Abstract Introduction The tumor microenvironment is a complex ecosystem of cancer, immune, and stromal cells. Within this environment, cancer progression is coordinated by both cell-intrinsic and -extrinsic factors. Targeting signaling interactions between cancer and immune cells has proven to be an effective therapeutic strategy, as evidenced by the remarkable success of cancer immunotherapies. However, clinical responses vary across patients and tumor types. Developing effective cancer therapies and defining critical windows for clinical intervention requires a detailed understanding of the phenotypic and spatiotemporal dynamics of the cell types that make up the tumor microenvironment. Methods To enable high-resolution reconstruction of cell fate decisions in the tumor microenvironment, we developed PEtracer, a prime editing-based lineage tracing system compatible with single-cell sequencing and multimodal imaging, allowing for joint analysis of cell state and lineage in dissociated cells or in intact tissues with high spatial resolution. Results Using PEtracer coupled with single-cell and spatial transcriptomic profiling in syngeneic mouse models of cancer, we reconstructed the growth of individually seeded tumors in vivo, uncovered distinct modules of cell-intrinsic and -extrinsic factors that coordinate tumor growth, and mapped the recruitment and differentiation dynamics of tumor-infiltrating immune cells. Conclusion PEtracer enables high-resolution reconstruction of cell state and lineage dynamics within the tumor microenvironment in vivo. By leveraging prime editing-based lineage recording and multimodal imaging, PEtracer allows systematic dissection of how tumor, immune, and stromal cells evolve during cancer progression and therapeutic response. The resulting spatially resolved multimodal datasets provide a foundation for predictive models of tumor microenvironment dynamics. Funding Source National Institutes of Health Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Our ability to edit genomes lags behind our capacity to sequence them, but the growing understanding of CRISPR biology and its application to genome, epigenome and transcriptome engineering is narrowing this gap. In this Review, we discuss recent developments of various CRISPR-based systems that can transiently or permanently modify the genome and the transcriptome. The discovery of further CRISPR enzymes and systems through functional metagenomics has meaningfully broadened the applicability of CRISPR-based editing. Engineered Cas variants offer diverse capabilities such as base editing, prime editing, gene insertion and gene regulation, thereby providing a panoply of tools for the scientific community. We highlight the strengths and weaknesses of current CRISPR tools, considering their efficiency, precision, specificity, reliance on cellular DNA repair mechanisms and their applications in both fundamental biology and therapeutics. Finally, we discuss ongoing clinical trials that illustrate the potential impact of CRISPR systems on human health.

The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived haematopoietic stem cells (HSCs)1. Perturbations to this process underlie diverse diseases, but the clonal contributions to human haematopoiesis and how this changes with age remain incompletely understood. Although recent insights have emerged from barcoding studies in model systems2-5, simultaneous detection of cell states and phylogenies from natural barcodes in humans remains challenging. Here we introduce an improved, single-cell lineage-tracing system based on deep detection of naturally occurring mitochondrial DNA mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs and map the physiological state and output of clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as both differences in total HSC output and biases towards the production of different mature cell types. We also find that the diversity of HSC clones decreases markedly with age, leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides a clonally resolved and cell-state-aware atlas of human haematopoiesis at single-cell resolution, showing an unappreciated functional diversity of human HSC clones and, more broadly, paving the way for refined studies of clonal dynamics across a range of tissues in human health and disease.

Tumor progression is driven by dynamic interactions between cancer cells and their surrounding microenvironment. Investigating the spatiotemporal evolution of tumors can provide crucial insights into how intrinsic changes within cancer cells and extrinsic alterations in the microenvironment cooperate to drive different stages of tumor progression. Here, we integrate high-resolution spatial transcriptomics and evolving lineage tracing technologies to elucidate how tumor expansion, plasticity, and metastasis co-evolve with microenvironmental remodeling in a Kras;p53-driven mouse model of lung adenocarcinoma. We find that rapid tumor expansion contributes to a hypoxic, immunosuppressive, and fibrotic microenvironment that is associated with the emergence of pro-metastatic cancer cell states. Furthermore, metastases arise from spatially-confined subclones of primary tumors and remodel the distant metastatic niche into a fibrotic, collagen-rich microenvironment. Together, we present a comprehensive dataset integrating spatial assays and lineage tracing to elucidate how sequential changes in cancer cell state and microenvironmental structures cooperate to promote tumor progression.

