In vivo interaction screening reveals liver-derived constraints to metastasis.

It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.

Integrated compact regulators of protein activity enable control of signaling pathways and genome-editing in vivo.

Viral proteases and clinically safe inhibitors were employed to build integrated compact regulators of protein activity (iCROP) for post-translational regulation of functional proteins by tunable proteolytic activity. In the absence of inhibitor, the co-localized/fused protease cleaves a target peptide sequence introduced in an exposed loop of the protein of interest, irreversibly fragmenting the protein structure and destroying its functionality. We selected three proteases and demonstrated the versatility of the iCROP framework by validating it to regulate the functional activity of ten different proteins. iCROP switches can be delivered either as mRNA or DNA, and provide rapid actuation kinetics with large induction ratios, while remaining strongly suppressed in the off state without inhibitor. iCROPs for effectors of the NF-κB and NFAT signaling pathways were assembled and confirmed to enable precise activation/inhibition of downstream events in response to protease inhibitors. In lipopolysaccharide-treated mice, iCROP-sr-IκBα suppressed cytokine release ("cytokine storm") by rescuing the activity of IκBα, which suppresses NF-κB signaling. We also constructed compact inducible CRISPR-(d)Cas9 variants and showed that iCROP-Cas9-mediated knockout of the PCSK9 gene in the liver lowered blood LDL-cholesterol levels in mice. iCROP-based protein switches will facilitate protein-level regulation in basic research and translational applications.

Transcriptional linkage analysis with in vivo AAV-Perturb-seq.

The ever-growing compendium of genetic variants associated with human pathologies demands new methods to study genotype-phenotype relationships in complex tissues in a high-throughput manner1,2. Here we introduce adeno-associated virus (AAV)-mediated direct in vivo single-cell CRISPR screening, termed AAV-Perturb-seq, a tuneable and broadly applicable method for transcriptional linkage analysis as well as high-throughput and high-resolution phenotyping of genetic perturbations in vivo. We applied AAV-Perturb-seq using gene editing and transcriptional inhibition to systematically dissect the phenotypic landscape underlying 22q11.2 deletion syndrome3,4 genes in the adult mouse brain prefrontal cortex. We identified three 22q11.2-linked genes involved in known and previously undescribed pathways orchestrating neuronal functions in vivo that explain approximately 40% of the transcriptional changes observed in a 22q11.2-deletion mouse model. Our findings suggest that the 22q11.2-deletion syndrome transcriptional phenotype found in mature neurons may in part be due to the broad dysregulation of a class of genes associated with disease susceptibility that are important for dysfunctional RNA processing and synaptic function. Our study establishes a flexible and scalable direct in vivo method to facilitate causal understanding of biological and disease mechanisms with potential applications to identify genetic interventions and therapeutic targets for treating disease.

Fast, multiplexable and efficient somatic gene deletions in adult mouse skeletal muscle fibers using AAV-CRISPR/Cas9.

Molecular screens comparing different disease states to identify candidate genes rely on the availability of fast, reliable and multiplexable systems to interrogate genes of interest. CRISPR/Cas9-based reverse genetics is a promising method to eventually achieve this. However, such methods are sorely lacking for multi-nucleated muscle fibers, since highly efficient nuclei editing is a requisite to robustly inactive candidate genes. Here, we couple Cre-mediated skeletal muscle fiber-specific Cas9 expression with myotropic adeno-associated virus-mediated sgRNA delivery to establish a system for highly effective somatic gene deletions in mice. Using well-characterized genes, we show that local or systemic inactivation of these genes copy the phenotype of traditional gene-knockout mouse models. Thus, this proof-of-principle study establishes a method to unravel the function of individual genes or entire signaling pathways in adult skeletal muscle fibers without the cumbersome requirement of generating knockout mice.

CRISPR for neuroscientists.

