BASELINE: a CRISPR base editing platform for mammalian-scale single-cell lineage tracing

Production of Cell Models with Differential Zygosity Using CRISPR Base Editors.

Single-cell and in vivo profiling reveal heterogeneous and organ-specific CRISPR-Cas9 off-target and translocation outcomes

It is shown that off-target editing and translocations vary widely between individual cells and organs, motivating the development of more sensitive and organ-specific safety assays for CRISPR therapies.

In vivo reconstruction of the cell lineage history of a developing mouse with DNA Typewriter, from zygote to late organogenesis

The complete cell lineage of C. elegans, mapped over four decades ago, was tractable because the animal is small, transparent, and lineage invariant1. Most animals are none of these, having orders of magnitude more cells, being opaque, and developing with substantial stochasticity2. Since 2016, genome editing-based lineage tracing has opened the door to dense cell lineage reconstruction in such organisms3–8, but delivering on that promise has proven technically challenging. Here we apply DNA Typewriter9–11, a prime editing-based recorder that writes stochastic symbolic insertions to an engineered genomic TAPE in strictly sequential order, to trace mouse development from zygote (E0) to late organogenesis (E13.5). We introduce constructs encoding a prime editor, engineered prime editing guide RNAs (epegRNAs), and Pol-III-driven circularized TAPE RNA (circTAPE) into wildtype zygotes by pronuclear injection (PNI), then assay embryos by single-nucleus transcriptional profiling (sci-RNA-seq3)12 with paired circTAPE recovery. From one E13.5 embryo bearing ∼7 integrations of a constitutively expressed prime editor and 11 integrations of 6-unit circTAPE, we recover ∼1.75M single-nucleus transcriptomes and reconstruct a time-calibrated phylogeny of 1,340,794 annotated cells with parsimony-based node support. The first cell division is marked unequivocally, and although the resulting blastomeres contribute asymmetrically to the embryo proper, they are fate-neutral and serve as internal replicates that reproduce every finding. A modest cohort of pre-gastrulation founders dominates the embryo, with inequality exceeding neutral expectation within one to two cell cycles of founder allocation, yet these founders remain broadly multipotent; a second phase of clonal dominance arises in specific lineages during organogenesis. At the finest scale, sibling cells share cell type 9-fold in excess of chance, reaching 68- to 107-fold for cell types arising from spatially restricted founder pools, while the recent differentiations of organogenesis are legible in the heterotypic structure of terminal clades. From clade co-occurrence alone, we recover germ-layer organization and a dated hierarchy of cell-type couplings with branch points from E8.5 onwards. Finally, by integrating these data with our single-cell time-series of mouse development13, we impute transcriptional states and annotations for the majority of internal nodes and recover established state histories for diverse cell types. All data are made freely available, together with NextCell, an interactive browser for this annotated cellular phylogeny of mouse development from zygote to late organogenesis.

Genome-wide CRISPR screening identifies cellular factors controlling nonviral genome editing efficiency

To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies. Low editing efficiency of nonviral delivery in post mitotic tissues presents a challenge to the field of gene therapy. Here, authors dissect the genetic regulators of nonviral delivery in post mitotic retinal epithelial cells describe strategies for improved base editor delivery and editing.

Genome-Wide CRISPR Screening Identifies Cellular Factors Controlling Nonviral Genome Editing Efficiency

A novel genome-wide CRISPR screening strategy that will facilitate the systematic engineering of novel nonviral genome editing delivery methods, where the identified novel gene hits can be further used to increase editing efficiency for other therapeutically relevant cell types.

From Design to Single-Cell Cloning: A Complete RNP-Based CRISPR-Cas9 Protocol for Precision Gene Correction in Human iPSCs.

Precise genome editing of induced pluripotent stem cells (iPSCs) using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) has opened unprecedented avenues for advancements in regenerative medicine and disease modelling. However, the establishment of isogenic single-cell-derived iPSC populations, particularly upon CRISPR-Cas9 gene editing modifications, is one of the major challenges still associated with these advanced methodologies relying on low-rate editing events and requiring defined clonogenicity. In response, we have developed a systematic, comprehensive and efficient workflow combining generation, genotyping and expansion of high-quality monoclonal iPSC lines following CRISPR-Cas9 genome editing. In particular, the protocol incorporates optimized single-cell cloning procedures for two commercially available dispensing platforms, one based on microfluidic imaging and the other on impedance technology, together with rapid droplet digital PCR (ddPCR)-based screening of non-homologous end joining (NHEJ) and homology-directed repair (HDR) outcomes. By combining gentle single-cell handling with advanced genotyping methodologies, the protocol enables efficient early assessment of editing outcomes before commitment to labour-intensive clonal derivation, thereby accelerating project timelines, minimising cell stress and loss, preserving genetic fidelity and supporting scalability. Coupled with precisely defined culture conditions tailored for post-seeding recovery, these approaches aim to improve iPSC viability and clonal outgrowth, achieving at least 60% in 96-well plate format within 10 days. Importantly, the protocol goes beyond step-by-step experimental instructions by providing comprehensive design strategies, decision-making criteria, and practical advice for avoiding and addressing the common pitfalls and unintended consequences of these advanced methods. Collectively, this integrated end-to-end approach provides a robust and high-throughput framework for the reliable production of monoclonal genome-edited iPSCs, thereby advancing translational research and the development of iPSC-based regenerative therapies and disease models.