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The Cre/loxP system is a versatile and powerful tool that has been used to develop many kinds of genetically modified mice, such as conditional knockout mice and mutant protein-expressing mice through the excision of a STOP cassette. However, while numerous in vivo and in vitro applications of the Cre/loxP system have been reported, it remains difficult to target at one time more than one set of recognition sites in an identical single cell in mice using the Cre/loxP system. To overcome this barrier, we developed two novel site-specific recombination systems called VCre/VloxP and SCre/SloxP. These systems allow multiple independent site-specific recombination, for example, multiple targeted deletions in the same cell at different times. In this chapter, I describe the features of VCre/VloxP and SCre/SloxP, practical protocols and tips on how to use them in genomic engineering applications, potential problems in their use, and how problems can be identified and solved., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Transposons are mobile genetic elements evolved to execute highly efficient integration of their genes into the genomes of their host cells. These natural DNA transfer vehicles have been harnessed as experimental tools for stably introducing a wide variety of foreign DNA sequences, including selectable marker genes, reporters, shRNA expression cassettes, mutagenic gene trap cassettes, and therapeutic gene constructs into the genomes of target cells in a regulated and highly efficient manner. Given that transposon components are typically supplied as naked nucleic acids (DNA and RNA) or recombinant protein, their use is simple, safe, and economically competitive. Thus, transposons enable several avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture comprising the generation of pluripotent stem cells, the production of germline-transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species and therapy of genetic disorders in humans. This review describes the molecular mechanisms involved in transposition reactions of the three most widely used transposon systems currently available ( Sleeping Beauty , piggyBac, and Tol2 ), and discusses the various parameters and considerations pertinent to their experimental use, highlighting the state-of-the-art in transposon technology in diverse genetic applications.

Non-alcoholic fatty liver disease (NAFLD), which affects both adults and children, is the most common liver disorder worldwide. NAFLD is characterized by excess fat accumulation in the liver in the absence of significant alcohol use. NAFLD is strongly associated with obesity, insulin resistance, metabolic syndrome, as well as specific genetic polymorphisms. Severe NAFLD cases can further progress to cirrhosis, hepatocellular carcinoma (HCC), or cardiovascular complications. Here, we describe the pathophysiological features and critical genetic variants associated with NAFLD. Recent advances in genome-engineering technology have provided a new opportunity to generate in vitro and in vivo models that reflect the genetic abnormalities of NAFLD. We review the currently developed NAFLD models generated using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. We further discuss unique features of CRISPR/Cas9 and Cas9 variants, including base editors and prime editor, that are useful for replicating genetic features specific to NAFLD. We also compare advantages and limitations of currently available methods for delivering genome-editing tools necessary for optimal genome editing. This review should provide helpful guidance for selecting "good fit" genome-editing tools and appropriate gene-delivery methods for the successful development of NAFLD models and clinical therapeutics.

The emergence of genome editing technology based on the CRISPR/Cas system enabled revolutionary progress in genetic engineering. Double-strand breaks (DSBs), which can be induced by the CRISPR/Cas9 system, cause serious DNA damage that can be repaired by a homologous recombination (HR) system or the nonhomologous end joining (NHEJ) pathway. However, many bacterial species have a very weak HR system. Thus, the NHEJ pathway can be used in prokaryotes. Starting with a brief introduction of the mechanism of the NHEJ pathway, this review focuses on current research and details of applications of NHEJ in eukaryotes, which forms the theoretical basis for the application of the NHEJ system in prokaryotes.

Producing 'designer cells' with specific functions is potentially feasible in the near future. Recent developments, including whole-cell models, genome design algorithms and gene editing tools, have advanced the possibility of combining biological research and mathematical modelling to further understand and better design cellular processes. In this review, we will explore computational and experimental approaches used for metabolic and genome design. We will highlight the relevance of modelling in this process, and challenges associated with the generation of quantitative predictions about cell behaviour as a whole: although many cellular processes are well understood at the subsystem level, it has proved a hugely complex task to integrate separate components together to model and study an entire cell. We explore these developments, highlighting where computational design algorithms compensate for missing cellular information and underlining where computational models can complement and reduce lab experimentation. We will examine issues and illuminate the next steps for genome engineering., (© 2019 The Author(s).)

