RNA Modification & Editing

A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens

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Thu, 01/10/2019 - 00:00
Gasperini M, Hill AJ, McFaline-Figueroa JL, Martin B, Kim S, Zhang MD, Jackson D, Leith A, Schreiber J, Noble WS, Trapnell C, Ahituv N, Shendure J.
Cell. 2019 Jan 10;176(1-2):377-390.e19. doi: 10.1016/j.cell.2018.11.029. Epub 2019 Jan 3.
https://www.cell.com/cell/pdf/S0092-8674(18)31554-X.pdf
Reuben Hogan
Time
12:00pm

 

Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome.

RNA Modification & Editing
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reuhogan
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Multiplexed orthogonal genome editing and transcriptional activation by Cas12a

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Fri, 10/19/2018 - 00:00
Breinig M, Schweitzer AY, Herianto AM, Revia S, Schaefer L, Wendler L, Cobos Galvez A, Tschaharganeh DF.
Nat Methods. 2018 Dec 17. doi: 10.1038/s41592-018-0262-1. [Epub ahead of print]
Audre May
Time
12:00pm

 

CRISPR-Cas9-based combinatorial perturbation approaches for orthogonal knockout and gene activation have been impeded by complex vector designs and co-delivery of multiple constructs. Here, we demonstrate that catalytically active CRISPR-Cas12a fused to a transcriptional-activator domain enables flexible switching between genome editing and transcriptional activation by altering guide length. By leveraging Cas12a-mediated CRISPR-RNA array processing, we illustrate that Cas12a-VPR enables simplified multiplexed knockout and transcriptional activation in vitro and in vivo.

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[email protected]
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Identification and mitigation of pervasive off-target activity in CRISPR-Cas9 screens for essential non-coding elements

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Fri, 01/18/2019 - 00:00
Josh Tycko, Michael Wainberg, Georgi K Marinov, Oana Ursu, Gaelen T Hess, Braeden K Ego, Aradhana, Amy Li, Alisa Truong, Alexandro E Trevino, Kaitlyn Spees, David Yao, Irene M Kaplow, Peyton G Greenside, David W Morgens, Douglas H Phanstiel, Michael P Snyder, Lacramioara Bintu, William J Greenleaf, Anshul Kundaje, Michael C Bassik
bioRxiv preprint first posted online Jan. 18, 2019; doi: http://dx.doi.org/10.1101/520569. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
https://www.biorxiv.org/content/biorxiv/early/2019/01/18/520569.full.pdf
Theodore Roth
Time
12:00pm

Pooled CRISPR-Cas9 screens have recently emerged as a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we conducted a genome-scale screen for essential CTCF loop anchors in the K562 leukemia cell line. Surprisingly, the primary drivers of signal in this screen were single guide RNAs (sgRNAs) with low specificity scores. After removing these guides, we found that there were no CTCF loop anchors critical for cell growth. We also observed this effect in an independent screen fine-mapping the core motifs in enhancers of the GATA1 gene. We then conducted screens in parallel with CRISPRi and CRISPRa, which do not induce DNA damage, and found that an unexpected and distinct set of off-targets also caused strong confounding growth effects with these epigenome-editing platforms. Promisingly, strict filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and allowed for the identification of essential enhancers, which we validated extensively. Together, our results show off-target activity can severely limit identification of essential functional motifs by active Cas9, while strictly filtered CRISPRi screens can be reliably used for assaying larger regulatory elements.

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theoroth
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Predicting the mutations generated by repair of Cas9-induced double-strand breaks

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Tue, 11/27/2018 - 00:00
Allen F, Crepaldi L, Alsinet C, Strong AJ, Kleshchevnikov V, De Angeli P, Páleníková P, Khodak A, Kiselev V, Kosicki M, Bassett AR, Harding H, Galanty Y, Muñoz-Martínez F, Metzakopian E, Jackson SP, Parts L.
Nat Biotechnol. 2018 Nov 27. doi: 10.1038/nbt.4317. [Epub ahead of print]
Neil Tay
Time
12:00pm

 

The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.

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neiltay
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A Cas9 with Complete PAM Recognition for Adenine Dinucleotides

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Thu, 09/27/2018 - 00:00
Noah Jakimo , Pranam Chatterjee , Lisa Nip , & Joseph M Jacobson
bioRxiv preprint first posted online Sep. 27, 2018; doi: http://dx.doi.org/10.1101/429654. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
Stacey Phan
Time
12:00pm

CRISPR-associated (Cas) DNA-endonucleases are remarkably effective tools for genome engineering, but have limited target ranges due to their protospacer adjacent motif (PAM) requirements. We demonstrate a critical expansion of the targetable sequence space for a Type-IIA CRISPR-associated enzyme through identification of the natural 5’-NAA-3’ PAM specificity of a Streptococcus macacae Cas9 (Smac Cas9). We further recombine protein domains between Smac Cas9 and its well-established ortholog from Streptococcus pyogenes (Spy Cas9), as well as an “increased" nucleolytic variant (iSpy Cas9), to achieve consistent mediation of gene modification and base editing. In a comparison to previously reported Cas9 and Cas12a enzymes, we show that our hybrids recognize all adenine dinucleotide PAM sequences and possess robust editing efficiency in human cells.