Programmable C•G-to-G•C base editors (CGBEs) have broad scientific and therapeutic potential, but their editing outcomes have proved difficult to predict and their editing efficiency and product purity are often low. We describe a suite of engineered CGBEs paired with machine learning models to enable efficient, high-purity C•G-to-G•C base editing. We performed a CRISPR interference (CRISPRi) screen targeting DNA repair genes to identify factors that affect C•G-to-G•C editing outcomes and used these insights to develop CGBEs with diverse editing profiles. We characterized ten promising CGBEs on a library of 10,638 genomically integrated target sites in mammalian cells and trained machine learning models that accurately predict the purity and yield of editing outcomes ( R = 0.90) using these data. These CGBEs enable correction to the wild-type coding sequence of 546 disease-related transversion single-nucleotide variants (SNVs) with >90% precision (mean 96%) and up to 70% efficiency (mean 14%). Computational prediction of optimal CGBE–single-guide RNA pairs enables high-purity transversion base editing at over fourfold more target sites than achieved using any single CGBE variant.

Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB 1 . We used a custom adenine base editor (ABE8e-NRCH) 2 , 3 to convert the SCD allele ( HBB S ) into Makassar β-globin ( HBB G ), a non-pathogenic variant 4 , 5 . Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBB S to HBB G . Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBB G was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBB S base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse 6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBB S -to- HBB G editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBB S , generates benign HBB G , and minimizes the undesired consequences of double-strand DNA breaks.

SummaryThe targeted deletion, replacement, integration, or inversion of DNA sequences at specified locations in the genome could be used to study or treat many human genetic diseases. Here, we describe twin prime editing (twinPE), a method for the programmable replacement or excision of DNA sequence at endogenous human genomic sites without requiring double-strand DNA breaks. TwinPE uses a prime editor (PE) protein and two prime editing guide RNAs (pegRNAs) that template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, resulting in the replacement of endogenous DNA sequence between the PE-induced nick sites with pegRNA-encoded sequences. We show that twinPE in human cells can perform precise deletions of at least 780 bp and precise replacements of genomic DNA sequence with new sequences of at least 108 bp. By combining single or multiplexed twinPE with site-specific serine recombinases either in separate steps or in a single step, we demonstrate targeted integration of gene-sized DNA plasmids (>5,000 bp) into safe-harbor loci including AAVS1, CCR5, and ALB in human cells. To our knowledge, these results represent the first RNA-programmable insertion of gene-sized DNA sequences into targeted genomic sites of unmodified human cells without requiring double-strand breaks or homology-directed repair. Twin PE combined with recombinases also mediated a 40,167-bp inversion at IDS that corrects a common Hunter syndrome allele. TwinPE expands the capabilities of precision gene editing without requiring double-strand DNA breaks and synergizes with other tools to enable the correction or complementation of large or complex pathogenic alleles in human cells.

The targeted deletion, replacement, integration or inversion of genomic sequences could be used to study or treat human genetic diseases, but existing methods typically require double-strand DNA breaks (DSBs) that lead to undesired consequences, including uncontrolled indel mixtures and chromosomal abnormalities. Here we describe twin prime editing (twinPE), a DSB-independent method that uses a prime editor protein and two prime editing guide RNAs (pegRNAs) for the programmable replacement or excision of DNA sequences at endogenous human genomic sites. The two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the prime-editor-induced nick sites. When combined with a site-specific serine recombinase, twinPE enabled targeted integration of gene-sized DNA plasmids (>5,000 bp) and targeted sequence inversions of 40 kb in human cells. TwinPE expands the capabilities of precision gene editing and might synergize with other tools for the correction or complementation of large or complex human pathogenic alleles. Prime editing of large DNA sequences is achieved with two pegRNAs and site-specific recombinases.

Study design: Preclinical study Population: Fibroblasts derived from people with Hutchinson-Gilford progeria syndrome and mouse models of the genetic disease Key result: Single injection of an adenine base editor (ABE) using a viral vector corrects up to 90% of the mutations in fibroblasts and ameliorates vascular disease and extends lifespan in mice Next steps: ABE could be a feasible therapeutic strategy for progeria in humans

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder with features of accelerated aging. The majority of HGPS cases are caused by a de novo point mutation in the LMNA gene (c.1824C>T; p.G608G) resulting in progerin, a toxic lamin A protein variant. Children with HGPS typically die from coronary artery diseases or strokes at an average age of 14.6 years. Endothelial dysfunction is a known driver of cardiovascular pathogenesis; however, it is currently unknown how progerin antagonizes normal angiogenic function in HGPS. Here, we use human iPSC-derived endothelial cell (iPSC-EC) models to study angiogenesis in HGPS. We cultured normal and HGPS iPSC-ECs under both static and fluidic culture conditions. HGPS iPSC-ECs show reduced endothelial nitric oxide synthase (eNOS) expression and activity compared with normal controls and concomitant decreases in intracellular nitric oxide (NO) level, which result in deficits in capillary-like microvascular network formation. Furthermore, the expression of matrix metalloproteinase 9 (MMP-9) was reduced in HGPS iPSC-ECs, while the expression of tissue inhibitor metalloproteinases 1 and 2 (TIMP1 and TIMP2) was upregulated relative to healthy controls. Finally, we used an adenine base editor (ABE7.10max-VRQR) to correct the pathogenic c.1824C>T allele in HGPS iPSC-ECs. Remarkably, ABE7.10max-VRQR correction of the HGPS mutation significantly reduced progerin expression to a basal level, rescued nuclear blebbing, increased intracellular NO level, normalized the misregulated TIMPs, and restored angiogenic competence in HGPS iPSC-ECs. Together, these results provide molecular insights of endothelial dysfunction in HGPS and suggest that ABE could be a promising therapeutic approach for correcting HGPS-related cardiovascular phenotypes.

