Computational pipeline for designing guide RNAs for mismatch-CRISPRi.

CRISPR interference is an increasingly popular method for perturbing gene expression. Guided by single-guide RNAs (sgRNAs), nuclease-deficient Cas9 proteins bind to specific DNA sequences and hinder transcription. Specificity is achieved through complementarity of the sgRNAs to the DNA. Changing complementarity by introducing single-nucleotide mismatches can be exploited to tune knockdown. Here, we present a computational pipeline to identify sgRNAs targeting specific genes in a bacterial genome, filter them, and titrate their activity by introducing mismatches. For complete details on the use and execution of this protocol, please refer to Hawkins et al. (2020).

Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis.

Essential genes are the hubs of cellular networks, but lack of high-throughput methods for titrating gene expression has limited our understanding of the fitness landscapes against which their expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate defined levels of CRISPRi activity and demonstrated its broad applicability. Using libraries of mismatched sgRNAs predicted to span the full range of knockdown levels, we characterized the expression-fitness relationships of most essential genes in Escherichia coli and Bacillus subtilis. We find that these relationships vary widely from linear to bimodal but are similar within pathways. Notably, despite ∼2 billion years of evolutionary separation between E. coli and B. subtilis, most essential homologs have similar expression-fitness relationships with rare but informative differences. Thus, the expression levels of essential genes may reflect homeostatic or evolutionary constraints shared between the two organisms.

Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs.

A lack of tools to precisely control gene expression has limited our ability to evaluate relationships between expression levels and phenotypes. Here, we describe an approach to titrate expression of human genes using CRISPR interference and series of single-guide RNAs (sgRNAs) with systematically modulated activities. We used large-scale measurements across multiple cell models to characterize activities of sgRNAs containing mismatches to their target sites and derived rules governing mismatched sgRNA activity using deep learning. These rules enabled us to synthesize a compact sgRNA library to titrate expression of ~2,400 genes essential for robust cell growth and to construct an in silico sgRNA library spanning the human genome. Staging cells along a continuum of gene expression levels combined with single-cell RNA-seq readout revealed sharp transitions in cellular behaviors at gene-specific expression thresholds. Our work provides a general tool to control gene expression, with applications ranging from tuning biochemical pathways to identifying suppressors for diseases of dysregulated gene expression.

Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans1-6. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.

Let-7 microRNA-dependent control of leukotriene signaling regulates the transition of hematopoietic niche in mice.

Hematopoietic stem and progenitor cells arise from the vascular endothelium of the dorsal aorta and subsequently switch niche to the fetal liver through unknown mechanisms. Here we report that vascular endothelium-specific deletion of mouse Drosha (Drosha cKO), an enzyme essential for microRNA biogenesis, leads to anemia and death. A similar number of hematopoietic stem and progenitor cells emerge from Drosha-deficient and control vascular endothelium, but Drosha cKO-derived hematopoietic stem and progenitor cells accumulate in the dorsal aorta and fail to colonize the fetal liver. Depletion of the let-7 family of microRNAs is a primary cause of this defect, as it leads to activation of leukotriene B4 signaling and induction of the α4ß1 integrin cell adhesion complex in hematopoietic stem and progenitor cells. Inhibition of leukotriene B4 or integrin rescues maturation and migration of Drosha cKO hematopoietic stem and progenitor cells to the fetal liver, while it hampers hematopoiesis in wild-type animals. Our study uncovers a previously undefined role of innate leukotriene B4 signaling as a gatekeeper of the hematopoietic niche transition.Hematopoietic stem and progenitor cells are generated first from the vascular endothelium of the dorsal aorta and then the fetal liver but what regulates this switch is unknown. Here, the authors show that changing miRNA biogenesis and leukotriene B4 signaling in mice modulates this switch in the niche.

Fluorescent tagged episomals for stoichiometric induced pluripotent stem cell reprogramming.

BACKGROUND: Non-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the "Yamanaka reprogramming factors" (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors. METHODS: We modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors. RESULTS: The reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage. CONCLUSIONS: Together, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency.

A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria.

Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.

Bacterial CRISPR: accomplishments and prospects.

In this review we briefly describe the development of CRISPR tools for genome editing and control of transcription in bacteria. We focus on the Type II CRISPR/Cas9 system, provide specific examples for use of the system, and highlight the advantages and disadvantages of CRISPR versus other techniques. We suggest potential strategies for combining CRISPR tools with high-throughput approaches to elucidate gene function in bacteria.

Single-cell resolution of morphological changes in hemogenic endothelium.

