Tool For Precise Genome Editing Research Articles

High-Efficiency Genome Editing Based on Endogenous CRISPR-Cas System Enhances Cell Growth and Lactic Acid Production in Pediococcus acidilactici.

Pediococcus acidilactici is commonly used for pediocin production and lactic acid fermentation. However, a high-efficiency genome editing tool is unavailable for this species. In this study, we constructed endogenous subtype II-A CRISPR-Cas system-based genome interference plasmids which carried a "repeat-spacer-repeat" cassette in the pMG36e shuttle vector. These plasmids exhibited self-interference activities in P. acidilactici LA412. Then, the genome-editing plasmids were constructed by cloning the upstream/downstream donor DNA into the corresponding interference plasmids to exert high-efficiency markerless gene deletion, gene integration, and point mutation in P. acidilactici LA412. We found that endogenous CRISPR-mediated depletion of the native plasmids enhanced the cell growth and that integration of an l-lactate dehydrogenase gene into the chromosome enhanced both cell growth and lactic acid production. IMPORTANCE A rapid and precise genome editing tool will promote the practical application of Pediococcus acidilactici, one type of lactic acid bacterium with excellent stress tolerance and probiotic characteristics. This study established a high-efficiency endogenous CRISPR-Cas system-based genome editing tool for P. acidilactici and achieved different genetic manipulations, including gene deletion, gene insertion, mononucleotide mutation, and endogenous plasmid depletion. The engineered strain edited by this tool showed significant advantages in cell growth and lactic acid fermentation. Therefore, our tool can satisfy the requirements for genetic manipulations of P. acidilactici, thus making it a sophisticated chassis species for synthetic biology and bioindustry.

Integrating Biomaterials and Genome Editing Approaches to Advance Biomedical Science.

The recent discovery and subsequent development of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9) platform as a precise genome editing tool have transformed biomedicine. As these CRISPR-based tools have matured, multiple stages of the gene editing process and the bioengineering of human cells and tissues have advanced. Here, we highlight recent intersections in the development of biomaterials and genome editing technologies. These intersections include the delivery of macromolecules, where biomaterial platforms have been harnessed to enable nonviral delivery of genome engineering tools to cells and tissues in vivo. Further, engineering native-like biomaterial platforms for cell culture facilitates complex modeling of human development and disease when combined with genome engineering tools. Deeper integration of biomaterial platforms in these fields could play a significant role in enabling new breakthroughs in the application of gene editing for the treatment of human disease.

Genome editing of avian species: implications for animal use and welfare.

Avian species are used as model systems in research and have contributed to ground-breaking concepts in developmental biology, immunology, genetics, virology, cancer and cell biology. The chicken in particular is an important research model and an agricultural animal as a major contributor to animal protein resources for the global population. The development of genome editing methods, including CRISPR/Cas9, to mediate germline engineering of the avian genome will have important applications in biomedical, agricultural and biotechnological activities. Notably, these precise genome editing tools have the potential to enhance avian health and productivity by identifying and validating beneficial genetic variants in bird populations. Here, we present a concise description of the existing methods and current applications of the genome editing tools in bird species, focused on chickens, with attention on animal use and welfare issues for each of the techniques presented.

Base editing in rice: current progress, advances, limitations, and future perspectives.

Base editing is one of the promising genome editing tools for generating single-nucleotide changes in rice genome. Rice (Oryza sativa L.) is an important staple food crop, feeding half of the population around the globe. Developing new rice varieties with desirable agronomic traits is necessary for sustaining global food security. The use of genome editing technologies for developing rice varieties is pre-requisite in the present scenario. Among the genome editing technologies developed for rice crop improvement, base editing technology has emerged as an efficient and reliable tool for precise genome editing in rice plants. Base editing technology utilizes either adenosine or cytidine base editor for precise editing at the target region. A base editor (adenosine or cytidine) is a fusion of catalytically inactive CRISPR/Cas9 domain and adenosine or cytidine deaminase domain. In this review, authors have discussed the different adenine and cytosine base editors developed so far for precise genome editing of rice via base editing technology. We address the current progress, advances, limitations, as well as future perspectives of the base editing technology for rice crop improvement.

Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by the detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae Specifically, we demonstrate an approach for making targeted markerless gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed repair (HDR) is exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid, which is temperature sensitive for replication, by growing it at the nonpermissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The approaches developed here could potentially be adapted for use with other Gram-positive bacteria.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing more than 1 million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts to identify new vaccine candidates. Here, we introduced an easy-to-use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The approaches used here will advance genome editing projects in this important human pathogen.

Single Base Editing Using Cytidine Deaminase to Change Grain Size and Seed Coat Color in Rice.

The fast-moving CRISPR technology has allowed plant scientists to manipulate plant genomes in a targeted manner. So far, most of the applications were focused on gene knocking out by creating indels. However, more precise genome editing tools are demanded to assist the introduction of functional single nucleotide polymorphisms (SNPs) in breeding programs. The CRISPR base editing tools were developed to meet this need. In this chapter, we present a cytidine deaminase base editing method for editing the point mutations that control the grain size and seed coat color in rice.

High-throughput assessment of mutations generated by genome editing in induced pluripotent stem cells by high-resolution melting analysis

Background and aimsGenome editing of induced pluripotent stem cells (iPSCs) holds great potential for both disease modeling and regenerative medicine. Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 provides an efficient and precise genome editing tool, iPSCs are especially difficult to transfect, resulting in a small percentage of cells carrying the desired correction. A high-throughput method to identify edited clones is required to reduce the time and costs of such an approach. MethodsHere we assess high-resolution melting analysis (HRMA), a simple and efficient real-time polymerase chain reaction-based method, and compare it with more commonly used assays. Results and conclusionsOur data show that HRMA is a robust and highly sensitive method, allowing the cost-effective and time-saving screening of genome-edited iPSCs. Samples can be prepared directly from 96-well microtiter plates for high-throughput analysis, and amplicons can be further analyzed with downstream techniques for further confirmation, if needed.

Network-Based Prediction of Novel CRISPR-Associated Genes in Metagenomes.

A diversity of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity to bacteria and archaea through recording "memories" of past viral infections. Recently, many novel CRISPR-associated proteins have been discovered via computational studies, but those studies relied on biased and incomplete databases of assembled genomes. We avoided these biases and applied a network theory approach to search for novel CRISPR-associated genes by leveraging subtle ecological cooccurrence patterns identified from environmental metagenomes. We validated our method using existing annotations and discovered 32 novel CRISPR-associated gene families. These genes span a range of putative functions, with many potentially regulating the response to infection.IMPORTANCE Every branch on the tree of life, including microbial life, faces the threat of viral pathogens. Over the course of billions of years of coevolution, prokaryotes have evolved a great diversity of strategies to defend against viral infections. One of these is the CRISPR adaptive immune system, which allows microbes to "remember" past infections in order to better fight them in the future. There has been much interest among molecular biologists in CRISPR immunity because this system can be repurposed as a tool for precise genome editing. Recently, a number of comparative genomics approaches have been used to detect novel CRISPR-associated genes in databases of genomes with great success, potentially leading to the development of new genome-editing tools. Here, we developed novel methods to search for these distinct classes of genes directly in environmental samples ("metagenomes"), thus capturing a more complete picture of the natural diversity of CRISPR-associated genes.

Improving Editing Efficiency for the Sequences with NGH PAM Using xCas9-Derived Base Editors

The development of CRISPR/Cas9-mediated base editors (BEs) provided a versatile tool for precise genome editing. The recently developed xCas9-derived base editors (xBEs) that recognize the NG PAM substantially expand the targeting scope in the genome, while their editing efficiency needs to be improved. Here, we described an improved version of xBEs by fusing the BPNLS and Gam to the N terminus of xBEs (BPNLS-Gam-xBE3 and BPNLS-xABE), and this version of base editor displayed higher targeting efficiency for the majority of detected sites. By using this improved version of xBEs, we successfully created and corrected pathogenic mutations at genomic sites with the NGN protospacer-adjacent motif in human cells. Lastly, we used BPNLS-Gam-xBE3 to model pathogenic mutations in discarded human tripronuclear (3PN) zygotes, and no obvious off-targets and indels were detected. Taken together, the data in our study offer an efficient tool for precise genome editing and, thus, an enriched base editing toolkit.

