Large-scale interrogation of genome structure is crucial for understanding how genomic organization influences cellular function, yet existing methods are limited by the low density of achievable modifications or the toxicity of methods. Here we address this gap by presenting a versatile approach that combines gene editing and recombinase technologies. The protocol serves two critical purposes: (1) facilitating the introduction of hundreds to thousands of precise genomic edits per cell and (2) enabling the creation of a controlled platform to systematically investigate the effects of induced genomic rearrangements. Specifically, the method leverages prime editing to insert recombinase recognition sites (for example, loxP) into repetitive genomic regions, such as LINE-1 elements, thereby enabling extensive genetic modifications in human cells. This scale of genome editing has not previously been attainable and supports a wide range of studies, including genome-wide functional analyses and essentiality mapping. Inducing controlled rearrangements with recombinase and tracking cell survival under selective conditions allows direct mapping of genome architecture to cellular fitness, opening new opportunities for genome-wide functional screens and rational synthetic genome design. Unlike methods that rely on double-strand breaks or random transposon insertion, this Protocol supports a programmable installation of thousands of recombination sites at repeat elements, offering denser and more predictable substrates for controlled genome rearrangement. The full protocol takes ~12–18 weeks to complete and requires intermediate to advanced expertise in genome editing, mammalian cell culture and sequencing analysis. This large-scale genome structure interrogation approach uses prime editing to insert recombinase recognition sites into repetitive genomic regions. It enables thousands of precise genomic edits to be installed per cell with systematic investigation of their effects.

Programmable technologies that sense nucleic acid signatures in living cells and trigger cellular functions hold promise for biotechnology and medicine. Here, we develop SONAR (Sensing Of Nucleic acids using ASOs and Reverse-transcriptases), a platform that detects target DNA and RNA sequences and triggers controlled gene expression in human cells. SONAR operates through circularizable single-stranded DNA (ssDNA) sensors that, upon hybridization with complementary DNA or reverse-transcribed RNA, undergo target-dependent ligation via cellular ligases, subsequently driving expression of genetic payloads. For RNA sensing, we employ antisense oligonucleotides (ASOs) to prime targeted reverse transcription, generating complementary DNA that promotes ssDNA circularization. We demonstrate SONAR's ability to detect ssDNA, exogenous and endogenous RNA, couple sensing to programmable expression of diverse protein payloads, including reporters, recombinases, and genome editors, and enable enrichment and clonal recovery of target-positive cells from mixed populations. This platform establishes a versatile framework for targeted nucleic acid detection and inducible gene expression, with broad potential applications in diagnostics, therapeutics, and synthetic biology.

Abstract Programmable intracellular production of short functional RNAs underlies diverse applications ranging from gene regulation to genome engineering, but is often constrained by cloning workflows, RNA synthesis, or viral-vector delivery. Here, we introduce a single-stranded DNA (ssDNA)-driven intracellular transcription strategy in which short ssDNA templates function as transcriptional substrates when paired with orthogonal bacteriophage RNA polymerases (RNAPs). By embedding phage promoters within short hairpin structures to create a locally double-stranded recognition site, we enable robust RNAP-dependent transcription from ssDNA in mammalian cells. We demonstrate that ssDNA templates can drive production of functional CRISPR guides, supporting adenine base editing at reporter and endogenous loci and enabling CRISPR-based transcriptional activation across distinct human cell lines. These results establish ssDNA templates as compact, synthetically accessible inputs for RNA production in mammalian cells, with broad utility in synthetic biology and genome engineering.

