Genome Engineering Tools Research Articles

Bxb1 phage recombinase assists genome engineering in Drosophila melanogaster.

Rapid and reliable genome modifications provide the basis for detailed in vivo functional analysis of any genomic entity (gene, regulatory DNA, non-coding RNA, etc). With the advent of CRISPR/Cas9 genome editing technology, manipulation of a particular genomic locus has become a routine undertaking in variety of model organisms, including the fruit fly Drosophila melanogaster. To further diversify the available tools for genome engineering, we successfully harnessed the phage recombinase Bxb1 to perform recombinase-mediated cassette exchange (RMCE) in D. melanogaster. We demonstrate that Bxb1 possesses highly efficient recombinase activity and could be used alone or in conjunction with other currently available recombinases for creating platforms for cassette exchange of targeted loci.

Genome Engineering and Agriculture: Opportunities and Challenges.

In recent years, plant biotechnology has witnessed unprecedented technological change. Advances in high-throughput sequencing technologies have provided insight into the location and structure of functional elements within plant DNA. At the same time, improvements in genome engineering tools have enabled unprecedented control over genetic material. These technologies, combined with a growing understanding of plant systems biology, will irrevocably alter the way we create new crop varieties. As the first wave of genome-edited products emerge, we are just getting a glimpse of the immense opportunities the technology provides. We are also seeing its challenges and limitations. It is clear that genome editing will play an increased role in crop improvement and will help us to achieve food security in the coming decades; however, certain challenges and limitations must be overcome to realize the technology's full potential.

Dual Recombinase-Mediated Cassette Exchange by Tyrosine Site-Specific Recombinases.

Recombinase-mediated cassette exchange, or RMCE, is a genome engineering tool that can be used to swap DNA fragments of interest between two DNA molecules. In a variation of RMCE, called dual RMCE, the exchange of DNA fragments is mediated by two recombinases in contrast to one recombinase in the classic RMCE reaction. Under optimal conditions, the efficiency of dual RMCE can be quite high: up to ~45% of the transfected cells depending on the recombinase pair used to mediate the replacement reaction. Here we describe protocols for preparing for, performing, and optimizing the parameters of dual RMCE.

Use of the Cas9 Orthologs from Streptococcus thermophilus and Staphylococcus aureus for Non-Homologous End-Joining Mediated Site-Specific Mutagenesis in Arabidopsis thaliana.

Since the discovery of the CRISPR/Cas system and its in vivo application for site-specific targeted mutagenesis, this technique is wildly used in a great variety of organisms, such as many plant species. Commonly used for this application is the Cas9 enzyme from Streptococcus pyogenes. Here, we describe the application of two Cas9 orthologs from Streptococcus thermophilus and Staphylococcus aureus for targeted non-homologous end-joining mediated mutagenesis in Arabidopsis thaliana. With both orthologs, we could show efficient inheritance of the induced mutations. As both Cas9 orthologs are smaller in size than the enzyme of S. pyogenes and as the Protospacer adjacent motifs (PAMs) differ between all orthologs, they are attractive alternative tools for genome engineering in plants.

Butanol 생합성 Clostridium 속 미생물 대사공학용 게놈 편집 도구 개발

Global warming caused from the heavy consumption of fossil fuel is one of the biggest problems to be solved. Biofuel has been gained more attention as an alternative to reduce the consumption of fossil fuel. Recently, butanol produced from the genus Clostridium has been considered as one of the promising alternatives for gasoline, fossil based fuel. Nevertheless, the lack of the genome-engineering tools for the genus Clostridium is the major hurdle for the economic production of butanol. More recently, genome engineering tools have been developed for metabolic engineering of butanol-producing Clostridium species, which includes genome scale network model and genome editing tools on the basis of mobile group II introns and CRISPR/Cas system. In this study, the genome engineering tools for butanol-producing Clostridium species have been reviewed with a brief future perspective.

Applications of ZFN, TALEN and CRISPR/Cas9 techniques in disease modeling and gene therapy

Precise and effective modification of complex genomes at the predicted loci has long been an important goal for scientists. However, conventional techniques for manipulating genomes in diverse organisms and cells have lagged behind the rapid advance in genomic studies. Such genome engineering tools have featured low efficiency and off-targeting. The newly developed custom-designed nucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system have conferred genome modification with ease of customization, flexibility and high efficiency, which may impact biological research and studies on pathogenesis of human diseases. These novel techniques can edit the genomic DNA with high efficiency and specificity in a rich variety of organisms and cell types including the induced pluripotent stem cells (iPSCs), which has conferred them with the potential for revealing the pathogenesis and treatment of many human diseases. This review has briefly introduced the mechanisms of ZFN, TALENs and CRISPR/Cas9 system, and compared the efficiency and specificity of such approaches. In addition, the application of ZFN, TALENs and CRISPR/Cas9 mediated genome modification for human disease modeling and gene therapy was also discussed.

