Site-specific Genome Modification Research Articles

CRISPR/Cas9 Based Site-Specific Modification of FAD2 cis-Regulatory Motifs in Peanut (Arachis hypogaea L).

Peanut (Arachis hypogaea L.) seed is a rich source of edible oil, comprised primarily of monounsaturated oleic acid and polyunsaturated linoleic acid, accounting for 80% of its fatty acid repertoire. The conversion of oleic acid to linoleic acid, catalyzed by Fatty Acid Desaturase 2 (FAD2) enzymes, is an important regulatory point linked to improved abiotic stress responses while the ratio of these components is a significant determinant of commercial oil quality. Specifically, oleic acid has better oxidative stability leading to longer shelf life and better taste qualities while also providing nutritional based health benefits. Naturally occurring FAD2 gene knockouts that lead to high oleic acid levels improve oil quality at the potential expense of plant health though. We undertook a CRISPR/Cas9 based site-specific genome modification approach designed to downregulate the expression of two homeologous FAD2 genes in seed while maintaining regulation in other plant tissues. Two cis-regulatory elements the RY repeat motif and 2S seed protein motif in the 5′UTR and associated intron of FAD2 genes are potentially important for regulating seed-specific gene expression. Using hairy root and stable germ line transformation, differential editing efficiencies were observed at both CREs when targeted by single gRNAs using two different gRNA scaffolds. The editing efficiencies also differed when two gRNAs were expressed simultaneously. Additionally, stably transformed seed exhibited an increase in oleic acid levels relative to wild type. Taken together, the results demonstrate the immense potential of CRISPR/Cas9 based approaches to achieve high frequency targeted edits in regulatory sequences for the generation of novel transcriptional alleles, which may lead to fine tuning of gene expression and functional genomic studies in peanut.

Genetic Technologies for Sustainable Management of Insect Pests and Disease Vectors

Recent advancements in genetic and genome editing research, augmented by the discovery of new molecular tools such as CRISPR, have revolutionised the field of genetic engineering by enabling precise site-specific genome modifications with unprecedented ease. These technologies have found a vast range of applications, including the development of novel methods for the control of vector and pest insects. According to their genetic makeup and engineering, these tools can be tuned to impose different grades of impact on the targeted populations. Here, we review some of the most recent genetic control innovations under development, describing their molecular mechanisms and performance, highlighting the sustainability potentials of such interventions.

Keratinocyte-specific knockout mice models via Cre–loxP recombination system

The Cre recombinase/loxP (Cre/loxP) system has emerged as a useful tool in genetic manipulations. Generally, any DNA sequence of interest can be deleted when flanked with loxP sites. The conditional (timely or spatially controlled) expression of Cre recombinase enables to determine, where (e.g. in which cell type or tissue) and when (at which time of life or of developmental stage of cells/tissues) the deletion of the floxed DNA sequence should occur. Therefore, two mouse lines are usually needed for conditional site-specific genome modification. This review aims to provide a general overview of the Cre/loxP system, elaborate on the different keratinocyte specific knockout mice used to study gene function in keratinocytes, and finally demonstrate the generation of a conditional knockout mice. Knockout mice have been efficient and powerful approach to understand the functions of specific genes in development and physiological homeostasis. However, deletion of the specific genes which are concerned in stages of development induces to embryonic lethality. To overcome this obstacle, Cre/loxP system has been used to delete a gene in a specific organ or tissue, or at a specific embryonic developmental stage. Conditional knockout mice have allowed extensive research of the epidermis and helped elucidate roles of many gene functions in keratinocytes.

Gene therapy using haematopoietic stem and progenitor cells.

Haematopoietic stem and progenitor cell (HSPC) gene therapy has emerged as an effective treatment modality for monogenic disorders of the blood system such as primary immunodeficiencies and β-thalassaemia. Medicinal products based on autologous HSPCs corrected using lentiviral and gammaretroviral vectors have now been approved for clinical use, and the site-specific genome modification of HSPCs using gene editing techniques such as CRISPR-Cas9 has shown great clinical promise. Preclinical studies have shown engineered HSPCs could also be used to cross-correct non-haematopoietic cells in neurodegenerative metabolic diseases. Here, we review the most recent advances in HSPC gene therapy and discuss emerging strategies for using HSPC gene therapy for a range of diseases.

Plant Genome Engineering for Targeted Improvement of Crop Traits.

