A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as $\textit{DOT1L}$, $\textit{BCL2}$, and $\textit{MEN1}$, and many other genes including clinically actionable candidates. We validate selected genes using genetic and pharmacological inhibition, and chose $\textit{KAT2A}$ as a candidate for downstream study. $\textit{KAT2A}$ inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs of diverse genotypes while sparing normal hemopoietic stem-progenitor cells. Our results propose that KAT2A inhibition should be investigated as a therapeutic strategy in AML and provide a large number of genetic vulnerabilities of this leukemia that can be pursued in downstream studies.This work was funded by the Kay Kendall Leukaemia Fund (KKLF) and the Wellcome Trust (WT098051). G.S.V. is funded by a Wellcome Trust Senior Fellowship in Clinical Science (WT095663MA) and work in his laboratory is funded by Bloodwise. C.P. is funded by a Kay Kendall Leukaemia Fund Intermediate Fellowship (KKL888)

Measurement of B0d - B0d-bar mixing rate from the time evolution of dilepton events at the Upsilon(4S)

We report a determination of the B0d - B0d-bar mixing parameter Delta-m_d based on the time evolution of dilepton yields in Upsilon(4S) decays. The measurement is based on a 5.9 /fb data sample collected by the Belle detector at KEKB. The proper-time difference distributions for same-sign and opposite-sign dilepton events are simultaneously fitted to an expression containing Delta-m_d as a free parameter. Using both muons and electrons, we obtain Delta-m_d = 0.463 +- 0.008(stat.) +- 0.016(sys.) ps^{-1} This is the first determination of Delta-m_d from time evolution measurements at the Upsilon(4S). We also place limits on possible CPT violations.Comment: 12 pages, 2 figure

Measurement of the CP Violation Parameter sin(2phi_1) in B^0_d Meson Decays

We present a measurement of the Standard Model CP violation parameter sin(2phi_1) based on a 10.5 fb^{-1} data sample collected at the Upsilon(4S) resonance with the Belle detector at the KEKB asymmetric e+e- collider. One neutral B meson is reconstructed in the J/psi K_S, psi(2S) K_S, chi_{c1} K_S, eta_c K_S, J/psi K_L or J/psi pi^0 CP-eigenstate decay channel and the flavor of the accompanying B meson is identified from its charged particle decay products. From the asymmetry in the distribution of the time interval between the two B-meson decay points, we determine sin(2phi_1) = 0.58 +0.32-0.34 (stat) +0.09-0.10 (syst).Comment: LaTex, 13 pages, 3 figures, submitted to P.R.

Mutagenesis of diploid mammalian genes by gene entrapment

The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 × 10(−5) to 1.2 × 10(−4) per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells

Belle II Technical Design Report

The Belle detector at the KEKB electron-positron collider has collected almost 1 billion Y(4S) events in its decade of operation. Super-KEKB, an upgrade of KEKB is under construction, to increase the luminosity by two orders of magnitude during a three-year shutdown, with an ultimate goal of 8E35 /cm^2 /s luminosity. To exploit the increased luminosity, an upgrade of the Belle detector has been proposed. A new international collaboration Belle-II, is being formed. The Technical Design Report presents physics motivation, basic methods of the accelerator upgrade, as well as key improvements of the detector.Comment: Edited by: Z. Dole\v{z}al and S. Un

CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases

High-throughput mapping of regulatory DNA

Quantifying the effects of cis-regulatory DNA on gene expression is a major challenge. Here, we present the multiplexed editing regulatory assay (MERA), a high-throughput CRISPR-Cas9–based approach that analyzes the functional impact of the regulatory genome in its native context. MERA tiles thousands of mutations across ~40 kb of cis-regulatory genomic space and uses knock-in green fluorescent protein (GFP) reporters to read out gene activity. Using this approach, we obtain quantitative information on the contribution of cis-regulatory regions to gene expression. We identify proximal and distal regulatory elements necessary for expression of four embryonic stem cell–specific genes. We show a consistent contribution of neighboring gene promoters to gene expression and identify unmarked regulatory elements (UREs) that control gene expression but do not have typical enhancer epigenetic or chromatin features. We compare thousands of functional and nonfunctional genotypes at a genomic location and identify the base pair–resolution functional motifs of regulatory elements.National Institutes of Health (U.S.) (1U01HG007037

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

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.Paul & Daisy Soros Fellowships for New Americans (New York, N.Y.)McGovern Institute for Brain Research at MIT (Friends of McGovern Institute Fellowship)Massachusetts Institute of Technology. Poitras Center for Affective Disorders ResearchUnited States. Department of Energy (Computational Science Graduate Fellowship)National Institute of Mental Health (U.S.) (5DP1-MH100706)National Institute of Mental Health (U.S.) (1R01-MH110049)New York Stem Cell FoundationPoitras FoundationSimons FoundationPaul G. Allen Family FoundationVallee FoundationTom HarrimanB. Metcalf