Whole genome engineering is now feasible with the aid of genome editing and synthesis tools. Synthesizing a genome from scratch allows modifications of the genomic structure and function to an extent that was hitherto not possible, which will finally lead to new insights into the basic principles of life and enable valuable applications. With several recent genome synthesis projects as examples, the technical details to synthesize a genome and applications of synthetic genome are addressed in this perspective. A series of ongoing or future synthetic genomics projects, including the different genomes to be synthesized in GP-write, synthetic minimal genome, massively recoded genome, chimeric genome and synthetic genome with expanded genetic alphabet, are also discussed here with a special focus on theoretical and technical impediments in the design and synthesis process. Synthetic genomics will become a commonplace to engineer pathways and genomes according to arbitrary sets of design principles with the development of high-efficient, low-cost genome synthesis and assembly technologies.

The discovery and mechanistic understanding of target-specific genome engineering technologies has led to extremely effective and specific genome editing in higher organisms. Target-specific genetic modification technology is expected to have a leading position in future gene therapy development, and has a ripple effect on various basic and applied studies. However, several problems remain and hinder efficient and specific editing of target genomic loci. The issues are particularly critical in precise targeted insertion of external DNA sequences into genomes. Here, we discuss some recent efforts to overcome such problems and present a perspective of future genome editing technologies.

The faithful execution of cell division requires the coordinated action of hundreds of gene products. Precisely perturbing these gene products in cells is central to understanding their functions during normal cell division, and the contributions of their disruption to disease. Here, we describe experimental approaches for using CRISPR/Cas9 for gene disruption and modification, with a focus on human cell culture. We describe strategies for inducible gene disruption to generate acute knockouts of essential cell division genes, which can be modified for the chronic elimination of nonessential genes. We also describe strategies for modifying the genome to generate protein fusions to report on and modify protein behavior. These tools facilitate investigation of protein function, dissection of protein assembly networks, and analyses of disease-associated mutations., (© 2018 Elsevier Inc. All rights reserved.)

High quality DNA design tools are becoming increasingly important as synthetic biology continues to increase the rate and throughput of building and testing genetic constructs. To make effective use of expanded build and test capacity, genotype design tools must not only be efficient enough to allow for many designs to be easily created, but also expressive enough to support the complex design patterns required by scientists on the frontier of genome engineering. Genotype Specification Language (GSL) is a language-based design tool invented at Amyris that enables scientists to quickly create DNA designs using a familiar syntax. This syntax provides a layer of abstraction that moves users away from reading and writing raw DNA sequences toward composing designs in terms of functional parts . GSL increases the speed at which scientists can design DNA constructs, provides a precise and reproducible representation of parts, and achieves these goals while maintaining design flexibility. Finally, the GSL compiler can emit information such as the exact final DNA sequence of the design as well as the reagents (primers and template information) required to physically build the constructs. Since its open-source release in February 2016, the GSL compiler can be freely downloaded and used by genome engineers to efficiently specify genetic designs. This chapter briefly introduces GSL syntax and design principles before examining specific examples of genome engineering tasks with accompanying GSL code.

Genome editing to introduce specific mutations or to knock out genes in model cell systems has become an efficient platform for research in the fields of molecular biology, genetics, and cell biology. With recent rapid improvements in genome editing techniques, bench-top manipulation of the genome in cell culture has become progressively easier. The application of this knowledge to erythroid cell culture systems now allows the rapid analysis of the downstream effects of virtually any engineered gene disruption or modification in cell systems. Here, we describe a CRISPR/Cas9-based approach to making genomic modifications in erythroid lineage cells which we have successfully used in both murine (MEL) and human (K562) erythroleukaemia immortalized cell lines.

Drosophila has long been a premier model for the development and application of cutting-edge genetic approaches. The CRISPR-Cas system now adds the ability to manipulate the genome with ease and precision, providing a rich toolbox to interrogate relationships between genotype and phenotype, to delineate and visualize how the genome is organized, to illuminate and manipulate RNA, and to pioneer new gene drive technologies. Myriad transformative approaches have already originated from the CRISPR-Cas system, which will likely continue to spark the creation of tools with diverse applications. Here, we provide an overview of how CRISPR-Cas gene editing has revolutionized genetic analysis in Drosophila and highlight key areas for future advances., (Copyright © 2018 by the Genetics Society of America.)

DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering., (Copyright © 2016 Vrljicak et al.)

Reverse genetics-the creation of mutations in preselected target genes-has until recently been a bottleneck in many Drosophila projects. The advent of clustered, regularly interspaced, short palindromic repeat (CRISPR) genome engineering systems has transformed this situation. A short time after the in vitro demonstration of target site cleavage by the RNA-guided endonuclease CRISPR-associated nuclease 9 (Cas9) (Jinek et al., Science 337:816-821, 2012), hundreds of fly researchers are using CRISPR technology to generate loss-of-function mutant alleles in specific genes, as well as to create specific point mutations or tagged protein products. It appears that most target genes can be edited with remarkably high efficiency, with engineered strains often available a few weeks after conception of a project. Here, we provide a step-by-step protocol for creating loss-of-function mutations in Drosophila using transgenic Cas9 sources, which is based on optimized reagents and procedures that have been evaluated in our laboratory. We also provide guidance on extending this protocol to produce precise genomic alterations by homology-directed repair in the presence of a donor sequence. Additional information and updates are available from our website, www.crisprflydesign.org .

What a living organism looks like and how it works and what are its components-all this is encoded on DNA, the genetic blueprint. Consequently, the way to change an organism is to change its genetic information. Since the first pieces of recombinant DNA have been used to transform cells in the 1970s, this approach has been enormously extended. Bigger and bigger parts of the genetic information have been exchanged or added over the years. Now we are at a point where the construction of entire chromosomes becomes a reachable goal and first examples appear. This development leads to fundamental new questions, for example, about what is possible and desirable to build or what construction rules one needs to follow when building synthetic chromosomes. Here we review the recent progress in the field, discuss current challenges and speculate on the appearance of future synthetic chromosomes., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Genome engineering relies on DNA-modifying enzymes that are able to locate a DNA sequence of interest and initiate a desired genome rearrangement. Currently, the field predominantly utilizes site-specific DNA nucleases that depend on the host DNA repair machinery to complete a genome modification task. We show here that genome engineering approaches that employ target-specific variants of the self-sufficient, versatile site-specific DNA recombinase Flp can be developed into promising alternatives. We demonstrate that the Flp variant evolved to recombine an FRT-like sequence, FL-IL10A, which is located upstream of the human interleukin-10 gene, and can target this sequence in the model setting of Chinese hamster ovary and human embryonic kidney 293 cells. This target-specific Flp variant is able to perform the integration reaction and, when paired with another recombinase, the dual recombinase-mediated cassette exchange reaction. The efficiency of the integration reaction in human cells can be enhanced by 'humanizing' the Flp variant gene and by adding the nuclear localization sequence to the recombinase., (© 2015 FEBS.)

The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe a method using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome.

Custom-designed nucleases afford a powerful reverse genetic tool for direct gene disruption and genome modification in vivo. Among various applications of the nucleases, homologous recombination (HR)-mediated genome editing is particularly useful for inserting heterologous DNA fragments, such as GFP, into a specific genomic locus in a sequence-specific fashion. However, precise HR-mediated genome editing is still technically challenging in zebrafish. Here, we establish a GFP reporter system for measuring the frequency of HR events in live zebrafish embryos. By co-injecting a TALE nuclease and GFP reporter targeting constructs with homology arms of different size, we defined the length of homology arms that increases the recombination efficiency. In addition, we found that the configuration of the targeting construct can be a crucial parameter in determining the efficiency of HR-mediated genome engineering. Implementing these modifications improved the efficiency of zebrafish knock-in generation, with over 10% of the injected F0 animals transmitting gene-targeting events through their germline. We generated two HR-mediated insertion alleles of sox2 and gfap loci that express either superfolder GFP (sfGFP) or tandem dimeric Tomato (tdTomato) in a spatiotemporal pattern that mirrors the endogenous loci. This efficient strategy provides new opportunities not only to monitor expression of endogenous genes and proteins and follow specific cell types in vivo, but it also paves the way for other sophisticated genetic manipulations of the zebrafish genome., (© 2014. Published by The Company of Biologists Ltd.)

