Nat Biomed Eng 2018 Jul 9;2(7):540-554. Epub 2018 Apr 9.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Understanding neurological diseases requires tractable genetic systems. Engineered 3D neural tissues are an attractive choice, but how the cellular transcriptomic profiles in these tissues are affected by the encapsulating materials and are related to the human-brain transcriptome is not well understood. Here, we report the characterization of the effects of culturing conditions on the transcriptomic profiles of induced neuronal cells, as well as a method for the rapid generation of 3D co-cultures of neuronal and astrocytic cells from the same pool of human embryonic stem cells. By comparing the gene-expression profiles of neuronal cells in culture conditions relevant to the developing human brain, we found that modifying the degree of crosslinking of composite hydrogels can tune expression patterns so they correlate with those of specific brain regions and developmental stages. Moreover, by using single-cell sequencing, we show that our engineered tissues recapitulate transcriptional patterns of cell types in the human brain. The analysis of culturing conditions will inform the development of 3D neural tissues for use as tractable models of brain diseases.
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http://dx.doi.org/10.1038/s41551-018-0219-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6157920PMC
July 2018

A genome-wide net to catch and understand cancer.

Sci Transl Med 2018 08;10(453)

New York Genome Center, New York, NY 10013, USA.

Genome-scale forward genetic screens elucidate the genetic basis of therapeutic resistance, tumor evolution, and metastasis in diverse human cancers.
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http://dx.doi.org/10.1126/scitranslmed.aat8288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180290PMC
August 2018

GFAP Mutations in Astrocytes Impair Oligodendrocyte Progenitor Proliferation and Myelination in an hiPSC Model of Alexander Disease.

Cell Stem Cell 2018 Aug;23(2):239-251.e6

Division of Stem Cell Biology Research, Department of Developmental and Stem Cell Biology, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA; Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA. Electronic address:

Alexander disease (AxD) is a leukodystrophy that primarily affects astrocytes and is caused by mutations in the astrocytic filament gene GFAP. While astrocytes are thought to have important roles in controlling myelination, AxD animal models do not recapitulate critical myelination phenotypes and it is therefore not clear how AxD astrocytes contribute to leukodystrophy. Here, we show that AxD patient iPSC-derived astrocytes recapitulate key features of AxD pathology such as GFAP aggregation. Moreover, AxD astrocytes inhibit proliferation of human iPSC-derived oligodendrocyte progenitor cells (OPCs) in co-culture and reduce their myelination potential. CRISPR/Cas9-based correction of GFAP mutations reversed these phenotypes. Transcriptomic analyses of AxD astrocytes and postmortem brains identified CHI3L1 as a key mediator of AxD astrocyte-induced inhibition of OPC activity. Thus, this iPSC-based model of AxD not only recapitulates patient phenotypes not observed in animal models, but also reveals mechanisms underlying disease pathology and provides a platform for assessing therapeutic interventions.
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http://dx.doi.org/10.1016/j.stem.2018.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230521PMC
August 2018

Two Angelman families with unusually advanced neurodevelopment carry a start codon variant in the most highly expressed UBE3A isoform.

Am J Med Genet A 2018 07 7;176(7):1641-1647. Epub 2018 May 7.

Division of Genetics and Genomics, Boston Children's Hospital, Boston, Massachusetts.

We present three children from two unrelated families with Angelman syndrome (AS) whose developmental skills are far more advanced than any other non-mosaic AS individual ever reported. All have normal gait and use syntactic language spontaneously to express their needs. All of them have a c.2T > C (p.Met1Thr) variant in UBE3A, which abrogates the start codon of isoform 1, but not of isoforms 2 and 3. This variant was maternally inherited in one set of siblings, but de novo in the other child from the unrelated family. This report underscores the importance of considering AS in the differential diagnosis even in the presence of syntactic speech.
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http://dx.doi.org/10.1002/ajmg.a.38831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117199PMC
July 2018

Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments.

Nat Protoc 2018 05 12;13(5):946-986. Epub 2018 Apr 12.

Molecular Pathology Unit and Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts, as well as computational data analysis that can be performed in 1-2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.
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http://dx.doi.org/10.1038/nprot.2018.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182299PMC
May 2018

Target Discovery for Precision Medicine Using High-Throughput Genome Engineering.

Adv Exp Med Biol 2017 ;1016:123-145

New York Genome Center, 101 Avenue of the Americas, New York, NY, 10013, USA.

Over the past few years, programmable RNA-guided nucleases such as the CRISPR/Cas9 system have ushered in a new era of precision genome editing in diverse model systems and in human cells. Functional screens using large libraries of RNA guides can interrogate a large hypothesis space to pinpoint particular genes and genetic elements involved in fundamental biological processes and disease-relevant phenotypes. Here, we review recent high-throughput CRISPR screens (e.g. loss-of-function, gain-of-function, and targeting noncoding elements) and highlight their potential for uncovering novel therapeutic targets, such as those involved in cancer resistance to small molecular drugs and immunotherapies, tumor evolution, infectious disease, inborn genetic disorders, and other therapeutic challenges.
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http://dx.doi.org/10.1007/978-3-319-63904-8_7DOI Listing
April 2018

High-Throughput Approaches to Pinpoint Function within the Noncoding Genome.

