Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras.

The genetic dependencies of human cancers widely vary. Here, we catalog this heterogeneity and use it to identify functional gene interactions and genotype-dependent liabilities in cancer. By using genome-wide CRISPR-based screens, we generate a gene essentiality dataset across 14 human acute myeloid leukemia (AML) cell lines. Sets of genes with correlated patterns of essentiality across the lines reveal new gene relationships, the essential substrates of enzymes, and the molecular functions of uncharacterized proteins. Comparisons of differentially essential genes between Ras-dependent and -independent lines uncover synthetic lethal partners of oncogenic Ras. Screens in both human AML and engineered mouse pro-B cells converge on a surprisingly small number of genes in the Ras processing and MAPK pathways and pinpoint PREX1 as an AML-specific activator of MAPK signaling. Our findings suggest general strategies for defining mammalian gene networks and synthetic lethal interactions by exploiting the natural genetic and epigenetic diversity of human cancer cells.

Machine-learning-optimized Cas12a barcoding enables the recovery of single-cell lineages and transcriptional profiles.

The development of CRISPR-based barcoding methods creates an exciting opportunity to understand cellular phylogenies. We present a compact, tunable, high-capacity Cas12a barcoding system called dual acting inverted site array (DAISY). We combined high-throughput screening and machine learning to predict and optimize the 60-bp DAISY barcode sequences. After optimization, top-performing barcodes had ∼10-fold increased capacity relative to the best random-screened designs and performed reliably across diverse cell types. DAISY barcode arrays generated ∼12 bits of entropy and ∼66,000 unique barcodes. Thus, DAISY barcodes-at a fraction of the size of Cas9 barcodes-achieved high-capacity barcoding. We coupled DAISY barcoding with single-cell RNA-seq to recover lineages and gene expression profiles from ∼47,000 human melanoma cells. A single DAISY barcode recovered up to ∼700 lineages from one parental cell. This analysis revealed heritable single-cell gene expression and potential epigenetic modulation of memory gene transcription. Overall, Cas12a DAISY barcoding is an efficient tool for investigating cell-state dynamics.

Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects.

Several existing technologies enable short genomic alterations including generating indels and short nucleotide variants, however, engineering more significant genomic changes is more challenging due to reduced efficiency and precision. Here, we developed RecT Editor via Designer-Cas9-Initiated Targeting (REDIT), which leverages phage single-stranded DNA-annealing proteins (SSAP) RecT for mammalian genome engineering. Relative to Cas9-mediated homology-directed repair (HDR), REDIT yielded up to a 5-fold increase of efficiency to insert kilobase-scale exogenous sequences at defined genomic regions. We validated our REDIT approach using different formats and lengths of knock-in templates. We further demonstrated that REDIT tools using Cas9 nickase have efficient gene-editing activities and reduced off-target errors, measured using a combination of targeted sequencing, genome-wide indel, and insertion mapping assays. Our experiments inhibiting repair enzyme activities suggested that REDIT has the potential to overcome limitations of endogenous DNA repair steps. Finally, our REDIT method is applicable across cell types including human stem cells, and is generalizable to different Cas9 enzymes.

Computationally guided high-throughput engineering of an anti-CRISPR protein for precise genome editing in human cells.

The application of CRISPR-Cas systems to genome editing has revolutionized experimental biology and is an emerging gene and cell therapy modality. CRISPR-Cas systems target off-target regions within the human genome, which is a challenge that must be addressed. Phages have evolved anti-CRISPR proteins (Acrs) to evade CRISPR-Cas-based immunity. Here, we engineer an Acr (AcrIIA4) to increase the precision of CRISPR-Cas-based genome targeting. We developed an approach that leveraged (1) computational guidance, (2) deep mutational scanning, and (3) highly parallel DNA repair measurements within human cells. In a single experiment, ∼10,000 Acr variants were tested. Variants that improved editing precision were tested in additional validation experiments that revealed robust enhancement of gene editing precision and synergy with a high-fidelity version of Cas9. This scalable high-throughput screening framework is a promising methodology to engineer Acrs to increase gene editing precision, which could be used to improve the safety of gene editing-based therapeutics.

Efficient and multiplexed somatic genome editing with Cas12a mice.

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations in areas ranging from neuroscience to cancer biology and beyond. However, existing models are limited in their ability to create multiple targeted edits. Thus, our understanding of the complex genetic interactions that underlie development, homeostasis, and disease remains incomplete. Cas12a is an RNA-guided endonuclease with unique attributes that enable simple targeting of multiple genes with crRNA arrays containing tandem guides. To accelerate and expand the generation of complex genotypes in somatic cells, we generated transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a). In these mice, enAsCas12a-mediated somatic genome editing robustly generated compound genotypes, as exemplified by the initiation of diverse cancer types driven by homozygous inactivation of trios of tumor suppressor genes. We further integrated these modular crRNA arrays with clonal barcoding to quantify the size and number of tumors with each array, as well as the efficiency of each crRNA. These Cas12a alleles will enable the rapid generation of disease models and broadly facilitate the high-throughput investigation of coincident genomic alterations in somatic cells in vivo .

Functional mapping of epigenetic regulators uncovers coordinated tumor suppression by the HBO1 and MLL1 complexes.