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.

Hutchinson-Gilford progeria syndrome (HGPS or progeria) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T; p.G608G) in LMNA, the gene that encodes nuclear lamin A. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children with progeria to approximately 14 years1-4. Adenine base editors (ABEs) convert targeted A•T base pairs to G•C base pairs with minimal by-products and without requiring double-strand DNA breaks or donor DNA templates5,6. Here we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured fibroblasts derived from children with progeria and in a mouse model of HGPS. Lentiviral delivery of the ABE to fibroblasts from children with HGPS resulted in 87-91% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced levels of progerin and correction of nuclear abnormalities. Unbiased off-target DNA and RNA editing analysis did not detect off-target editing in treated patient-derived fibroblasts. In transgenic mice that are homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (around 20-60% across various organs six months after injection), restoration of normal RNA splicing and reduction of progerin protein levels. In vivo base editing rescued the vascular pathology of the mice, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single injection of ABE-expressing AAV9 at postnatal day 14 improved vitality and greatly extended the median lifespan of the mice from 215 to 510 days. These findings demonstrate the potential of in vivo base editing as a possible treatment for HGPS and other genetic diseases by directly correcting their root cause.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The success of base editors for the study and treatment of genetic diseases depends on the ability to deliver them in vivo to the relevant cell types. Delivery via adeno-associated viruses (AAVs) is limited by AAV packaging capacity, which precludes the use of full-length base editors. Here, we report the application of dual AAVs for the delivery of split cytosine and adenine base editors that are then reconstituted by trans-splicing inteins. Optimized dual AAVs enable in vivo base editing at therapeutically relevant efficiencies and dosages in the mouse brain (up to 59% of unsorted cortical tissue), liver (38%), retina (38%), heart (20%) and skeletal muscle (9%). We also show that base editing corrects, in mouse brain tissue, a mutation that causes Niemann–Pick disease type C (a neurodegenerative ataxia), slowing down neurodegeneration and increasing lifespan. The optimized delivery vectors should facilitate the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest. Optimized adeno-associated viruses delivering split cytosine base editors and adenine base editors with trans-splicing inteins can edit brain, liver, retina, heart and skeletal-muscle tissues at therapeutically relevant efficiencies.

The development of new CRISPR–Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR–Cas-derived genome editing agents—nucleases, base editors, transposases/recombinases and prime editors—are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.

Most genetic variants that contribute to disease(1) are challenging to correct efficiently and without excess by products(2-5). Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few by products, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer by products than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expandsthe scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background: Children afflicted with Hutchinson-Gilford Progeria Syndrome (HGPS) die from progressive vascular disease at an average age of 14. Despite elucidation of the genetic cause—C1824T point mutation that activates a cryptic splice site in the LMNA gene —no cure exists. Adenine DNA base editor (ABE), a genome editing enzyme that mediates programmable conversion of A:T to G:C base pairs represents a new class of genome therapy with the potential to cure HGPS by directly correcting the causal mutation. Objective: To determine whether ABE-mediated correction of the C1824T HGPS mutation can halt or ameliorate disease progression in patient-derived cells and in a humanized mouse model of disease. Methods and Results: We first tested ABE in primary, patient-derived fibroblasts using a newly derived ABEmax variant that has been optimized for expression in mammalian cells and engineered to target non-NGG PAMs. Expression of ABEmax with a single guide (sg)RNA targeting the C1824T site resulted in 38% and 61% correction of the HGPS mutation in two separate fibroblast lines 10 days after lentivirus infection. Minimal indels were detected at the locus. We next used an established human transgenic model of HGPS in which mice harbor 2 copies of the human HGPS allele to test in vivo base editing. A split AAV system was used to package and deliver the large payload of the ABEmax and sgRNA cassette. Intraperitoneal injection of split AAV-ABEmax into double copy mutant mice resulted in significant LMNA sequence correction in liver (68±6.7%), heart (24±5.3%), skeletal muscle (25±7.0%) and aorta (16±5.6%). Preliminary data from one ABEmax treated mouse also demonstrated partial restoration of wild type LMNA mRNA and reduced expression of the mutant protein progerin. Conclusions: These results provide the first proof-of-concept data for DNA base editing as a novel treatment strategy to directly reverse the mutation that causes HGPS, a genetic disease associated with high cardiovascular morbidity and mortality.