Genome engineering technologies provide an entry point into understanding and controlling the function of genetic elements in health and disease. The discovery and development of the microbial defense system CRISPR-Cas yielded a treasure trove of genome engineering technologies and revolutionized the biomedical sciences. Comprising diverse RNA-guided enzymes and effector proteins that evolved or were engineered to manipulate nucleic acids and cellular processes, the CRISPR toolbox provides precise control over biology. Virtually all biological systems are amenable to genome engineering-from cancer cells to the brains of model organisms to human patients-galvanizing research and innovation and giving rise to fundamental insights into health and powerful strategies for detecting and correcting disease. In the field of neuroscience, these tools are being leveraged across a wide range of applications, including engineering traditional and non-traditional transgenic animal models, modeling disease, testing genomic therapies, unbiased screening, programming cell states, and recording cellular lineages and other biological processes. In this primer, we describe the development and applications of CRISPR technologies while highlighting outstanding limitations and opportunities.

Microbial medics diagnose and treat gut inflammation.

Engineered microbes show potential for diagnosing and treating diseases. In this issue of Cell Host & Microbe, Zou et al. develop an "intelligent" bacterial strain that detects and monitors an inflammation biomarker in the gut and responds by releasing an immunomodulator, thereby combining diagnosis and therapy for intestinal inflammation.

Noninvasive assessment of gut function using transcriptional recording sentinel cells.

Transcriptional recording by CRISPR spacer acquisition from RNA endows engineered Escherichia coli with synthetic memory, which through Record-seq reveals transcriptome-scale records. Microbial sentinels that traverse the gastrointestinal tract capture a wide range of genes and pathways that describe interactions with the host, including quantitative shifts in the molecular environment that result from alterations in the host diet, induced inflammation, and microbiome complexity. We demonstrate multiplexed recording using barcoded CRISPR arrays, enabling the reconstruction of transcriptional histories of isogenic bacterial strains in vivo. Record-seq therefore provides a scalable, noninvasive platform for interrogating intestinal and microbial physiology throughout the length of the intestine without manipulations to host physiology and can determine how single microbial genetic differences alter the way in which the microbe adapts to the host intestinal environment.

miR-137 and miR-122, two outer subventricular zone non-coding RNAs, regulate basal progenitor expansion and neuronal differentiation.

Cortical expansion in primate brains relies on enlargement of germinal zones during a prolonged developmental period. Although most mammals have two cortical germinal zones, the ventricular zone (VZ) and subventricular zone (SVZ), gyrencephalic species display an additional germinal zone, the outer subventricular zone (oSVZ), which increases the number and diversity of neurons generated during corticogenesis. How the oSVZ emerged during evolution is poorly understood, but recent studies suggest a role for non-coding RNAs, which allow tight genetic program regulation during development. Here, using in vivo functional genetics, single-cell RNA sequencing, live imaging, and electrophysiology to assess progenitor and neuronal properties in mice, we identify two oSVZ-expressed microRNAs (miRNAs), miR-137 and miR-122, which regulate key cellular features of cortical expansion. miR-137 promotes basal progenitor self-replication and superficial layer neuron fate, whereas miR-122 decreases the pace of neuronal differentiation. These findings support a cell-type-specific role of miRNA-mediated gene expression in cortical expansion.

An In Vivo CRISPR Screen Identifies Stepwise Genetic Dependencies of Metastatic Progression.

Blood-borne metastasis of breast cancer involves a series of tightly regulated sequential steps, including the growth of a primary tumor lesion, intravasation of circulating tumor cells (CTC), and adaptation in various distant metastatic sites. The genes orchestrating each of these steps are poorly understood in physiologically relevant contexts, owing to the rarity of experimental models that faithfully recapitulate the biology, growth kinetics, and tropism of human breast cancer. Here, we conducted an in vivo loss-of-function CRISPR screen in newly derived CTC xenografts, unique in their ability to spontaneously mirror the human disease, and identified specific genetic dependencies for each step of the metastatic process. Validation experiments revealed sensitivities to inhibitors that are already available, such as PLK1 inhibitors, to prevent CTC intravasation. Together, these findings present a new tool to reclassify driver genes involved in the spread of human cancer, providing insights into the biology of metastasis and paving the way to test targeted treatment approaches. SIGNIFICANCE: A loss-of-function CRISPR screen in human CTC-derived xenografts identifies genes critical for individual steps of the metastatic cascade, suggesting novel drivers and treatment opportunities for metastatic breast cancers.