Epigenetic dysregulation is widespread in cancer. However, the specific epigenetic regulators and the processes they control to drive cancer phenotypes are poorly understood. Here, we employed a novel, scalable and high-throughput in vivo method to perform iterative functional screens of over 250 epigenetic regulatory genes within autochthonous oncogenic KRAS-driven lung tumors. We identified multiple novel epigenetic tumor suppressor and tumor dependency genes. We show that a specific HBO1 complex and the MLL1 complex are among the most impactful tumor suppressive epigenetic regulators in lung. The histone modifications generated by the HBO1 complex are frequently absent or reduced in human lung adenocarcinomas. The HBO1 and MLL1 complexes regulate chromatin accessibility of shared genomic regions, lineage fidelity and the expression of canonical tumor suppressor genes. The HBO1 and MLL1 complexes are epistatic during lung tumorigenesis, and their functional correlation is conserved in human cancer cell lines. Together, these results demonstrate the value of quantitative methods to generate a phenotypic roadmap of epigenetic regulatory genes in tumorigenesis in vivo .

Multiplexed screens identify RAS paralogues HRAS and NRAS as suppressors of KRAS-driven lung cancer growth.

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the regulators of RAS signalling is incomplete. Here to uncover the impact of diverse KRAS-interacting proteins on lung cancer growth, we combined multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumour barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung cancer models, we show that HRAS and NRAS are suppressors of KRASG12D-driven tumour growth in vivo and confirm these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogues interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signalling. Furthermore, HRAS and NRAS mutations identified in oncogenic KRAS-driven human tumours partially abolished this effect. By comparing the tumour-suppressive effects of HRAS and NRAS in oncogenic KRAS- and oncogenic BRAF-driven lung cancer models, we confirm that RAS paralogues are specific suppressors of KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role RAS paralogue imbalance in oncogenic KRAS-driven lung cancer.

dCas9-based gene editing for cleavage-free genomic knock-in of long sequences.

Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR-Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9-SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9-SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9-SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering.

CRISPR-Cas12a System With Synergistic Phage Recombination Proteins for Multiplex Precision Editing in Human Cells.

The development of CRISPR-based gene-editing technologies has brought an unprecedented revolution in the field of genome engineering. Cas12a, a member of the Class 2 Type V CRISPR-associated endonuclease family distinct from Cas9, has been repurposed and developed into versatile gene-editing tools with distinct PAM recognition sites and multiplexed gene targeting capability. However, with current CRISPR/Cas12a technologies, it remains a challenge to perform efficient and precise genome editing of long sequences in mammalian cells. To address this limitation, we utilized phage recombination enzymes and developed an efficient CRISPR/Cas12a tool for multiplexed precision editing in mammalian cells. Through protein engineering, we were able to recruit phage recombination proteins to Cas12a to enhance its homology-directed repair efficiencies. Our phage-recombination-assisted Cas12a system achieved up to 3-fold improvements for kilobase-scale knock-ins in human cells without compromising the specificity of the enzyme. The performance of this system compares favorably against Cas9 references, the commonly used enzyme for gene-editing tasks, with improved specificity. Additionally, we demonstrated multi-target editing with similar improved activities thanks to the RNA-processing activity of the Cas12a system. This compact, multi-target editing tool has the potential to assist in understanding multi-gene interactions. In particular, it paves the way for a gene therapy method for human diseases that complements existing tools and is suitable for polygenic disorders and diseases requiring long-sequence corrections.

A Functional Taxonomy of Tumor Suppression in Oncogenic KRAS-Driven Lung Cancer.

Cancer genotyping has identified a large number of putative tumor suppressor genes. Carcinogenesis is a multistep process, but the importance and specific roles of many of these genes during tumor initiation, growth, and progression remain unknown. Here we use a multiplexed mouse model of oncogenic KRAS-driven lung cancer to quantify the impact of 48 known and putative tumor suppressor genes on diverse aspects of carcinogenesis at an unprecedented scale and resolution. We uncover many previously understudied functional tumor suppressors that constrain cancer in vivo. Inactivation of some genes substantially increased growth, whereas the inactivation of others increases tumor initiation and/or the emergence of exceptionally large tumors. These functional in vivo analyses revealed an unexpectedly complex landscape of tumor suppression that has implications for understanding cancer evolution, interpreting clinical cancer genome sequencing data, and directing approaches to limit tumor initiation and progression. SIGNIFICANCE: Our high-throughput and high-resolution analysis of tumor suppression uncovered novel genetic determinants of oncogenic KRAS-driven lung cancer initiation, overall growth, and exceptional growth. This taxonomy is consistent with changing constraints during the life history of cancer and highlights the value of quantitative in vivo genetic analyses in autochthonous cancer models.This article is highlighted in the In This Issue feature, p. 1601.

Identification and characterization of essential genes in the human genome.

Large-scale genetic analysis of lethal phenotypes has elucidated the molecular underpinnings of many biological processes. Using the bacterial clustered regularly interspaced short palindromic repeats (CRISPR) system, we constructed a genome-wide single-guide RNA library to screen for genes required for proliferation and survival in a human cancer cell line. Our screen revealed the set of cell-essential genes, which was validated with an orthogonal gene-trap-based screen and comparison with yeast gene knockouts. This set is enriched for genes that encode components of fundamental pathways, are expressed at high levels, and contain few inactivating polymorphisms in the human population. We also uncovered a large group of uncharacterized genes involved in RNA processing, a number of whose products localize to the nucleolus. Last, screens in additional cell lines showed a high degree of overlap in gene essentiality but also revealed differences specific to each cell line and cancer type that reflect the developmental origin, oncogenic drivers, paralogous gene expression pattern, and chromosomal structure of each line. These results demonstrate the power of CRISPR-based screens and suggest a general strategy for identifying liabilities in cancer cells.