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phanstacey
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Accurate classification of BRCA1 variants with saturation genome editing

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Wed, 09/12/2018 - 00:00
Findlay GM, Daza RM, Martin B, Zhang MD, Leith AP, Gasperini M, Janizek JD, Huang X, Starita LMShendure J
Nature. 2018 Sep 12. doi: 10.1038/s41586-018-0461-z. [Epub ahead of print]
http://www.nature.com/articles/s41586-018-0461-z.pdf
Theodore Roth
Time
12:00pm

 

Variants of uncertain significance fundamentally limit the clinical utility of genetic information. The challenge they pose is epitomized by BRCA1, a tumour suppressor gene in which germline loss-of-function variants predispose women to breast and ovarian cancer. Although BRCA1 has been sequenced in millions of women, the risk associated with most newly observed variants cannot be definitively assigned. Here we use saturation genome editing to assay 96.5% of all possible single-nucleotide variants (SNVs) in 13 exons that encode functionally critical domains of BRCA1. Functional effects for nearly 4,000 SNVs are bimodally distributed and almost perfectly concordant with established assessments of pathogenicity. Over 400 non-functional missense SNVs are identified, as well as around 300 SNVs that disrupt expression. We predict that these results will be immediately useful for the clinical interpretation of BRCA1 variants, and that this approach can be extended to overcome the challenge of variants of uncertain significance in additional clinically actionable genes.

RNA Modification & Editing
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theoroth
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An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities

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Mon, 07/30/2018 - 00:00
Gehrke JM, Cervantes O, Clement MK, Wu Y, Zeng J, Bauer DE, Pinello L Joung JK.
Nat Biotechnol. 2018 Jul 30. doi: 10.1038/nbt.4199. [Epub ahead of print]
https://www.nature.com/articles/nbt.4199.pdf
Cristina Rodriguez Mateo
Time
12:00pm

 

Base editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations. However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystandermutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy. In direct comparisons with the widely used base editor 3 (BE3) fusion in human cells, our eA3A-BE3 fusion exhibits similar activities on cytidines in TC motifs but greatly reduced editing on cytidines in other sequence contexts. eA3A-BE3 corrects a human β-thalassemia promoter mutation with much higher (>40-fold) precision than BE3. We also demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.

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crirodmat
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"Bacteriophage Cooperation Suppresses CRISPR-Cas3 and Cas9 Immunity" and "Anti-CRISPR Phages Cooperate to Overcome CRISPR-Cas Immunity"

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Thu, 08/09/2018 - 00:00
First Paper: Borges AL, Zhang JY, Rollins MF, Osuna BA, Wiedenheft B, Bondy-Denomy J

Second Paper: Landsberger M, Gandon S, Meaden S, Rollie C, Chevallereau A, Chabas H, Buckling A, Westra ER, van Houte S4.
Cell. 2018 Aug 9;174(4):917-925.e10. doi: 10.1016/j.cell.2018.06.013. Epub 2018 Jul 19. Second Paper: Cell. 2018 Aug 9;174(4):908-916.e12. doi: 10.1016/j.cell.2018.05.058. Epub 2018 Jul 19.
https://reader.elsevier.com/reader/sd/00E39846AC6FAC99C31768B25F6081AC89E63BB9B8130AE8A4E393545475FFC3BD7A99AF389FA3E88B5E3C535138D36A
John Gagnon
Time
12:00pm

 

Bacteria utilize CRISPR-Cas adaptive immune systems for protection from bacteriophages (phages), and some phages produce anti-CRISPR (Acr) proteins that inhibit immune function. Despite thorough mechanistic and structural information for some Acr proteins, how they are deployed and utilized by a phage during infection is unknown. Here, we show that Acr production does not guarantee phage replication when faced with CRISPR-Cas immunity, but instead, infections fail when phage population numbers fall below a critical threshold. Infections succeed only if a sufficient Acr dose is contributed to a single cell by multiple phage genomes. The production of Acr proteins by phage genomes that fail to replicate leave the cell immunosuppressed, which predisposes the cell for successful infection by other phages in the population. This altruistic mechanism for CRISPR-Cas inhibition demonstrates inter-virus cooperation that may also manifest in other host-parasite interactions.

Abstract

Some phages encode anti-CRISPR (acr) genes, which antagonize bacterial CRISPR-Cas immune systems by binding components of its machinery, but it is less clear how deployment of these acr genes impacts phage replication and epidemiology. Here, we demonstrate that bacteria with CRISPR-Cas resistance are still partially immune to Acr-encoding phage. As a consequence, Acr-phages often need to cooperate in order to overcome CRISPR resistance, with a first phage blocking the host CRISPR-Cas immune system to allow a second Acr-phage to successfully replicate. This cooperation leads to epidemiological tipping points in which the initial density of Acr-phage tips the balance from phage extinction to a phage epidemic. Furthermore, both higher levels of CRISPR-Cas immunity and weaker Acr activities shift the tipping points toward higher initial phage densities. Collectively, these data help elucidate how interactions between phage-encoded immune suppressors and the CRISPR systems they target shape bacteria-phage population dynamics.

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jgagnon
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p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells

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Sun, 07/01/2018 - 00:00
Ihry RJ, Worringer KA, Salick MR, Frias E, Ho D, Theriault K, Kommineni S, Chen J, Sondey M, Ye C, Randhawa R, Kulkarni T, Yang Z, McAllister G, Russ C, Reece-Hoyes J, Forrester W, Hoffman GR2, Dolmetsch R, Kaykas A.
Nat Med. 2018 Jul;24(7):939-946. doi: 10.1038/s41591-018-0050-6. Epub 2018 Jun 11.
Hui Li
Time
12:00pm

CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells1-3. Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells3-13. Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction3. The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations14, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

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huili
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