Cancer metastasis is a major contributor to patient morbidity and mortality1, yet the factors that determine the organs where cancers can metastasize are incompletely understood. Here we quantify the absolute levels of 124 metabolites in multiple tissues in mice and investigate how this relates to the ability of breast cancer cells to grow in different organs. We engineered breast cancer cells with broad metastatic potential to be auxotrophic for specific nutrients and assessed their ability to colonize different tissue sites. We then asked how tumour growth in different tissues relates to nutrient availability and tumour biosynthetic activity. We find that single nutrients alone do not define the sites where breast cancer cells can grow as metastases. In addition, we identify purine synthesis as a requirement for tumour growth and metastasis across many tissues and find that this phenotype is independent of tissue nucleotide availability or tumour de novo nucleotide synthesis activity. These data suggest that a complex interplay between multiple nutrients within the microenvironment dictates potential sites of metastatic cancer growth, and highlights the interdependence between extrinsic environmental factors and intrinsic cellular properties in influencing where breast cancer cells can grow as metastases.

Abstract Nutrient availability varies across tissues and shapes how cancer cells use metabolism to proliferate and survive, creating context-specific vulnerabilities that might be targeted for improved therapy. Because access to lipids is constrained in some tissue environments, we sought to identify metabolic pathways required for proliferation under lipid-depleted conditions. Specifically, we performed a CRISPR/Cas9 loss-of-function screen targeting metabolic synthesis genes in human cancer cells cultured in lipid-replete versus lipid-depleted media. The screen identified methionine synthase (MTR), an enzyme linking the folate and methionine cycles, as a top hit required for proliferation in lipid-depleted conditions. Genetic validation confirmed that MTR knockout (KO) cell proliferation is impaired in lipid-depleted compared to lipid-replete conditions. Follow-up pharmacologic studies revealed that inhibition of methionine adenosyltransferase 2A (MAT2A), an enzyme in the methionine cycle that generates S-adenosylmethionine (SAM), also reduced proliferation in lipid-depleted conditions, suggesting that cells become broadly dependent on the methionine/folate cycle when lipids are scarce.To determine how methionine/folate cycle perturbations alter intracellular metabolism in lipid-depleted conditions, we performed LC-MS-based metabolomics. MTR KO in lipid-depleted media caused depletion of multiple nucleotide species, and supplementation with purine nucleotides or folinic acid fully rescued proliferation, consistent with impaired folate-dependent nucleotide synthesis. MTR KO cells also exhibited elevated markers of DNA damage, supporting a model in which MTR loss limits nucleotide availability under lipid-depleted conditions, leading to DNA damage. In contrast, MAT2A-inhibited cells were not rescued by nucleotides or folinic acid. Instead, supplementation with phosphatidylcholine, a major membrane phospholipid synthesized in a SAM-dependent manner, restored proliferation in lipid-depleted media. These results suggest that perturbing different nodes of the methionine cycle triggers distinct metabolic liabilities: MTR loss primarily limits nucleotide availability, whereas MAT2A inhibition restricts SAM-dependent phospholipid synthesis. Together, these findings reveal that cancer cells rely more heavily on methionine and folate metabolism when lipids are scarce and uncover two mechanistically distinct vulnerabilities that emerge in low-lipid environments with potential relevance for treating cancer. Citation Format: Diya Lakshmi Ramesh, Keene L. Abbott, Ryan Elbashir, Edrees H. Rashan, Raphael Ferreira, Matthew G. Vander Heiden. Cancer cells are sensitive to methionine cycle perturbation in low-lipid environments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 540.

Base editors enable precise genome modification but are constrained by bystander edits that limit their applicability. Existing strategies to enhance precision often compromise efficiency and remain highly sequence dependent. Here we present a parallel engineering approach that optimizes both guide RNAs and the deaminase enzyme to minimize bystander editing without sacrificing activity. We designed a library of 3'-extended guide RNAs and identified context-dependent variants that improved specificity. Using a precision-driven phage-assisted evolution system and protein language models, we evolved adenine base editor variants two- to threefold more precise than adenine base editor ABE8e while maintaining high efficiency across a library of thousands of human pathogenic contexts in vitro. Our findings establish a scalable framework for precision engineering of base editors, addressing a major challenge in genome editing.