Progress in Developing Bacterial Spot Resistance in Tomato

Bacterial spot (BS), caused by four species of Xanthomonas: X. euvesicatoria, X. vesicatoria, X. perforans and X. gardneri in tomato (Solanum lycopersicum L.) results in severe loss in yield and quality by defoliation and the appearance of lesions on fruits, respectively. The combined industry standard for BS control (foliar applications Actigard® rotated with copper plus mancozeb) does not offer sufficient protection, especially when weather conditions favor disease spread. Development of tomato cultivars with BS resistance is thus an important measure to minimize losses. Hypersensitive and non-hypersensitive resistance has been identified in different wild accessions and cultivated tomato relatives and has been transferred to cultivated tomato. However, complete resistance is yet to be obtained. With the advent of next generation sequencing and precise genome editing tools, the genetic regions that confer resistance to bacterial spot can be targeted and enriched through gene pyramiding in a new commercial cultivar which may confer higher degree of horizontal resistance to multiple strains of Xanthomonas causing bacterial spot in tomato.

CRISPR-Cas9 therapies in experimental mouse models of cancer.

The CRISPR-Cas9, a part of the defence mechanism from bacteria, has rapidly become the simplest, fastest and the most precise genome-editing tool available. The therapeutic applications of CRISPR are boundless: correction of mutations in several disorders, inactivation of oncogenes and viral oncoproteins, and activation of tumor suppressor genes. In this review, we expose recent advances concerning animal models of cancer that use CRISPR-Cas9, addressing also the current efforts to develop CRISPR-Cas9-based therapies, focusing on proof-of-concept studies. Finally, the review exposes some of the main challenges that this genome-editing tool faces. The key issue remains: does CRISPR-Cas9 have real potential for therapeutic application or will it just remain a wonderful research tool?

Exposing cancer with CRISPR-Cas9: from genetic identification to clinical therapy

Cancer genomes commonly harbor many genetic aberrations which are often associated with carcinogenesis, progression, metastasis and tolerance. The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9), has been extensively utilized as a precise genome editing tool for genetic screens of cancer. Comprehensive CRISPR-Cas9 libraries and databases have been established for cancer researches, and some essential genes, druggable targets or biomarkers have been identified by CRISPR-Cas9 technology. The identified genes that are helpful to uncover genotype-phenotype interactions by establishing cell or animal models with gene knockout or knock-in, and the druggable targets or biomarkers provide an approach to cancer treatment or diagnosis. Furthermore, combined with pharmaceutical delivery system, CRISPR-Cas9 system has been directly applied for cancer gene therapy and some exciting results have been reported from different research groups. Furthermore, immune cells edited with the CRISPR-Cas9 system have been developed for cancer immunotherapies, 11 of which have entered phase I/II stage clinical trials up to now. This review focused on the application of the CRISPR-Cas9 system for cancer research, including oncogene identification, genetic screen, druggable targets, cell or animal models, gene therapy, cell therapy, and clinical trials. In addition, some concerns about translational CRISPR-Cas9 therapeutics are also addressed for cancer therapy. Altogether, the CRISPR-Cas9 system is a powerful tool for cancer research and a promising approach to cancer therapy.

Exposing cancer with CRISPR-Cas9: from genetic identification to clinical therapy

: Cancer genomes commonly harbor many genetic aberrations which are often associated with carcinogenesis, progression, metastasis and tolerance. The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9), has been extensively utilized as a precise genome editing tool for genetic screens of cancer. Comprehensive CRISPR-Cas9 libraries and databases have been established for cancer researches, and some essential genes, druggable targets or biomarkers have been identified by CRISPR-Cas9 technology. The identified genes that are helpful to uncover genotype-phenotype interactions by establishing cell or animal models with gene knockout or knock-in, and the druggable targets or biomarkers provide an approach to cancer treatment or diagnosis. Furthermore, combined with pharmaceutical delivery system, CRISPR-Cas9 system has been directly applied for cancer gene therapy and some exciting results have been reported from different research groups. Furthermore, immune cells edited with the CRISPR-Cas9 system have been developed for cancer immunotherapies, 11 of which have entered phase I/II stage clinical trials up to now. This review focused on the application of the CRISPR-Cas9 system for cancer research, including oncogene identification, genetic screen, druggable targets, cell or animal models, gene therapy, cell therapy, and clinical trials. In addition, some concerns about translational CRISPR-Cas9 therapeutics are also addressed for cancer therapy. Altogether, the CRISPR-Cas9 system is a powerful tool for cancer research and a promising approach to cancer therapy.