Temporal controls over inter-areal cortical projection neuron fate diversity.

Interconnectivity between neocortical areas is critical for sensory integration and sensorimotor transformations1-6. These functions are mediated by heterogeneous inter-areal cortical projection neurons (ICPN), which send axon branches across cortical areas as well as to subcortical targets7-9. Although ICPN are anatomically diverse10-14, they are molecularly homogeneous15, and how the diversity of their anatomical and functional features emerge during development remains largely unknown. Here we address this question by linking the connectome and transcriptome in developing single ICPN of the mouse neocortex using a combination of multiplexed analysis of projections by sequencing16,17 (MAPseq, to identify single-neuron axonal projections) and single-cell RNA sequencing (to identify corresponding gene expression). Focusing on neurons of the primary somatosensory cortex (S1), we reveal a protracted unfolding of the molecular and functional differentiation of motor cortex-projecting ([Formula: see text]) ICPN compared with secondary somatosensory cortex-projecting ([Formula: see text]) ICPN. We identify SOX11 as a temporally differentially expressed transcription factor in [Formula: see text] versus [Formula: see text] ICPN. Postnatal manipulation of SOX11 expression in S1 impaired sensorimotor connectivity and disrupted selective exploratory behaviours in mice. Together, our results reveal that within a single cortical area, different subtypes of ICPN have distinct postnatal paces of molecular differentiation, which are subsequently reflected in distinct circuit connectivities and functions. Dynamic differences in the expression levels of a largely generic set of genes, rather than fundamental differences in the identity of developmental genetic programs, may thus account for the emergence of intra-type diversity in cortical neurons.

Multiplexed Genome Engineering with Cas12a.

Genome engineering technologies based on CRISPR-Cas systems are fueling efforts to study genotype-phenotype relationships in a high-throughput and multiplexed fashion. While many genome engineering technologies exist and provide a means to efficiently manipulate one or a few genes in a singular context-knockout, inhibition, or activation in a constitutive, conditional, or inducible manner-progress towards engineering complex cellular programs has been hampered by the lack of technologies that can integrate these functions within a unified framework. To address this challenge, our lab created single transcript CRISPR-Cas12a (SiT-Cas12a), which enables conditional, inducible, orthogonal, and massively multiplexed genome engineering of dozens, to potentially hundreds, of genomic targets in eukaryotic cells simultaneously-providing a novel way to interrogate and engineer complex genetic programs. In this chapter, we outline the utility of SiT-Cas12a in human cells and describe experimental procedures for executing massively multiplexed genome engineering experiments-including strategies for designing and assembling customized multiplexed CRISPR guide RNA arrays as well as validating and analyzing CRISPR guide RNA array processing and genome engineering outcomes.

Regulation of purine metabolism connects KCTD13 to a metabolic disorder with autistic features.

Genetic variation of the 16p11.2 deletion locus containing the KCTD13 gene and of CUL3 is linked with autism. This genetic connection suggested that substrates of a CUL3-KCTD13 ubiquitin ligase may be involved in disease pathogenesis. Comparison of Kctd13 mutant (Kctd13 -/- ) and wild-type neuronal ubiquitylomes identified adenylosuccinate synthetase (ADSS), an enzyme that catalyzes the first step in adenosine monophosphate (AMP) synthesis, as a KCTD13 ligase substrate. In Kctd13 -/- neurons, there were increased levels of succinyl-adenosine (S-Ado), a metabolite downstream of ADSS. Notably, S-Ado levels are elevated in adenylosuccinate lyase deficiency, a metabolic disorder with autism and epilepsy phenotypes. The increased S-Ado levels in Kctd13 -/- neurons were decreased by treatment with an ADSS inhibitor. Lastly, functional analysis of human KCTD13 variants suggests that KCTD13 variation may alter ubiquitination of ADSS. These data suggest that succinyl-AMP metabolites accumulate in Kctd13 -/- neurons, and this observation may have implications for our understanding of 16p11.2 deletion syndrome.

Engineered bacteria to report gut function: technologies and implementation.