We lack tools to edit DNA sequences at scales necessary to study 99% of the human genome that is noncoding. To address this gap, we applied CRISPR prime editing to insert recombination handles into repetitive sequences, up to 1697 per cell line, which enables generating large-scale deletions, inversions, translocations, and circular DNA. Recombinase induction produced more than 100 stochastic megabase-sized rearrangements in each cell. We tracked these rearrangements over time to measure selection pressures, finding a preference for shorter variants that avoided essential genes. We characterized 29 clones with multiple rearrangements, finding an impact of deletions on expression of genes in the variant but not on nearby genes. This genome-scrambling strategy enables large deletions, sequence relocations, and the insertion of regulatory elements to explore genome dispensability and organization.

Cancer cell metabolism is significantly influenced by microenvironmental factors such as nutrient availability, which can vary across different tissues and introduce exploitable therapeutic vulnerabilities. Notably, lipid availability is particularly low in the brain compared to other tissues. To identify metabolic vulnerabilities of cells in lipid-poor environments, we performed a CRISPR/Cas9 screen of metabolic synthesis genes in human cancer cells cultured in lipid-rich versus lipid-poor media. Through this screen, we observed a novel metabolic dependency in the cells when grown in lipid-depleted conditions. We verified this differential dependency by knockout (KO) of this target gene, which resulted in slower cell proliferation in lipid-depleted media. Additionally, wild-type cells displayed heightened sensitivity to inhibitors of other proteins in the pathway in lipid-depleted conditions, thereby phenocopying the KO. Polar metabolomics analyses by LC-MS revealed depletion of several nucleotide species in the KO cells cultured in lipid-depleted conditions, and supplementation with purine nucleotides rescued the proliferation of KO cells cultured in lipid-depleted media. We hypothesized that this nucleotide depletion could in turn result in nucleotide imbalance and DNA damage, and Western Blot analyses confirmed elevated DNA damage levels in the KO cells in lipid-depleted media. Our model is that in low-lipid environments, the KO redirects metabolic resources toward lipid synthesis, causing nucleotide imbalance, DNA damage, and cell death. Overall, our findings indicate that cancer cells exhibit novel metabolic vulnerabilities when exposed to lipid-depleted environments, which could be leveraged for treating tumors growing in lipid-poor organs such as the brain. Diya L. Ramesh, Keene L. Abbott, Raphael Ferreira, Matthew G. Vander Heiden. Cancer cells exhibit novel metabolic vulnerabilities when exposed to low lipid environments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4154.

Acquiring nucleotides is essential for all dividing cells, and failure to maintain sufficient levels and balance of nucleotides impairs DNA replication. Eukaryotic cells meet nucleotide demands through either de novo synthesis or salvage of nucleotide precursors from the extracellular environment. Although it is known that levels of salvageable nucleotide precursors vary across tissues, the contribution of nucleotide salvage to total nucleotide acquisition in malignant cells remains underexplored. Using a mouse model of B-cell acute lymphoblastic leukemia (B-ALL), we formulated a mouse-plasma-like medium (MPM) to investigate how cells balance nucleotide acquisition strategies in nutrient environments that mimic mouse plasma during leukemogenesis. We used stable-isotope tracing and LC-MS to measure the contribution of de novo synthesis and salvage pathways for nucleotide acquisition in B-ALL cells. We found that cells cultured in MPM preferentially acquire some nucleotide species through salvage pathways, and genetic perturbation of nucleotide salvage pathways leads to deoxynucleotide triphosphate (dNTP) depletion, induction of replication stress, and reduced proliferation. We found that the dependence on nucleotide salvage arises because physiological levels of the vitamin folate, a molecule used in de novo nucleotide synthesis, are low enough to constrain de novo nucleotide biosynthesis in physiological conditions, driving increased reliance on salvage pathways. Accordingly, culturing cancer cells in conventional cell culture media with folate levels found in physiological conditions attenuates cell proliferation due to decreased de novo nucleotide synthesis. In vivo, genetically perturbing pyrimidine nucleotide salvage slows B-ALL proliferation, and this is further exacerbated when mice were fed folate-depleted diets. On the other hand, dietary folate supplementation increased the proliferation of salvage deficient cells in vivo. Together, these findings demonstrate that physiological folate availability limits de novo nucleotide synthesis in B-ALL, creating dependence on nucleotide salvage pathways. This work informs potential combination therapy strategies targeting folate metabolism and nucleotide salvage. Ryan Elbashir, Keene L. Abbott, Ahmed Ali, Diya L. Ramesh, Michelle Wu, Brian T. Do, Anya Shevzov-Zebrun, Tenzin Kunchok, Millenia Waite, Wontaek Chung, Chelsea Zhang, Sharanya Sivanand, Azucena Ramos, Jacob A. Hansen, Raphael Ferreira, Alexander Muir, Michael H. Hemann, Matthew G. Vander Heiden. Physiological nutrient levels reveal nucleotide salvage as a dependency in B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3800.