To improve food security, plant biology research aims to improve crop yield and tolerance to biotic and abiotic stress, as well as increasing the nutrient contents of food. Conventional breeding systems have allowed breeders to produce improved varieties of many crops; for example, hybrid grain crops show dramatic improvements in yield. However, many challenges remain and emerging technologies have the potential to address many of these challenges. For example, site-specific nucleases such as TALENs and CRISPR/Cas systems, which enable high-efficiency genome engineering across eukaryotic species, have revolutionized biological research and its applications in crop plants. These nucleases have been used in diverse plant species to generate a wide variety of site-specific genome modifications through strategies that include targeted mutagenesis and editing for various agricultural biotechnology applications. Moreover, CRISPR/Cas genome-wide screens make it possible to discover novel traits, expand the range of traits, and accelerate trait development in target crops that are key for food security. Here, we discuss the development and use of various site-specific nuclease systems for different plant genome-engineering applications. We highlight the existing opportunities to harness these technologies for targeted improvement of traits to enhance crop productivity and resilience to climate change. These cutting-edge genome-editing technologies are thus poised to reshape the future of agriculture and food security.

Changing the Way of Entrance: Highly Efficient Transfer of mRNA and siRNA via Fusogenic Nano-Carriers.

Transferring nucleic acids into mammalian cells heavily influences life science for decades. While first applications mainly dealt with DNA transfer for various purposes as e.g., plasmid encoded protein expression or generation of mutant strains, subsequent applications additionally transferred RNA molecules of mainly small lengths for specific knockdown (RNAi) or site-specific genome modification (gRNA). Significant improvements in full length mRNA generation and extension of mRNA lifetimes additionally allows their use for transient expression in latest times. For all of these types of nucleic acids the most common cell incorporation method is based on complexation and subsequent endosomal uptake. This so-called lipofection can be used theoretically for almost any mammalian cell type and a tremendous number of different product compositions exist in order to deal with drawbacks as transfer efficiency, cell type selectivity, endosomal degradation, slow uptake and cytotoxicity. In contrast, new methods transfer complexed RNA molecules directly into the cytoplasm using liposomal nano-carriers that fuse with cellular plasma membranes immediately upon contact to free functional nucleic acids directly into the cytoplasm. Here, we compare both methods in detail with special focus on robustness, short- and long-term cytotoxicity, efficiency and functionality for various types of transferred RNA. Our data clearly indicate that direct RNA incorporation via fusogenic nano-carriers circumvents most endosomal uptake-based challenges, making it to a most promising alternative for nucleic acid transfer.

Targeted Genome Engineering and Its Application in Trait Improvement of Crop Plants

Targeted genome engineering refers to technologies that are used for site-specific genome modifications such as knockout, knockin and transcriptional regulation of genes of interest in organisms. Site-specific recombination system, zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9) technologies are the representatives of targeted genome engineering and have been widely used in crop basic and applied research. In this review, we introduce the basic information and action modes of these different genome engineering technologies, summarize the recent progresses of targeted genome engineering technologies and their applications in crop improvement, and propose perspectives for genome engineering-mediated modifications of crop plants in the future.

CRISPR-Cas System: History and Prospects as a Genome Editing Tool in Microorganisms.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR or more precisely CRISPR-Cas) system has proven to be a highly efficient and simple tool for achieving site-specific genome modifications in comparison to Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). The discovery of bacterial defense system that uses RNA-guided DNA cleaving enzymes for producing double-strand breaks along CRISPR has provided an exciting alternative to ZFNs and TALENs for gene editing & regulation, as the CRISPR-associated (Cas) proteins remain the same for different gene targets and only the short sequence of the guide RNA needs to be changed to redirect the site-specific cleavage. Therefore, in recent years the CRISPR-Cas system has emerged as a revolutionary engineering tool for carrying out precise and controlled genetic modifications in many microbes such as Escherichia coli, Staphylococcus aureus, Lactobacillus reuteri, Clostridium beijerinckii, Streptococcus pneumonia, and Saccharomyces cerevisiae. Though, concerns about CRISPR-Cas effectiveness in interlinked gene modifications and off-target effects need to be addressed. Nevertheless, it holds a great potential to speed up the pace of gene function discovery by interacting with previously intractable organisms and by raising the extent of genetic screens. Therefore, the potential applications of this system in microbial adaptive immune system, genome editing, gene regulations, functional genomics & biosynthesis along ethical issues, and possible harmful effects have been reviewed.

The Use of CRISPR-Cas9 Technology to Reveal Important Aspects of Human Airway Biology.