Advances in synthetic biology and microbiology have enabled the creation of engineered bacteria which can sense and report on intracellular and extracellular signals. When deployed in vivo these whole-cell bacterial biosensors can act as sentinels to monitor biomolecules of interest in human health and disease settings. This is particularly interesting in the context of the gut microbiota, which interacts extensively with the human host throughout time and transit of the gut and can be accessed from feces without requiring invasive collection. Leveraging rational engineering approaches for genetic circuits as well as an expanding catalog of disease-associated biomarkers, bacterial biosensors can act as non-invasive and easy-to-monitor reporters of the gut. Here, we summarize recent engineering approaches applied in vivo in animal models and then highlight promising technologies for designing the next generation of bacterial biosensors.

Recording transcriptional histories using Record-seq.

It is difficult to elucidate the transcriptional history of a cell using current experimental approaches, as they are destructive in nature and therefore describe only a moment in time. To overcome these limitations, we recently established Record-seq, a technology that enables transcriptional recording by CRISPR spacer acquisition from RNA. The recorded transcriptomes are recovered by SENECA, a method that selectively amplifies expanded CRISPR arrays, followed by deep sequencing. The resulting CRISPR spacers are aligned to the host genome, thereby enabling transcript quantification and associated analyses. Here, we describe the experimental procedures of the Record-seq workflow as well as subsequent data analysis. Beginning with the experimental design, Record-seq data can be obtained and analyzed within 1-2 weeks.

Mapping human cell phenotypes to genotypes with single-cell genomics.

The cumulative activity of all of the body's cells, with their myriad interactions, life histories, and environmental experiences, gives rise to a condition that is distinctly human and specific to each individual. It is an enduring goal to catalog our human cell types, to understand how they develop, how they vary between individuals, and how they fail in disease. Single-cell genomics has revolutionized this endeavor because sequencing-based methods provide a means to quantitatively annotate cell states on the basis of high-information content and high-throughput measurements. Together with advances in stem cell biology and gene editing, we are in the midst of a fascinating journey to understand the cellular phenotypes that compose human bodies and how the human genome is used to build and maintain each cell. Here, we will review recent advances into how single-cell genomics is being used to develop personalized phenotyping strategies that cross subcellular, cellular, and tissue scales to link our genome to our cumulative cellular phenotypes.

Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts.

The ability to modify multiple genetic elements simultaneously would help to elucidate and control the gene interactions and networks underlying complex cellular functions. However, current genome engineering technologies are limited in both the number and the type of perturbations that can be performed simultaneously. Here, we demonstrate that both Cas12a and a clustered regularly interspaced short palindromic repeat (CRISPR) array can be encoded in a single transcript by adding a stabilizer tertiary RNA structure. By leveraging this system, we illustrate constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using up to 25 individual CRISPR RNAs delivered on a single plasmid. Our method provides a powerful platform to investigate and orchestrate the sophisticated genetic programs underlying complex cell behaviors.

Author Correction: Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.

In the published version of this paper, Step 64 of the Procedure reads, "Refer to Steps 37-39 for NGS analysis of the sgRNA distribution." This step should refer the reader to Steps 35-39. This text has not been corrected in the original paper.

Transcriptional recording by CRISPR spacer acquisition from RNA.

The ability to record transcriptional events within a cell over time would help to elucidate how molecular events give rise to complex cellular behaviours and states. However, current molecular recording technologies capture only a small set of defined stimuli. Here we use CRISPR spacer acquisition to capture and convert intracellular RNAs into DNA, enabling DNA-based storage of transcriptional information. In Escherichia coli, we show that defined stimuli, such as an RNA virus or arbitrary sequences, as well as complex stimuli, such as oxidative stress, result in quantifiable transcriptional records that are stored within a population of cells. We demonstrate that the transcriptional records enable us to classify and describe complex cellular behaviours and to identify the precise genes that orchestrate differential cellular responses. In the future, CRISPR spacer acquisition-mediated recording of RNA followed by deep sequencing (Record-seq) could be used to reconstruct transcriptional histories that describe complex cell behaviours or pathological states.