While protein-coding genes are characterized increasingly well, 99% of the human genome is non-coding and poorly understood. This gap is due to a lack of tools for engineering variants that affect sequence to the necessary extent. To bridge this gap, we have developed a toolbox to create deletions, inversions, translocations, and extrachromosomal circular DNA at scale by highly multiplexed insertion of recombinase recognition sites into repetitive sequences with CRISPR prime editing. Using this strategy, we derived stable human cell lines with several thousand clonal insertions, the highest number of novel sequences inserted into single human genomes. Subsequent recombinase induction generated an average of more than one hundred megabase-sized rearrangements per cell, and thousands across the whole population. The ability to detect rearrangements as they are generated and to track their abundance over time allowed us to measure the selection pressures acting on different types of structural changes. We observed a consolidation towards shorter variants that preferentially delete growth-inhibiting genes and a depletion of translocations. We isolated and characterized 21 clones with multiple recombinase-induced rearrangements. These included viable haploid clones with deletions that span hundreds of kilobases as well as triploid HEK293T clones with aneuploidies and fold back chromosomes. We mapped the impact of these genetic changes on gene expression to decipher how structural variants affect gene regulation. The genome scrambling strategy developed here makes it possible to delete megabases of sequence, move sequences between and within chromosomes, and implant regulatory elements into new contexts which will shed light on the genome organization principles of humans and other species. ### Competing Interest Statement G.M.C., J.K., L.P., R.F., T.E. filed a patent application on work presented here. G.C. is a co-founder of Editas Medicine and has other financial interests listed at: . T.V. has received PhD studentship funding from AstraZeneca.

Cancer metastasis is a major contributor to patient morbidity and mortality1, yet the factors that determine the organs where cancers can metastasize are incompletely understood. In this study, we quantify the absolute levels of over 100 nutrients available across multiple tissues in mice and investigate how this relates to the ability of breast cancer cells to grow in different organs. We engineered breast cancer cells with broad metastatic potential to be auxotrophic for specific nutrients and assessed their ability to colonize different organs. We then asked how tumor growth in different tissues relates to nutrient availability and tumor biosynthetic activity. We find that single nutrients alone do not define the sites where breast cancer cells can grow as metastases. Additionally, we identify purine synthesis as a requirement for tumor growth and metastasis across many tissues and find that this phenotype is independent of tissue nucleotide availability or tumor de novo nucleotide synthesis activity. These data suggest that a complex interplay of multiple nutrients within the microenvironment dictates potential sites of metastatic cancer growth, and highlights the interdependence between extrinsic environmental factors and intrinsic cellular properties in influencing where breast cancer cells can grow as metastases.

