Nicolas Bray

Near-optimal probabilistic RNA-seq quantification

We present kallisto, an RNA-seq quantification program that is two orders of magnitude faster than previous approaches and achieves similar accuracy. Kallisto pseudoaligns reads to a reference, producing a list of transcripts that are compatible with each read while avoiding alignment of individual bases. We use kallisto to analyze 30 million unaligned paired-end RNA-seq reads in <10 min on a standard laptop computer. This removes a major computational bottleneck in RNA-seq analysis.

Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells

Hematopoietic stem cells from patients with sickle cell disease can be edited by CRISPR/Cas9 and maintain the edits in vivo. Hammering out the sickle cell mutation Sickle cell disease is a genetic disorder caused by a mutation in one of the hemoglobin genes, which causes deformation of red blood cells and results in occlusion of blood vessels, severe pain crises, and progressive organ injury. To correct the mutation that causes this disease, DeWitt et al. modified hematopoietic stem cells from sickle cell disease patients using a CRISPR/Cas9 gene editing approach. The authors showed that the corrected cells successfully engrafted in a mouse model and produced enough normal hemoglobin to have a potential clinical benefit in the setting of sickle cell disease. Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the β-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.

Differential analysis of RNA-seq incorporating quantification uncertainty

We describe sleuth (http://pachterlab.github.io/sleuth), a method for the differential analysis of gene expression data that utilizes bootstrapping in conjunction with response error linear modeling to decouple biological variance from inferential variance. sleuth is implemented in an interactive shiny app that utilizes kallisto quantifications and bootstraps for fast and accurate analysis of data from RNA-seq experiments.

MAVID multiple alignment server

MAVID is a multiple alignment program suitable for many large genomic regions. The MAVID web server allows biomedical researchers to quickly obtain multiple alignments for genomic sequences and to subsequently analyse the alignments for conserved regions. MAVID has been successfully used for the alignment of closely related species such as primates and also for the alignment of more distant organisms such as human and fugu. The server is fast, capable of aligning hundreds of kilobases in less than a minute. The multiple alignment is used to build a phylogenetic tree for the sequences, which is subsequently used as a basis for identifying conserved regions in the alignment. The server can be accessed at http://baboon.math.berkeley.edu/mavid/.

Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair

Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9)-based therapeutics, especially those that can correct gene mutations via homology-directed repair, have the potential to revolutionize the treatment of genetic diseases. However, it is challenging to develop homology-directed repair-based therapeutics because they require the simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.Gold nanoparticles carrying Cas9 ribonucleoprotein and donor DNA, and complexed with endosomal disruptive polymers, correct the DNA mutation that causes Duchenne muscular dystrophy in mice, with minimal off-target effects.

Disabling Cas9 by an anti-CRISPR DNA mimic

Natural inhibitors of Cas9 pretend to be DNA and block target binding, and using them in human cells can reduce off-target events. CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9–single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo–electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

Discovery of stimulation-responsive immune enhancers with CRISPR activation

The majority of genetic variants associated with common human diseases map to enhancers, non-coding elements that shape cell-type-specific transcriptional programs and responses to extracellular cues. Systematic mapping of functional enhancers and their biological contexts is required to understand the mechanisms by which variation in non-coding genetic sequences contributes to disease. Functional enhancers can be mapped by genomic sequence disruption, but this approach is limited to the subset of enhancers that are necessary in the particular cellular context being studied. We hypothesized that recruitment of a strong transcriptional activator to an enhancer would be sufficient to drive target gene expression, even if that enhancer was not currently active in the assayed cells. Here we describe a discovery platform that can identify stimulus-responsive enhancers for a target gene independent of stimulus exposure. We used tiled CRISPR activation (CRISPRa) to synthetically recruit a transcriptional activator to sites across large genomic regions (more than 100 kilobases) surrounding two key autoimmunity risk loci, CD69 and IL2RA. We identified several CRISPRa-responsive elements with chromatin features of stimulus-responsive enhancers, including an IL2RA enhancer that harbours an autoimmunity risk variant. Using engineered mouse models, we found that sequence perturbation of the disease-associated Il2ra enhancer did not entirely block Il2ra expression, but rather delayed the timing of gene activation in response to specific extracellular signals. Enhancer deletion skewed polarization of naive T cells towards a pro-inflammatory T helper (TH17) cell state and away from a regulatory T cell state. This integrated approach identifies functional enhancers and reveals how non-coding variation associated with human immune dysfunction alters context-specific gene programs.

Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9–sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption.

Pseudoalignment for metagenomic read assignment

Motivation: Read assignment is an important first step in many metagenomic analysis workflows, providing the basis for identification and quantification of species. However ambiguity among the sequences of many strains makes it difficult to assign reads at the lowest level of taxonomy, and reads are typically assigned to taxonomic levels where they are unambiguous. We explore connections between metagenomic read assignment and the quantification of transcripts from RNA‐Seq data in order to develop novel methods for rapid and accurate quantification of metagenomic strains. Results: We find that the recent idea of pseudoalignment introduced in the RNA‐Seq context is highly applicable in the metagenomics setting. When coupled with the Expectation‐Maximization (EM) algorithm, reads can be assigned far more accurately and quickly than is currently possible with state of the art software, making it possible and practical for the first time to analyze abundances of individual genomes in metagenomics projects. Availability and Implementation: Pipeline and analysis code can be downloaded from http://github.com/pachterlab/metakallisto Contact: lpachter@math.berkeley.edu

Erratum: Near-optimal probabilistic RNA-seq quantification

Nat. Biotechnol. 34, 525–527 (2016); published online 4 April 2016; corrected after print 27 July 2016 In the version of this article initially published, in the HTML version only, the equation “αtN > 0.01” was written as “αtN > 0.01.” In addition, in the Figure 1 legend, the formatting of thenodes was incorrect (v_1, etc.