Transcription activator-like effectors (TALEs) are immensely powerful new tools for genome engineering that can be directed to bind to almost any DNA sequence of choice. They originate from the Xanthomonas species of plant pathogenic bacteria and, in nature, these proteins increase the virulence of Xanthomonas. However, in 2009, the DNA binding code of TALEs was deciphered and, subsequently, TALE proteins have been exploited for many diverse applications. Custom TALEs that target almost any required DNA sequence can be readily constructed in < 1 week. One major application is gene editing: TALEs fused with the Fok I endonuclease catalytic domain can induce double-stranded breaks at a chosen genomic location, similar to zinc finger nucleases. Designer TALE transcription factors have also been developed by linking TALEs to a transcription AD, such as VP64. More recently, TALEs have been developed that can repress transcription, bind methylated DNA or act as fluorescent chromatin probes. In the present review, we describe the assembly of designer TALEs, their expanding range of current and potential future applications, and briefly discuss alternatives, namely, zinc finger nucleases and clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat associated protein 9., (© 2014 FEBS.)

Genome engineering has revolutionised genetic analysis in many organisms. Here we describe a simple and efficient technique to generate and detect novel mutations in desired target genes in Drosophila melanogaster. We target double strand breaks to specific sites within the genome by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed synthetic guide RNA into Drosophila embryos. The small insertion and deletion mutations that result from inefficient non-homologous end joining at this site are detected by high resolution melt analysis of whole flies and individual wings, allowing stable lines to be made within 1 month., (Copyright © 2014. Published by Elsevier Inc.)

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is poised to transform developmental biology by providing a simple, efficient method to precisely manipulate the genome of virtually any developing organism. This RNA-guided nuclease (RGN)-based approach already has been effectively used to induce targeted mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. Illustrating the adaptability of RGNs, the genomes of >20 different plant and animal species as well as multiple cell lines and primary cells have been successfully modified. Here we review the current and potential uses of RGNs to investigate genome function during development., (© 2014 Harrison et al.; Published by Cold Spring Harbor Laboratory Press.)

A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms., Competing Interests: The authors declare no conflict of interest.

Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae Significant advancements in the past few years have bolstered R. toruloides engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism.IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeasts production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.

Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for Clostridium beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting pta and buk, two essential genes for acetate and butyrate production, respectively. pta and buk single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is

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 in CXCR4 and PD-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.

Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by traditional phosphoramidite column-based approaches. Here, we describe a novel method to amplify oligos from microarray chips for direct use in MAGE to perturb thousands of genomic sites simultaneously. We demonstrated the feasibility of large-scale mutagenesis by inserting T7 promoters upstream of 2585 operons in E. coli using this method, which we call Microarray-Oligonucleotide (MO)-MAGE. The resulting mutant library was characterized by high-throughput sequencing to show that all attempted insertions were estimated to have occurred at an average frequency of 0.02% per locus with 0.4 average insertions per cell. MO-MAGE enables cost-effective large-scale targeted genome engineering that should be useful for a variety of applications in synthetic biology and metabolic engineering.

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

Given the vast behavioral repertoire and biological complexity of even the simplest organisms, accurately predicting phenotypes in novel environments and unveiling their biological organization is a challenging endeavor. Here, we present an integrative modeling methodology that unifies under a common framework the various biological processes and their interactions across multiple layers. We trained this methodology on an extensive normalized compendium for the gram-negative bacterium Escherichia coli, which incorporates gene expression data for genetic and environmental perturbations, transcriptional regulation, signal transduction, and metabolic pathways, as well as growth measurements. Comparison with measured growth and high-throughput data demonstrates the enhanced ability of the integrative model to predict phenotypic outcomes in various environmental and genetic conditions, even in cases where their underlying functions are under-represented in the training set. This work paves the way toward integrative techniques that extract knowledge from a variety of biological data to achieve more than the sum of their parts in the context of prediction, analysis, and redesign of biological systems.