Genetic engineering strategies for biotic and abiotic stress tolerance and quality enhancement in horticultural crops: a comprehensive review.

Genetic engineering technique offers myriads of applications in improvement of horticultural crops for biotic and abiotic stress tolerance, and produce quality enhancement. During last two decades, a large number of transgenic horticultural crops has been developed and more are underway. A number of genes including natural and synthetic Cry genes, protease inhibitors, trypsin inhibitors and cystatin genes have been used to incorporate insect and nematode resistance. For providing protection against fungal and bacterial diseases, various genes like chitinase, glucanase, osmotin, defensin and pathogenesis-related genes are being transferred to many horticultural crops world over. RNAi technique has been found quite successful in inducing virus resistance in horticultural crops in addition to coat protein genes. Abiotic stresses such as drought, heat and salinity adversely affect production and productivity of horticultural crops and a number of genes encoding for biosynthesis of stress protecting compounds including mannitol, glycine betaine and heat shock proteins have been employed for abiotic stress tolerance besides various transcription factors like DREB1, MAPK, WRKY, etc. Antisense gene and RNAi technologies have revolutionized the pace of improvement of horticultural crops, particularly ornamentals for color modification, increasing shelf-life and reducing post-harvest losses. Precise genome editing tools, particularly CRISPR/Cas9, have been efficiently applied in tomato, petunia, citrus, grape, potato and apple for gene mutation, repression, activation and epigenome editing. This review provides comprehensive overview to draw the attention of researchers for better understanding of genetic engineering advancements in imparting biotic and abiotic stress tolerance as well as on improving various traits related to quality, texture, plant architecture modification, increasing shelf-life, etc. in different horticultural crops.

Centromeric DNA Facilitates Nonconventional Yeast Genetic Engineering.

Many nonconventional yeast species have highly desirable features that are not possessed by model yeasts, despite that significant technology hurdles to effectively manipulate them lay in front. Scheffersomyces stipitis is one of the most important exemplary nonconventional yeasts in biorenewables industry, which has a high native xylose utilization capacity. Recent study suggested its much better potential than Saccharomyces cerevisiae as a well-suited microbial biomanufacturing platform for producing high-value compounds derived from shikimate pathway, many of which are associated with potent nutraceutical or pharmaceutical properties. However, the broad application of S.stipitis is hampered by the lack of stable episomal expression platforms and precise genome-editing tools. Here we report the success in pinpointing the centromeric DNA as the partitioning element to guarantee stable extra-chromosomal DNA segregation. The identified centromeric sequence not only stabilized episomal plasmid, enabled homogeneous gene expression, increased the titer of a commercially relevant compound by 3-fold, and also dramatically increased gene knockout efficiency from <1% to more than 80% with the expression of CRISPR components on the new stable plasmid. This study elucidated that establishment of a stable minichromosome-like expression platform is key to achieving functional modifications of nonconventional yeast species in order to expand the current collection of microbial factories.

Sequence-Specific Removal of Shiga Toxin-Producing Escherichia Coli Using the Crispr-Cas9 System

