Publications by authors named "James K Nuñez"

11 Publications

  • Page 1 of 1

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing.

Cell 2021 Apr 9;184(9):2503-2519.e17. Epub 2021 Apr 9.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA. Electronic address:

A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
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http://dx.doi.org/10.1016/j.cell.2021.03.025DOI Listing
April 2021

Pervasive functional translation of noncanonical human open reading frames.

Science 2020 03;367(6482):1140-1146

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA.

Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.
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http://dx.doi.org/10.1126/science.aay0262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289059PMC
March 2020

Inducible and multiplex gene regulation using CRISPR-Cpf1-based transcription factors.

Nat Methods 2017 Dec 30;14(12):1163-1166. Epub 2017 Oct 30.

Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 nuclease fused to transcriptional activation domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.
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http://dx.doi.org/10.1038/nmeth.4483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909187PMC
December 2017

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response.

Cell 2016 Dec;167(7):1867-1882.e21

Department of Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.
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http://dx.doi.org/10.1016/j.cell.2016.11.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315571PMC
December 2016

CRISPR Immunological Memory Requires a Host Factor for Specificity.

Mol Cell 2016 06 19;62(6):824-833. Epub 2016 May 19.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics and Integrated Bioimaging, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address:

Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1-Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition.
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http://dx.doi.org/10.1016/j.molcel.2016.04.027DOI Listing
June 2016

Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

ACS Chem Biol 2016 Mar 9;11(3):681-8. Epub 2016 Feb 9.

Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720, United States.

The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.
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http://dx.doi.org/10.1021/acschembio.5b01019DOI Listing
March 2016

Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering.

Cell 2016 Jan;164(1-2):29-44

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute HHMI, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, Berkeley, CA 94720, USA. Electronic address:

Bacteria and archaea possess a range of defense mechanisms to combat plasmids and viral infections. Unique among these are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems, which provide adaptive immunity against foreign nucleic acids. CRISPR systems function by acquiring genetic records of invaders to facilitate robust interference upon reinfection. In this Review, we discuss recent advances in understanding the diverse mechanisms by which Cas proteins respond to foreign nucleic acids and how these systems have been harnessed for precision genome manipulation in a wide array of organisms.
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http://dx.doi.org/10.1016/j.cell.2015.12.035DOI Listing
January 2016

Foreign DNA capture during CRISPR-Cas adaptive immunity.

Nature 2015 Nov 21;527(7579):535-8. Epub 2015 Oct 21.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.

Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.
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http://dx.doi.org/10.1038/nature15760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662619PMC
November 2015

Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

Nature 2015 Mar 18;519(7542):193-8. Epub 2015 Feb 18.

1] Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA [2] Center for RNA Systems Biology, University of California, Berkeley, Berkeley, California 94720, USA [3] Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA [4] Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, USA [5] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.

Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.
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http://dx.doi.org/10.1038/nature14237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359072PMC
March 2015

Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity.

Nat Struct Mol Biol 2014 Jun 4;21(6):528-34. Epub 2014 May 4.

1] Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA. [2] Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA. [3] Department of Chemistry, University of California, Berkeley, Berkeley, California, USA. [4] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

The initial stage of CRISPR-Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR-Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1-Cas2 complex. Mutations that perturb Cas1-Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1-Cas2 complexes specify sites of CRISPR spacer integration.
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http://dx.doi.org/10.1038/nsmb.2820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4075942PMC
June 2014

Molecular basis for H3K36me3 recognition by the Tudor domain of PHF1.

Nat Struct Mol Biol 2012 Dec 11;19(12):1266-72. Epub 2012 Nov 11.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado, USA.

The PHD finger protein 1 (PHF1) is essential in epigenetic regulation and genome maintenance. Here we show that the Tudor domain of human PHF1 binds to histone H3 trimethylated at Lys36 (H3K36me3). We report a 1.9-Å resolution crystal structure of the Tudor domain in complex with H3K36me3 and describe the molecular mechanism of H3K36me3 recognition using NMR. Binding of PHF1 to H3K36me3 inhibits the ability of the Polycomb PRC2 complex to methylate Lys27 of histone H3 in vitro and in vivo. Laser microirradiation data show that PHF1 is transiently recruited to DNA double-strand breaks, and PHF1 mutants impaired in the H3K36me3 interaction exhibit reduced retention at double-strand break sites. Together, our findings suggest that PHF1 can mediate deposition of the repressive H3K27me3 mark and acts as a cofactor in early DNA-damage response.
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http://dx.doi.org/10.1038/nsmb.2435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3603146PMC
December 2012