Towards optimizing diversifying base editors for high-throughput studies of single- nucleotide variants.

Determining the phenotypic effects of single nucleotide variants is critical for understanding the genome and interpreting clinical sequencing results. Base editors, including diversifying base editors that create C>N mutations, are potent tools for installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing.

Development of compact transcriptional effectors using high-throughput measurements in diverse contexts.

Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector function for a library containing 5,092 nuclear protein Pfam domains across varied contexts. We also map context dependencies of effectors drawn from unannotated protein regions using a larger library tiling chromatin regulators and transcription factors.

Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems.

Gene therapy holds great therapeutic potential. Yet, controlling cargo expression in single cells is limited due to the variability of delivery methods. We implement an incoherent feedforward loop based on proteolytic cleavage of CRISPR-Cas activation or inhibition systems to reduce gene expression variability against the variability of vector delivery.

Cytotoxicity of Activator Expression in CRISPR-based Transcriptional Activation Systems.

CRISPR-based transcriptional activation (CRISPRa) has extensive research and clinical potential. Here, we show that commonly used CRISPRa systems can exhibit pronounced cytotoxicity. We demonstrate the toxicity of published and new CRISPRa vectors expressing the activation domains (ADs) of the transcription factors p65 and HSF1, components of the synergistic activation mediator (SAM) CRISPRa system.

Multicenter integrated analysis of noncoding CRISPRi screens.

The ENCODE Consortium's efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.

A genome-wide genetic screen uncovers determinants of human pigmentation.

Skin color, one of the most diverse human traits, is determined by the quantity, type, and distribution of melanin. In this study, we leveraged the light-scattering properties of melanin to conduct a genome-wide screen for regulators of melanogenesis. We identified 169 functionally diverse genes that converge on melanosome biogenesis, endosomal transport, and gene regulation, of which 135 represented previously unknown associations with pigmentation.

High-throughput functional characterization of combinations of transcriptional activators and repressors.

Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, whereas combining strong activators often results in weaker activation.

High-throughput discovery and characterization of viral transcriptional effectors in human cells.

Viruses encode transcriptional regulatory proteins critical for controlling viral and host gene expression. Given their multifunctional nature and high sequence divergence, it is unclear which viral proteins can affect transcription and which specific sequences contribute to this function. Using a high-throughput assay, we measured the transcriptional regulatory potential of over 60,000 protein tiles across ∼1,500 proteins from 11 coronaviruses and all nine human herpesviruses.

CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping.

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein.

Large-scale mapping and mutagenesis of human transcriptional effector domains.

Human gene expression is regulated by more than 2,000 transcription factors and chromatin regulators. Effector domains within these proteins can activate or repress transcription. However, for many of these regulators we do not know what type of effector domains they contain, their location in the protein, their activation and repression strengths, and the sequences that are necessary for their functions.

The sound of silence: Transgene silencing in mammalian cell engineering.

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos.

Modeling the efficacy of CRISPR gene drive for snail immunity on schistosomiasis control.

CRISPR gene drives could revolutionize the control of infectious diseases by accelerating the spread of engineered traits that limit parasite transmission in wild populations. Gene drive technology in mollusks has received little attention despite the role of freshwater snails as hosts of parasitic flukes causing 200 million annual cases of schistosomiasis. A successful drive in snails must overcome self-fertilization, a common feature of host snails which could prevents a drive's spread.

Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome.

Large serine recombinases (LSRs) are DNA integrases that facilitate the site-specific integration of mobile genetic elements into bacterial genomes. Only a few LSRs, such as Bxb1 and PhiC31, have been characterized to date, with limited efficiency as tools for DNA integration in human cells. In this study, we developed a computational approach to identify thousands of LSRs and their DNA attachment sites, expanding known LSR diversity by >100-fold and enabling the prediction of their insertion site specificities.

Adeno-Associated Virus Vector-Mediated Expression of Antirespiratory Syncytial Virus Antibody Prevents Infection in Mouse Airways.

Infants and older adults are especially vulnerable to infection by respiratory syncytial virus (RSV), which can cause significant illness and irreparable damage to the lower respiratory tract and for which an effective vaccine is not readily available. Palivizumab, a recombinant monoclonal antibody (mAb), is an approved therapeutic for RSV infection for use in high-risk infants only. Due to several logistical issues, including cost of goods and scale-up limitations, palivizumab is not approved for other populations that are vulnerable to severe RSV infections, such as older adults.

Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements.

Pooled CRISPR-Cas9 screens are 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 investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor.

Identification of Guide-Intrinsic Determinants of Cas9 Specificity.

Considerable effort has been devoted to developing a comprehensive understanding of CRISPR nuclease specificity. predictions and multiple genome-wide cellular and biochemical approaches have revealed a basic understanding of the Cas9 specificity profile. However, none of these approaches has delivered a model that allows accurate prediction of a CRISPR nuclease's ability to cleave a site based entirely on the sequence of the guide RNA (gRNA) and the target.

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells.

Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors.

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes.

The past several years have seen an explosion in development of applications for the CRISPR-Cas9 system, from efficient genome editing, to high-throughput screening, to recruitment of a range of DNA and chromatin-modifying enzymes. While homology-directed repair (HDR) coupled with Cas9 nuclease cleavage has been used with great success to repair and re-write genomes, recently developed base-editing systems present a useful orthogonal strategy to engineer nucleotide substitutions. Base editing relies on recruitment of cytidine deaminases to introduce changes (rather than double-stranded breaks and donor templates) and offers potential improvements in efficiency while limiting damage and simplifying the delivery of editing machinery.

Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements.

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer-promoter interactions, specifically at regions of shared DNA accessibility.

Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity.

Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing, with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods.

Toolbox for Exploring Modular Gene Regulation in Synthetic Biology Training.

We report a toolbox for exploring the modular tuning of genetic circuits, which has been specifically optimized for widespread deployment in STEM environments through a combination of bacterial strain engineering and distributable hardware development. The transfer functions of 16 genetic switches, programmed to express a GFP reporter under the regulation of the (acyl-homoserine lactone) AHL-sensitive luxR transcriptional activator, can be parametrically tuned by adjusting high/low degrees of transcriptional, translational, and post-translational processing. Strains were optimized to facilitate daily large-scale preparation and reliable performance at room temperature in order to eliminate the need for temperature controlled apparatuses, which are both cost-limiting and space-constraining.