The Role of UBR5 Mutations in the Pathogenesis of Mantle CELL Lymphoma

Intro: Mantle cell lymphoma (MCL) accounts for 6% of non-Hodgkin lymphomas and represents a particularly challenging disease with patient outcomes inferior to most other lymphoma subtypes. Using targeted capture sequencing of MCL biopsy samples, we recently reported frequent mutations (18%) in UBR5, a gene encoding an E3 ubiquitin-protein ligase that has not been previously implicated in lymphomagenesis. All mutations were clustered within 100bp in or around exon 58 of UBR5 and truncate the reading frame or change a key lysine residue. These mutations are predicted to result in the loss of the conserved cysteine residue in the HECT-domain, which is responsible for binding the ubiquitin co-factor. The recurrence and clustering of UBR5 mutations suggest their critical pathogenic involvement in a subgroup of MCL that might be therapeutically targetable. The aim of this study is to determine UBR5 mutation-associated proteome changes and altered cell signaling.Methods: As seen in MCL patients, mutations in exon 58 of UBR5 were introduced to three MCL cell lines (Granta-519, Jeko-1, and Mino) using the CRISPR-Cas9 genome engineering tool. First, mass spectrometry-based immunoprecipitation proteomics (IP-MS) was employed to identify differences in UBR5 interacting partners between UBR5 mutant and wildtype (WT) cells. Candidate UBR5 interacting proteins were validated by flow cytometry, western blotting, co-immunoprecipitation, and immunofluorescence. Next, global proteomes of UBR5 mutants and WT were analyzed by Tandem Mass Tag (TMT)-based mass spectrometry quantification to identify proteins with differential expression due to the UBR5 mutations.Results: The IP-MS analysis of WT vs UBR5 mutants revealed histone and cell cycle control proteins as candidate differential UBR5 interacting proteins (p<0.05). Particularly, histones H1, H4, and H2AFX, as well as the cell cycle genes CDC5L, BUB3, MAP4, RAD50 and CDK11B were identified as candidate UBR5 interacting partners. The global proteome analysis identified a set of differentially expressed genes (mutant vs wt; p<0.05) that are common among the MCL cell lines with the same direction of change. Gene ontology analysis of this set revealed DNA damage response, chromosome organization, and cell cycle response pathways as the predominant pathways affected. Moreover, our preliminary functional studies indicate constitutive phosphorylation of H2AFX in UBR5 mutants vs WT in line with the role of UBR5 in DNA damage response.Conclusions: Our results are consistent with UBR5 functioning as a key regulator of cell signalling and strongly suggest UBR5 as a novel regulator of histone modifications and DNA damage response. These findings provide an experimentally valid platform for further functional investigation and testing of target therapies for MCL harbouring UBR5 mutations. DisclosuresNo relevant conflicts of interest to declare.

Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida.

CRISPR/Cas9 systems are nowadays applied extensively to effect genome editing in various organisms including plants. CRISPR from Prevotella and Francisella 1 (Cpf1) is a newly characterized RNA-guided endonuclease that has two distinct features as compared to Cas9. First, Cpf1 utilizes a thymidine-rich protospacer adjacent motif (PAM) while Cas9 prefers a guanidine-rich PAM. Cpf1 could be used as a sequence-specific nuclease to target AT-rich regions of a genome that Cas9 had difficulty accessing. Second, Cpf1 generates DNA ends with a 5′ overhang, whereas Cas9 creates blunt DNA ends after cleavage. “Sticky” DNA ends should increase the efficiency of insertion of a desired DNA fragment into the Cpf1-cleaved site using complementary DNA ends. Therefore, Cpf1 could be a potent tool for precise genome engineering. To evaluate whether Cpf1 can be applied to plant genome editing, we selected Cpf1 from Francisella novicida (FnCpf1), which recognizes a shorter PAM (TTN) within known Cpf1 proteins, and applied it to targeted mutagenesis in tobacco and rice. Our results show that targeted mutagenesis had occurred in transgenic plants expressing FnCpf1 with crRNA. Deletions of the targeted region were the most frequently observed mutations. Our results demonstrate that FnCpf1 can be applied successfully to genome engineering in plants.