Mol Cell 2017 Oct;68(1):44-59

New York Genome Center, New York, NY, USA; Department of Biology, New York University, New York, NY, USA. Electronic address:

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nuclease system is a powerful tool for genome editing, and its simple programmability has enabled high-throughput genetic and epigenetic studies. These high-throughput approaches offer investigators a toolkit for functional interrogation of not only protein-coding genes but also noncoding DNA. Historically, noncoding DNA has lacked the detailed characterization that has been applied to protein-coding genes in large part because there has not been a robust set of methodologies for perturbing these regions. Although the majority of high-throughput CRISPR screens have focused on the coding genome to date, an increasing number of CRISPR screens targeting noncoding genomic regions continue to emerge. Here, we review high-throughput CRISPR-based approaches to uncover and understand functional elements within the noncoding genome and discuss practical aspects of noncoding library design and screen analysis.
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http://dx.doi.org/10.1016/j.molcel.2017.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701515PMC
October 2017

GUIDES: sgRNA design for loss-of-function screens.

Nat Methods 2017 08;14(9):831-832

New York Genome Center, New York, New York, USA.

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http://dx.doi.org/10.1038/nmeth.4423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870754PMC
August 2017

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

Nature 2017 08 11;548(7667):343-346. Epub 2017 Aug 11.

Department of Biological Engineering, MIT, Cambridge, Massachusetts 02139, USA.

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.
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http://dx.doi.org/10.1038/nature23451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706657PMC
August 2017

Identification of essential genes for cancer immunotherapy.

Nature 2017 08 7;548(7669):537-542. Epub 2017 Aug 7.

National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland 20892, USA.

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8 T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8 T cells with the resistance or non-responsiveness of cancer to immunotherapies.
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http://dx.doi.org/10.1038/nature23477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870757PMC
August 2017

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.

Nat Protoc 2017 04 23;12(4):828-863. Epub 2017 Mar 23.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

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.
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http://dx.doi.org/10.1038/nprot.2017.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526071PMC
April 2017

Transcription control by the ENL YEATS domain in acute leukaemia.

Nature 2017 03 1;543(7644):270-274. Epub 2017 Mar 1.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.
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http://dx.doi.org/10.1038/nature21688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497220PMC
March 2017

High-resolution interrogation of functional elements in the noncoding genome.

Science 2016 09;353(6307):1545-1549

Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

The noncoding genome affects gene regulation and disease, yet we lack tools for rapid identification and manipulation of noncoding elements. We developed a CRISPR screen using ~18,000 single guide RNAs targeting >700 kilobases surrounding the genes NF1, NF2, and CUL3, which are involved in BRAF inhibitor resistance in melanoma. We find that noncoding locations that modulate drug resistance also harbor predictive hallmarks of noncoding function. With a subset of regions at the CUL3 locus, we demonstrate that engineered mutations alter transcription factor occupancy and long-range and local epigenetic environments, implicating these sites in gene regulation and chemotherapeutic resistance. Through our expansion of the potential of pooled CRISPR screens, we provide tools for genomic discovery and for elucidating biologically relevant mechanisms of gene regulation.
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http://dx.doi.org/10.1126/science.aaf7613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5144102PMC
September 2016

CRISPR Screens to Discover Functional Noncoding Elements.

Trends Genet 2016 09 13;32(9):526-529. Epub 2016 Jul 13.

New York Genome Center, New York, NY 10013, USA; Center for Genomics and Systems Biology, Department of Biology, New York University, NY 10003, USA. Electronic address:

A major challenge in genomics is to identify functional elements in the noncoding genome. Recently, pooled clustered regularly interspersed palindromic repeat (CRISPR) mutagenesis screens of noncoding regions have emerged as a novel method for finding elements that impact gene expression and phenotype/disease-relevant biological processes. Here we review and compare different approaches for high-throughput dissection of noncoding elements.
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http://dx.doi.org/10.1016/j.tig.2016.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992445PMC
September 2016

Microfluidic neurite guidance to study structure-function relationships in topologically-complex population-based neural networks.

Sci Rep 2016 06 22;6:28384. Epub 2016 Jun 22.

Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 0213, USA.

The central nervous system is a dense, layered, 3D interconnected network of populations of neurons, and thus recapitulating that complexity for in vitro CNS models requires methods that can create defined topologically-complex neuronal networks. Several three-dimensional patterning approaches have been developed but none have demonstrated the ability to control the connections between populations of neurons. Here we report a method using AC electrokinetic forces that can guide, accelerate, slow down and push up neurites in un-modified collagen scaffolds. We present a means to create in vitro neural networks of arbitrary complexity by using such forces to create 3D intersections of primary neuronal populations that are plated in a 2D plane. We report for the first time in vitro basic brain motifs that have been previously observed in vivo and show that their functional network is highly decorrelated to their structure. This platform can provide building blocks to reproduce in vitro the complexity of neural circuits and provide a minimalistic environment to study the structure-function relationship of the brain circuitry.
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http://dx.doi.org/10.1038/srep28384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916598PMC
June 2016

Genome-scale CRISPR pooled screens.

Anal Biochem 2017 09 1;532:95-99. Epub 2016 Jun 1.

New York Genome Center, New York, NY 10013, USA; Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY 10012, USA. Electronic address:

Genome editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR) systems have ushered in a new era of targeted DNA manipulation. The easy programmability of CRISPR using short oligonucleotides enables rapid synthesis of large-scale libraries for functional genetic screens. Here we present fundamental concepts and methods for pooled CRISPR screens and review biological results from recent genome-scale loss-of-function and gain-of-function screens. We also discuss new frontiers in pooled screens, including novel effector domains for functional screens and applications in the noncoding genome.
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http://dx.doi.org/10.1016/j.ab.2016.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133192PMC
September 2017