The CRISPR-Cas9 technology is a powerful tool that enables site-specific genome modification (gene editing) and is increasingly used in research to generate gene knockout or knock-in in a variety of cells and organisms. This chapter provides a brief overview of this technology and describes a general methodology applicable to human airway biology research.

CRISPR is knocking on barn door.

Genome modification at specific loci in livestock species was only achievable by performing homologous recombination in somatic cells followed by somatic cell nuclear transfer. The difficulty and inefficiency of this method have slowed down the multiple applications of genome modification in farm animals. The discovery of site-specific endonucleases has provided a different and more direct route for targeted mutagenesis, as these enzymes allow the ablation (KO) or insertion (KI) of specific genomic sequences on a single step, directly applied to zygotes. Clustered regularly interspaced short palindromic repeats (CRISPR), the last site-specific endonuclease to be developed, is a RNA-guided endonuclease, easy to engineer and direct to a given target site. This technology has been successfully applied to rabbits, swine, goats, sheep and cattle, situating genome editing in livestock species at an attainable distance, thereby empowering scientist to develop a myriad of applications. Genetically modified livestock animals can be used as biomodels to study human or livestock physiology and disease, as bioreactors to produce complex proteins, or as organ donors for transplantation. Specifically on livestock production, genome editing in farm animals may serve to improve productive genetic traits, to improve various animal products, to confer resistance to diseases or to minimize the environmental impact on farming. In this review, we provide an overview of the current methods for site-specific genome modification in livestock species, discuss potential and already developed applications of genome edition in farm animals and debate about the possibilities for approval of products derived from gene-edited animals for human consumption.

An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing

Gene targeting is a powerful reverse genetics technique for site-specific genome modification. Intrinsic homologous recombination in the moss Physcomitrella patens permits highly effective gene targeting, a characteristic that makes this organism a valuable model for functional genetics. Functional characterization of domains located within a multi-domain protein depends on the ability to generate mutants harboring genetic modifications at internal gene positions while maintaining the reading-frames of the flanking exons. In this study, we designed and evaluated different gene targeting constructs for targeted gene manipulation of sequences corresponding to internal domains of the DEFECTIVE KERNEL1 protein in Physcomitrella patens. Our results show that gene targeting-associated mutagenesis of introns can have adverse effects on splicing, corrupting the normal reading frame of the transcript. We show that successful genetic modification of internal sequences of multi-exon genes depends on gene-targeting strategies which insert the selection marker cassette into the 5′ end of the intron and preserve the nucleotide sequence of the targeted intron.

Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti

Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells.

BackgroundCpf1 nucleases have recently been repurposed for site-specific genome modification. Two members of the Cpf1 family, the AsCpf1 from Acidaminococcus sp. and the LbCpf1 from Lachnospiraceae bacterium were shown to induce higher indel frequencies than SpCas9 when examining four randomly-selected target sequences for each type of nuclease. Whether they are a real match for Cas9 nucleases, however, remains to be verified.ResultsHere, we used AsCpf1 and LbCpf1 to induce homology directed repair, either single strand annealing (SSA) or homologous recombination (HR), in N2a mouse neuroblastoma cells. Exploiting a plasmid that contains two GFP halves with overlapping sequences and exploring 20 targets, on all but one both nucleases consistently performed with above 10 % efficiency. Several Cas9 nucleases have been previously characterised in order to find an orthogonal counterpart for the most widely used promiscuous SpCas9. Here, we found that AsCpf1 and LbCpf1 might be better candidates than three of the best such counterparts: Cas9 from Staphylococcus aureus, from Streptococcus thermophilus and from Neisseria meningitidis, when assessed for inducing efficient SSA mediated repair in N2a cells. When tested on genomic targets exploiting HR, both nucleases were able to induce the integration of a donor cassette with 1000 bp-long homologous arms. We also generated plasmids that express these Cpf1 nucleases together with their cognate crRNAs and that are equipped with type IIS restriction enzyme sites to facilitate spacer cloning.ConclusionsOur results suggest that employing As- or LbCpf1 nuclease to induce homology directed repair in N2a cells, although is less effective at present than employing SpCas9, it is an equally or more effective tool than the most frequently used orthogonal Cas9 counterparts of SpCas9. These findings support the position of Cpf1 nucleases on the side of SpCas9 on the palette of effective genome engineering tools.ReviewersThis article was reviewed by Eugene Koonin, Haruhiko Siomi and Jean-Yves Masson.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-016-0147-0) contains supplementary material, which is available to authorized users.

Progress of targeted genome modification approaches in higher plants.

