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Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases., Competing Interests: Declaration of interests D.R.L. and G.A.N. have filed patent applications on gene-editing technologies through the Broad Institute of MIT and Harvard. D.R.L. is a consultant and equity owner of Beam Therapeutics, Pairwise Plants, Prime Medicine, Chroma Medicine and Nvelop Therapeutics, companies that use or deliver genome editing or genome engineering technologies. R.J.X. is the co-founder of Jnana Therapeutics and Celsius Therapeutics, the director of Moonlake Immunotherapeutics and a scientific advisory board member to Nestle, all unrelated to the present work. V.G.S. serves as an advisor to and/or has equity in Branch Biosciences, Ensoma, Novartis, Forma, and Cellarity, all unrelated to the present work., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPCs). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into a CD3δ SCID patient's HSPCs resulted in a 71.2% ± 7.85% (n = 3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate the preclinical efficacy of ABE in HSPCs for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients., Competing Interests: Declaration of interests G.M.C. and C.S.S. are founders of Pluto Immunotherapeutics Inc. and serve as consultants to this company. D.R.L. is a consultant and equity holder of Prime Medicine, Beam Therapeutics, Pairwise Plants, and Chroma Medicine, companies that use gene editing or genome engineering., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery., Competing Interests: Declaration of interests The authors declare competing financial interests: S.B., A.R., and D.R.L. have filed patent applications on this work through the Broad Institute. K.M. is a consultant and equity holder of Verve Therapeutics and Variant Bio. K.P. is chief scientific officer of Polgenix, Inc. D.R.L. is a consultant and equity holder of Prime Medicine, Beam Therapeutics, Pairwise Plants, and Chroma Medicine, companies that use gene editing or genome engineering., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types., Competing Interests: Declaration of interests P.J.C., J.A.H., B.A., and D.R.L. have filed patent applications on aspects of this work through their respective institutions. J.A.H. is a consultant for Tessera Therapeutics. P.-F.C. is currently an employee of Tessera Therapeutics. B.A. was a member of a ThinkLab Advisory Board for, and holds equity in, Celsius Therapeutics. D.R.L. is a consultant and equity holder of Beam Therapeutics, Prime Medicine, Pairwise Plants, and Chroma Medicine., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein aggregation is a hallmark of many diseases but also underlies a wide range of positive cellular functions. This phenomenon has been difficult to study because of a lack of quantitative and high-throughput cellular tools. Here, we develop a synthetic genetic tool to sense and control protein aggregation. We apply the technology to yeast prions, developing sensors to track their aggregation states and employing prion fusions to encode synthetic memories in yeast cells. Utilizing high-throughput screens, we identify prion-curing mutants and engineer "anti-prion drives" that reverse the non-Mendelian inheritance pattern of prions and eliminate them from yeast populations. We extend our technology to yeast RNA-binding proteins (RBPs) by tracking their propensity to aggregate, searching for co-occurring aggregates, and uncovering a group of coalescing RBPs through screens enabled by our platform. Our work establishes a quantitative, high-throughput, and generalizable technology to study and control diverse protein aggregation processes in cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In experimental science, organisms are usually studied in isolation, but in the wild, they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose. [GAR(+)], a protein-based epigenetic element, allows yeast to circumvent this "glucose repression" and use multiple carbon sources in the presence of glucose. Some bacteria secrete a chemical factor that induces [GAR(+)]. [GAR(+)] is advantageous to bacteria because yeast cells make less ethanol and is advantageous to yeast because their growth and long-term viability is improved in complex carbon sources. This cross-kingdom communication is broadly conserved, providing a compelling argument for its adaptive value. By heritably transforming growth and survival strategies in response to the selective pressures of life in a biological community, [GAR(+)] presents a unique example of Lamarckian inheritance., (Copyright © 2014 Elsevier Inc. All rights reserved.)