Recent advances of molecular toolbox construction expand Pichia pastoris in synthetic biology applications.

Pichia pastoris: (reclassified as Komagataella phaffii), a methylotrophic yeast strain has been widely used for heterologous protein production because of its unique advantages, such as readily achievable high-density fermentation, tractable genetic modifications and typical eukaryotic post-translational modifications. More recently, P. pastoris as a metabolic pathway engineering platform has also gained much attention. In this mini-review, we addressed recent advances of molecular toolboxes, including synthetic promoters, signal peptides, and genome engineering tools that established for P. pastoris. Furthermore, the applications of P. pastoris towards synthetic biology were also discussed and prospected especially in the context of genome-scale metabolic pathway analysis.

PKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana.

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is widely used as a tool for genome engineering in various organisms. A complex consisting of Cas9 and single guide RNA (sgRNA) induces a DNA double-strand break in a sequence-specific manner, resulting in knockout. Some binary vectors for CRISPR/Cas9 in plants have been reported, but there is a problem with low efficiency. Here, we present a newly developed, highly efficient CRISPR/Cas9 vector for Arabidopsis thaliana, pKAMA-ITACHI Red (pKIR), harboring the RIBOSOMAL PROTEIN S5 A (RPS5A) promoter to drive Cas9. The RPS5A promoter maintains high constitutive expression at all developmental stages starting from the egg cell and including meristematic cells. Even in the T1 generation, pKIR induced null phenotypes in some genes: PHYTOENE DESATURASE 3 (PDS3), AGAMOUS (AG) and DUO POLLEN 1 (DUO1). Mutations induced by pKIR were carried in the germ cell line of the T1 generation. Surprisingly, in some lines, 100% of the T2 plants had the adh1 (ALCOHOL DEHYDROGENASE 1) null phenotype, indicating that pKIR strongly induced heritable mutations. Cas9-free T2 mutant plants were obtained by removing T2 seeds expressing a fluorescent marker in pKIR. Our results suggest that the pKIR system is a powerful molecular tool for genome engineering in Arabidopsis.

Bacterial genome engineering and synthetic biology: combating pathogens.

BackgroundThe emergence and prevalence of multidrug resistant (MDR) pathogenic bacteria poses a serious threat to human and animal health globally. Nosocomial infections and common ailments such as pneumonia, wound, urinary tract, and bloodstream infections are becoming more challenging to treat due to the rapid spread of MDR pathogenic bacteria. According to recent reports by the World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC), there is an unprecedented increase in the occurrence of MDR infections worldwide. The rise in these infections has generated an economic strain worldwide, prompting the WHO to endorse a global action plan to improve awareness and understanding of antimicrobial resistance. This health crisis necessitates an immediate action to target the underlying mechanisms of drug resistance in bacteria.ResearchThe advent of new bacterial genome engineering and synthetic biology (SB) tools is providing promising diagnostic and treatment plans to monitor and treat widespread recalcitrant bacterial infections. Key advances in genetic engineering approaches can successfully aid in targeting and editing pathogenic bacterial genomes for understanding and mitigating drug resistance mechanisms. In this review, we discuss the application of specific genome engineering and SB methods such as recombineering, clustered regularly interspaced short palindromic repeats (CRISPR), and bacterial cell-cell signaling mechanisms for pathogen targeting. The utility of these tools in developing antibacterial strategies such as novel antibiotic production, phage therapy, diagnostics and vaccine production to name a few, are also highlighted.ConclusionsThe prevalent use of antibiotics and the spread of MDR bacteria raise the prospect of a post-antibiotic era, which underscores the need for developing novel therapeutics to target MDR pathogens. The development of enabling SB technologies offers promising solutions to deliver safe and effective antibacterial therapies.

Generation of TALEN-mediated FH knockout rat model.