Transgene integration in plants is based on illegitimate recombination between non-homologous sequences. The low control of integration site and number of (trans/cis)gene copies might have negative consequences on the expression of transferred genes and their insertion within endogenous coding sequences. The first experiments conducted to use precise homologous recombination for gene integration commenced soon after the first demonstration that transgenic plants could be produced. Modern transgene targeting categories used in plant biology are: (a) homologous recombination-dependent gene targeting; (b) recombinase-mediated site-specific gene integration; (c) oligonucleotide-directed mutagenesis; (d) nuclease-mediated site-specific genome modifications. New tools enable precise gene replacement or stacking with exogenous sequences and targeted mutagenesis of endogeneous sequences. The possibility to engineer chimeric designer nucleases, which are able to target virtually any genomic site, and use them for inducing double-strand breaks in host DNA create new opportunities for both applied plant breeding and functional genomics. CRISPR is the most recent technology available for precise genome editing. Its rapid adoption in biological research is based on its inherent simplicity and efficacy. Its utilization, however, depends on available sequence information, especially for genome-wide analysis. We will review the approaches used for genome modification, specifically those for affecting gene integration and modification in higher plants. For each approach, the advantages and limitations will be noted. We also will speculate on how their actual commercial development and implementation in plant breeding will be affected by governmental regulations.

Astonishing advances in mouse genetic tools for biomedical research.

The humble house mouse has long been a workhorse model system in biomedical research. The technology for introducing site-specific genome modifications led to Nobel Prizes for its pioneers and opened a new era of mouse genetics. However, this technology was very time-consuming and technically demanding. As a result, many investigators continued to employ easier genome manipulation methods, though resulting models can suffer from overlooked or underestimated consequences. Another breakthrough, invaluable for the molecular dissection of disease mechanisms, was the invention of high-throughput methods to measure the expression of a plethora of genes in parallel. However, the use of samples containing material from multiple cell types could obfuscate data, and thus interpretations. In this review we highlight some important issues in experimental approaches using mouse models for biomedical research. We then discuss recent technological advances in mouse genetics that are revolutionising human disease research. Mouse genomes are now easily manipulated at precise locations thanks to guided endonucleases, such as transcription activator-like effector nucleases (TALENs) or the CRISPR/Cas9 system, both also having the potential to turn the dream of human gene therapy into reality. Newly developed methods of cell type-specific isolation of transcriptomes from crude tissue homogenates, followed by detection with next generation sequencing (NGS), are vastly improving gene regulation studies. Taken together, these amazing tools simplify the creation of much more accurate mouse models of human disease, and enable the extraction of hitherto unobtainable data.

Systematic Evaluation of Drosophila CRISPR Tools Reveals Safe and Robust Alternatives to Autonomous Gene Drives in Basic Research.

The Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated (CRISPR/Cas) technology allows rapid, site-specific genome modification in a wide variety of organisms . Proof-of-principle studies in Drosophila melanogaster have used various CRISPR/Cas tools and experimental designs, leading to significant uncertainty in the community about how to put this technology into practice. Moreover, it is unclear what proportion of genomic target sites can be modified with high efficiency. Here, we address these issues by systematically evaluating available CRISPR/Cas reagents and methods in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for somatic and germline genome engineering and conclude that optimized CRISPR with independent transgenes is as efficient, more versatile, and does not represent a biosafety risk.

Synthetic biology and therapeutic strategies for the degenerating brain: Synthetic biology approaches can transform classical cell and gene therapies, to provide new cures for neurodegenerative diseases.

Synthetic biology is an emerging engineering discipline that attempts to design and rewire biological components, so as to achieve new functions in a robust and predictable manner. The new tools and strategies provided by synthetic biology have the potential to improve therapeutics for neurodegenerative diseases. In particular, synthetic biology will help design small molecules, proteins, gene networks, and vectors to target disease-related genes. Ultimately, new intelligent delivery systems will provide targeted and sustained therapeutic benefits. New treatments will arise from combining ‘protect and repair’ strategies: the use of drug treatments, the promotion of neurotrophic factor synthesis, and gene targeting. Going beyond RNAi and artificial transcription factors, site-specific genome modification is likely to play an increasing role, especially with newly available gene editing tools such as CRISPR/Cas9 systems. Taken together, these advances will help develop safe and long-term therapies for many brain diseases in human patients.

Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice.

BackgroundThe type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation.FindingsHere, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL. After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA:Cas9 were generated by A. tumefaciens-mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon.ConclusionsOur results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Mouse genome engineering using designer nucleases.

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.