Explore the vast range of titles available.

Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call “serial enrichment for efficient recombineering” (SEER). By performing SEER in Escherichia coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5- to 10-fold in E. coli, while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.

\"James D. Watson, the Nobel laureate whose pioneering work helped unlock the mystery of DNA's structure, charts the greatest scientific journey of our time, from the discovery of the double helix to today's controversies to what the future may hold. [This edition has been] updated to include new findings in gene editing, epigenetics, agricultural chemistry, as well as two entirely new chapters on personal genomics and cancer research\"--Provided by publisher.

T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently “knock out” genes and “knock in” targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4⁺ T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved inCXCR4andPD-1(PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.

As scientists elsewhere start to catch up with China's vast genetic research programme, gene editing is fuelling an innovation economy that threatens to widen racial and economic inequality. Fundamental questions about science, health and social justice are at stake. Who gets access to gene-editing technologies? As countries loosen regulations around the globe, can we shape research agendas to promote an ethical and fair society? Professor Eben Kirksey takes us on a groundbreaking journey to meet the key scientists, lobbyists and entrepreneurs who are bringing cutting-edge genetic modification tools like CRISPR to your local clinic.

We developed an integrated engineering approach for the enhancement of microbial strains by combining in situ transposon mutagenesis, biosensor-guided selection, and multiplex automated genome engineering.Using naringenin (NRN) as a model high-value biochemical, we validated the effectiveness of this approach in strain improvement.From a single batch culture, we identified nine target genes whose individual knockouts (KOs) increased NRN production by up to 2.3-fold.The following high-throughput screening enabled the identification of combinatorial KOs that enhanced NRN production by up to 2.8-fold.Our integrated platform can accelerate the discovery and optimization of beneficial genetic perturbations that are difficult to predict through rational engineering alone, holding significant promise in the fields of biomanufacturing and biorefinery. Improving microbial strains is essential for the economic feasibility of bio-based chemical production; however, the intricate nature of metabolic networks and gene interactions makes identifying effective genetic engineering targets challenging. We developed iTARGET, an integrated approach combining in situ transposon mutagenesis, biosensor-guided selection, and multiplex automated genome engineering (MAGE) to identify novel and synergistic genetic targets that are challenging to predict through rational design. In the first phase, in situ transposon mutagenesis generated genetic diversity within a single batch culture, allowing biosensor-driven enrichment of high-producing mutants. Transposon sequencing (Tn-seq) was then performed to identify critical genomic targets. In the second phase, MAGE enabled the creation of combinatorial knockout (KO) libraries, and high-throughput screening revealed synergistic gene interactions. Applying iTARGET to naringenin (NRN) production enriched high-producing mutants, achieving a population-level titer 1.7-fold higher than that in the control. Next-generation sequencing identified nine unpredictable genetic targets, achieving a 2.3-fold titer increase with single KOs. Further combinatorial KOs revealed synergistic effects, with a double-KO mutant producing a 2.8-fold improvement. By integrating mutagenesis and selection into a single batch, iTARGET accelerates the discovery of challenging genetic targets and enables the exploration of synergistic gene interactions through high-throughput identification of combinatorial KOs, enhancing bio-based chemical production. [Display omitted] This study demonstrates the development of an integrated genome-engineering workflow (iTARGET) that combines in situ transposon mutagenesis, biosensor-guided enrichment, and multiplex genome editing, validated under controlled laboratory conditions in Escherichia coli, establishing Technology Readiness Level (TRL) 4. The approach systematically identified nonobvious knockout (KO) targets and beneficial gene combinations that increased naringenin production. To advance toward TRL 5, additional validation is needed in industrially relevant hosts, pilot-scale fermentations, and diverse operational environments, including adaptation to commercial production strains. Key gaps include the need for high-performance production-linked biosensors, improved multiplex editing efficiency in nonmodel organisms, and enrichment strategies that scale beyond fluorescence-activated cell sorting (FACS). With these advances and incorporation into biofoundry workflows, iTARGET could accelerate strain development for bio-based manufacturing. We developed an integrated platform that combines in situ transposon mutagenesis, biosensor-guided selection, and multiplex automated genome engineering. This approach significantly accelerates the identification of novel knockout target genes and their combinatorial effects, thereby facilitating efficient microbial engineering for enhancing valuable biochemical production.

The 5 May 2025 executive order (EO) \"Improving the safety and security of biological research\" established a federal funding pause for dangerous gain-of-function (DGoF) research, defined as seeking certain experimental outcomes and deemed capable of resulting in significant societal consequences. These moves place institutional biosafety committees central in the identification and self-reporting of DGoF. The previous federal review for research anticipated to result in enhanced potential pandemic pathogens involved a multidisciplinary board, including a bioethicist. From our experience on those boards and based on the EO's mandate to assess the significance of the societal consequences that might result from proposed DGoF research, we suggest a layered review process for the assessment of societal consequences to inform implementation of the EO. In the layered review, proposed research, initially identified based on anticipated experimental outcomes, is confirmed as DGoF through an assessment that is informed by ethical frameworks.

Traditionally, generation of new plants with improved or desirable features has relied on laborious and time-consuming breeding techniques. Genome-editing technologies have led to a new era of genome engineering, enabling an effective, precise, and rapid engineering of the plant genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a new genome-editing tool, extensively applied in various organisms, including plants. The use of CRISPR/Cas9 allows generating transgene-free genome-edited plants (“null segregants”) in a short period of time. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9 derived technologies for inducing mutations at target sites in the genome and controlling the expression of target genes. We highlight the major breakthroughs in applying CRISPR/Cas9 to plant engineering, and challenges toward the production of null segregants. We also provide an update on the efforts of engineering Cas9 proteins, newly discovered Cas9 variants, and novel CRISPR/Cas systems for use in plants. The application of CRISPR/Cas9 and related technologies in plant engineering will not only facilitate molecular breeding of crop plants but also accelerate progress in basic research.

Background The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species. Results In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses. Conclusions These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.

Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.