ObjectivesThe CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated genes) has emerged as a programmable and versatile tool for precise genome editing. The specificity of the CRISPR-Cas9 system is dictated by a 20-nucleiotide CRISPR guide RNA. The Cas9 protein is a double-stranded DNA nuclease guided by guide RNA to sequence-specific sites. The interaction between the Cas9 protein and the target DNA sequences leads to lethal cleavage of double-stranded DNA. Objectives of this study were to design and clone a guide RNA targeting Shiga toxin in pCRISPR and use a 2-plasmid platform to deliver this Shiga toxin specific CRISPR-Cas9 system into bacterial cells for specific killing of Shiga toxin-producing Escherichia coli.Materials and MethodsThe E. coli O157:H7 cells containing pCRISPR were cultured on tryptic soy agar (TSA) with 50 µg/ml of kanamycin, cells containing pCas9 were cultured on TSA with 50 µg/ml of chloramphenicol, and those containing both pCRISPR and pCas9 plasmids were cultured on TSA with 50 µg/ml of kanamycin and 50 µg/ml of chloramphenicol. Shiga toxin guide RNA was designed by screening Shiga toxin gene sequences for NGG on the 3’ side. The pCRISPR vector was digested with BsaI (10 units) and purified using a Monarch gel extraction kit (New England Biolabs). The designed guide RNA was ligated with the digested pCRISPR to create a new plasmid of pCRISPR w/stx. After ligation, the cloned region was sequenced by a Sanger Sequencing service (Genewiz).The pCas9 and pCRISPR w/stx were introduced into E. coli O157:H7 cells sequentially by electroporation: first the pCas9 was introduced into E. coli O157:H7 cells and then the pCRISPR w/stx was introduced into the recipient E. coli O157:H7 cells containing pCas9 plasmids. Briefly, E. coli O157:H7 cells were grown to a A600 of 0.4 to 0.6. Plasmids (around 100 ng each) were transformed into bacterial cells using 0.1 cm Gene Pulser cuvettes (Bio-Rad) and the Electroporator 2510 (Eppendorf) set at 1800 V. In addition to pCRISPR w/stx, pCRISPR cloned with a piece of oligo that does not target any DNA sequences in E. coli O157:H7 cells (pCRISPR w/oligo) and the original pCRISPR were used as controls. After electroporation, cells were plated onto TSA plates containing appropriate antibiotics for quantification.ResultsSanger sequencing confirmed that the guide RNA targeting Shiga toxin genes was successfully cloned in pCRISPR. When the newly created pCRISPR w/stx plasmid was introduced into the recipient E. coli O157:H7 cells containing pCas9 plasmids, an approximately 2 log lower concentration of E. coli O157:H7 cells was observed compared to that of the control plasmids of pCRISPR w/oligo and pCRISPR (Table 1).ConclusionThis study provides proof-of-concept evidence that introduction of a CRISPR-Cas9 system that specifically cleaves Shiga toxin genes in bacterial cells leads to death of Shiga toxin-producing E. coli. The CRISPR-Cas system could be further explored for improving meat safety by sequence-specific removal of pathogens that harbor target virulence or antibiotic resistance genes.

CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.).

The type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has been successfully applied to edit target genes in multiple plant species. However, it remains unknown whether this system can be used for genome editing in grape. In this study, we described genome editing and targeted gene mutation in ‘Chardonnay’ suspension cells and plants via the CRISPR/Cas9 system. Two single guide RNAs (sgRNAs) were designed to target distinct sites of the L-idonate dehydrogenase gene (IdnDH). CEL I endonuclease assay and sequencing results revealed the expected indel mutations at the target site, and a mutation frequency of 100% was observed in the transgenic cell mass (CM) as well as corresponding regenerated plants with expression of sgRNA1/Cas9. The majority of the detected mutations in transgenic CM were 1-bp insertions, followed by 1- to 3-nucleotide deletions. Off-target activities were also evaluated by sequencing the potential off-target sites, and no obvious off-target events were detected. Our results demonstrated that the CRISPR/Cas9 system is an efficient and specific tool for precise genome editing in grape.

Epigenetic Alterations at Genomic Loci Modified by Gene Targeting in Arabidopsis thaliana

Gene Targeting (GT) is the integration of an introduced vector into a specific chromosomal site, via homologous recombination. It is considered an effective tool for precise genome editing, with far-reaching implications in biological research and biotechnology, and is widely used in mice, with the potential of becoming routine in many species. Nevertheless, the epigenetic status of the targeted allele remains largely unexplored. Using GT-modified lines of the model plant Arabidopsis thaliana, we show that the DNA methylation profile of the targeted locus is changed following GT. This effect is non-directional as methylation can be either completely lost, maintained with minor alterations or show instability in the generations subsequent to GT. As DNA methylation is known to be involved in several cellular processes, GT-related alterations may result in unexpected or even unnoticed perturbations. Our analysis shows that GT may be used as a new tool for generating epialleles, for example, to study the role of gene body methylation. In addition, the analysis of DNA methylation at the targeted locus may be utilized to investigate the mechanism of GT, many aspects of which are still unknown.