Endothelial-to-hematopoietic transition (EHT) occurs within a population of hemogenic endothelial cells during embryogenesis, and leads to the formation of the adult hematopoietic system. Currently, the prospective identification of specific endothelial cells that will undergo EHT, and the cellular events enabling this transition, are not known. We set out to define precisely the morphological events of EHT, and to correlate cellular morphology with the expression of the transcription factors RUNX1 and SOX17. A novel strategy was developed to allow for correlation of immunofluorescence data with the ultrastructural resolution of scanning electron microscopy. The approach can identify single endothelial cells undergoing EHT, as identified by the ratio of RUNX1 to SOX17 immunofluorescence levels, and the morphological changes associated with the transition. Furthermore, this work details a new technical resource that is widely applicable for correlative analyses of single cells in their native tissue environments.

Repression of arterial genes in hemogenic endothelium is sufficient for haematopoietic fate acquisition.

Changes in cell fate and identity are essential for endothelial-to-haematopoietic transition (EHT), an embryonic process that generates the first adult populations of haematopoietic stem cells (HSCs) from hemogenic endothelial cells. Dissecting EHT regulation is a critical step towards the production of in vitro derived HSCs. Yet, we do not know how distinct endothelial and haematopoietic fates are parsed during the transition. Here we show that genes required for arterial identity function later to repress haematopoietic fate. Tissue-specific, temporally controlled, genetic loss of arterial genes (Sox17 and Notch1) during EHT results in increased production of haematopoietic cells due to loss of Sox17-mediated repression of haematopoietic transcription factors (Runx1 and Gata2). However, the increase in EHT can be abrogated by increased Notch signalling. These findings demonstrate that the endothelial haematopoietic fate switch is actively repressed in a population of endothelial cells, and that derepression of these programs augments haematopoietic output.

Targeted Transcriptional Repression in Bacteria Using CRISPR Interference (CRISPRi).

Clustered regularly interspersed short palindromic repeats (CRISPR) interference (CRISPRi) is a powerful technology for sequence-specifically repressing gene expression in bacterial cells. CRISPRi requires only a single protein and a custom-designed guide RNA for specific gene targeting. In Escherichia coli, CRISPRi repression efficiency is high (~300-fold), and there are no observable off-target effects. The method can be scaled up as a general strategy for the repression of many genes simultaneously using multiple designed guide RNAs. Here we provide a protocol for efficient guide RNA design, cloning, and assay of the CRISPRi system in E. coli. In principle, this protocol can be used to construct CRISPRi systems for gene repression in other species of bacteria.

Emergence of hematopoietic stem and progenitor cells involves a Chd1-dependent increase in total nascent transcription.

Lineage specification during development involves reprogramming of transcriptional states, but little is known about how this is regulated in vivo. The chromatin remodeler chomodomain helicase DNA-binding protein 1 (Chd1) promotes an elevated transcriptional output in mouse embryonic stem cells. Here we report that endothelial-specific deletion of Chd1 leads to loss of definitive hematopoietic progenitors, anemia, and lethality by embryonic day (E)15.5. Mutant embryos contain normal numbers of E10.5 intraaortic hematopoietic clusters that express Runx1 and Kit, but these clusters undergo apoptosis and fail to mature into blood lineages in vivo and in vitro. Hematopoietic progenitors emerging from the aorta have an elevated transcriptional output relative to structural endothelium, and this elevation is Chd1-dependent. In contrast, hematopoietic-specific deletion of Chd1 using Vav-Cre has no apparent phenotype. Our results reveal a new paradigm of regulation of a developmental transition by elevation of global transcriptional output that is critical for hemogenesis and may play roles in other contexts.

Generation of oligodendroglial cells by direct lineage conversion.

Transplantation of oligodendrocyte precursor cells (OPCs) is a promising potential therapeutic strategy for diseases affecting myelin. However, the derivation of engraftable OPCs from human pluripotent stem cells has proven difficult and primary OPCs are not readily available. Here we report the generation of induced OPCs (iOPCs) by direct lineage conversion. Forced expression of the three transcription factors Sox10, Olig2 and Zfp536 was sufficient to reprogram mouse and rat fibroblasts into iOPCs with morphologies and gene expression signatures resembling primary OPCs. More importantly, iOPCs gave rise to mature oligodendrocytes that could ensheath multiple host axons when co-cultured with primary dorsal root ganglion cells and formed myelin after transplantation into shiverer mice. We propose direct lineage reprogramming as a viable alternative approach for the generation of OPCs for use in disease modeling and regenerative medicine.

In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc finger nucleases.

The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically corrected, patient-derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene-corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug-resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN-induced break. Our approach delineates a roadmap for using ZFNs made by an open-source method to achieve efficient, transgene-free correction of monogenic disease mutations in patient-derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene-corrected human iPSCs that could be used for therapeutic applications.