Abstract The metastasis of cancer to the brain is a major contributor to patient morbidity and mortality. Prior work suggests that there is therapeutic potential in targeting metabolic processes within brain metastatic cancer, however how best to identify novel targets and select patient populations remains unclear. In this study, we determine what nutrients are available to breast cancer cells in the brain and how those nutrients are used. We also engineer breast cancer cells that are auxotrophic for specific nutrients and assess how this impacts tumor growth in the brain using various approaches. These methodologies include direct implantation into the brain and introduction into the circulation to evaluate tumor formation as brain metastases. Unexpectedly, we find that no single approach, including assessment of brain nutrient availability, tumor biosynthetic activity, and evaluation of genetic dependencies using in vivo CRISPR screens reliably predicts metabolic dependencies that broadly extend across models of breast cancer brain metastasis. Our findings underscore the necessity of a holistic approach in considering how best to identify and prioritize new targets for treating metastatic cancer. Citation Format: Keene L. Abbott, Sonu Subudhi, Raphael Ferreira, Yetiş Gültekin, Sophie C. Steinbuch, Sophie E. Honeder, Ashwin S Kumar, Michelle Wu, Diya Ramesh, Jacob Hansen, Lisa M. Riedmayr, Mark Duquette, Ahmed Ali, Nicole Henning, Sharanya Sivanand, Tenzin Kunchok, Millenia Waite, Brian T. Do, Virginia Spanoudaki, Francisco J. Sánchez-Rivera, George M. Church, Rakesh K Jain, Matthew G. Vander Heiden. Assessment of metabolic vulnerabilities of breast cancer brain metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A007.

As we enter the era of CRISPR medicines, base editors (BEs) emerged as one of the most promising tools to treat genetic associated diseases. However, unintended bystander editing beyond the target nucleotide poses a challenge to their translation into effective therapies. While many efforts have been made in the design of a universal enzyme with minimal bystander editing, the context dependent activity represents a major challenge for base editing-based therapies. In this work, we designed a sequence-specific guide RNA library with 3’-extensions and detected guides that were able to reduce bystander and increase editing efficiency in a context dependent manner. The best candidate was later used for phage assisted non-continuous evolution to find a new generation of precise base editors. Simultaneously, we use protein language models trained on massive protein sequence datasets to find the evolutionarily plausible mutational patterns that can improve deaminase activity and precision. Both strategies provide a collection of precise TadA variants that not only drastically reduced bystander edits, but also was not in detriment of on-target activity. Our findings introduce a guide/enzyme parallel engineering pipeline, which lays the foundation for the development of new personalized genome editing strategies, ultimately enhancing the safety and precision of this groundbreaking technology. ### Competing Interest Statement G.C. is a co-founder of Editas Medicine and has other financial interests listed at: .

Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.

The production of bio-based chemicals and fuels through microbial engineering offers a promising and sustainable alternative to petroleum-based fuels and chemicals, with the potential for scalability. However, engineering microbes and continuously evolving them to enhance the production of industrially relevant products is a complex and challenging task, requiring precise selection of genetic traits to achieve desired outcomes. In this study, we report the development of a novel counter-selectable growth-sensitive malonyl-CoA platform strain by coupling the malonyl-CoA repressor FapR from Bacillus subtilis to essential gene promoters involved in glucose growth and the plasma membrane arginine permease. This platform strain was then coupled with a CRISPR-dCas9 guide-RNA (gRNA) library, which after multiple rounds of dilutions and library sequencing, resulted in the enrichment for gRNAs that increased fluxes towards malonyl-CoA. The enriched gRNAs were validated for their effects on growth enhancement, gene regulation, and the production of an industrially relevant malonyl-CoA product, namely 3-hydroxypropionic acid. This study highlights an innovative approach to microbial engineering and opens up avenues for further exploration in the field of laboratory continuous evolution.

Control of cellular identity involves coordination of developmental programs with environmental factors such as nutrient availability, suggesting that modulating aspects of metabolism could enable therapeutically relevant changes in cell fate. We show that nucleotide depletion facilitates gene expression changes towards a new cell fate by perturbing DNA replication in models of acute myeloid leukemia, a cancer characterized by a differentiation blockade. This transition starts in S phase and is independent of replication stress signaling and DNA damage signaling pathways. Moreover, it occurs despite sustained oncogene-driven expression of the progenitor program and is accompanied by limited changes in chromatin accessibility. Altering lineage-determining transcription factor expression redirects cell fate progression towards an alternate fate upon replication stress, suggesting that perturbing DNA replication allows cells to mobilize primed maturation programs. Our work, along with other findings in diverse systems, suggests a conserved mechanism by which metabolic changes can orchestrate cell fate transitions.