Transcription activator-like effector nucleases (TALENs) are valuable tools for precise genome engineering of laboratory animals. Here we utilized this technique for efficient site-specific gene modification to create a fumarate hydratase (FH) gene knockout rat model, in which there was an 11 base-pair deletion in the first exon of the FH gene in 111 rats. 18 live-born targeted mutation offsprings were produced from 80 injected zygotes with 22.5% efficiency, indicating high TALEN knockout success in rat zygots. Only heterozygous deletion was observed in the offsprings. Sixteen pairs of heterozygous FH knockout (FH+/−) rats were arranged for mating experiments for six months without any homozygous KO rat identified. Sequencing from the pregnant rats embryo samples showed no homozygous FH KO, indicating that homozygous FH KO is embryonically lethal. Comparatively, the litter size was decreased in both male and female FH+/− KO rats. There was no behaviour difference between the FH+/− KO and the control rats except that the FH+/− KO male rats showed significantly higher body weight in the 16-week observation period. Clinical haematology and biochemical examinations showed hematopoietic and kidney dysfunction in the FH+/− KO rats. Small foci of anaplastic lesions of tubular epithelial cells around glomeruli were identified in the FH+/− kidney, and these anaplastic cells were comparatively positive for Ki67, p53 and Sox9, and such findings are most probably related to the kidney dysfunction reflected by the biochemical examinations of the rats. In conclusion, we have successfully established an FH+/− KO rat model, which will be useful for further functional FH studies.

Pseudorabies virus can escape from CRISPR-Cas9-mediated inhibition

The CRISPR-Cas9 system is a newly developed genome-engineering tool used to inhibit virus infection by targeting the conserved regions of the viral genomic DNA. In the present study, we constructed a cell line stably expressing Cas9 endonuclease and sgRNA targeting the conserved UL30 gene of pseudorabies virus (PRV). During the PRV infection, the CRISPR-Cas9 system was efficient in cleaving the UL30 gene in each passage. However, deletions and insertions occurred at low passages, while substitutions were frequently observed at high passages. Furthermore, copy numbers and virus titers of PRV were significantly increased in a passage-dependent manner, indicating that viral genomic replication and assembly were more effective at the high passages than at low passages. These results demonstrated that PRV could escape from CRISPR-Cas9-mediated inhibition. Therefore, whether the CRISPR-Cas9 system is suitable for antiviral application should be considered and carefully verified.

Structural Determinants of Sleeping Beauty Transposase Activity

Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.

Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans

Thoracic aortic aneurysms and dissections (TAAD) represent a substantial cause of morbidity and mortality worldwide. Many individuals presenting with an inherited form of TAAD do not have causal mutations in the set of genes known to underlie disease. Using whole-genome sequencing in two first cousins with TAAD, we identified a missense mutation in the lysyl oxidase (LOX) gene (c.893T > G encoding p.Met298Arg) that cosegregated with disease in the family. Using clustered regularly interspaced short palindromic repeats (CRISPR)/clustered regularly interspaced short palindromic repeats-associated protein-9 nuclease (Cas9) genome engineering tools, we introduced the human mutation into the homologous position in the mouse genome, creating mice that were heterozygous and homozygous for the human allele. Mutant mice that were heterozygous for the human allele displayed disorganized ultrastructural properties of the aortic wall characterized by fragmented elastic lamellae, whereas mice homozygous for the human allele died shortly after parturition from ascending aortic aneurysm and spontaneous hemorrhage. These data suggest that a missense mutation in LOX is associated with aortic disease in humans, likely through insufficient cross-linking of elastin and collagen in the aortic wall. Mutation carriers may be predisposed to vascular diseases because of weakened vessel walls under stress conditions. LOX sequencing for clinical TAAD may identify additional mutation carriers in the future. Additional studies using our mouse model of LOX-associated TAAD have the potential to clarify the mechanism of disease and identify novel therapeutics specific to this genetic cause.

Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering.

Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site-specific recombinases (SSRs) are valuable tools for genome engineering due to their exceptional ability to mediate precise excision, integration and inversion of genomic DNA in living systems. The ever-increasing complexity of genome manipulations and the desire to understand the DNA-binding specificity of these enzymes are driving efforts to identify novel SSR systems with unique properties. Here, we describe two novel tyrosine site-specific recombination systems designated Nigri/nox and Panto/pox. Nigri originates from Vibrio nigripulchritudo (plasmid VIBNI_pA) and recombines its target site nox with high efficiency and high target-site selectivity, without recombining target sites of the well established SSRs Cre, Dre, Vika and VCre. Panto, derived from Pantoea sp. aB, is less specific and in addition to its native target site, pox also recombines the target site for Dre recombinase, called rox. This relaxed specificity allowed the identification of residues that are involved in target site selectivity, thereby advancing our understanding of how SSRs recognize their respective DNA targets.

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Clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated proteins (CRISPR-Cas), a groundbreaking genome-engineering tool, has facilitated targeted trait improvement in plants. Recently, CRISPR-CRISPR from Prevotella and Francisella 1 (Cpf1) has emerged as a new tool for efficient genome editing, including DNA-free editing in plants, with higher efficiency, specificity, and potentially wider applications than CRISPR-Cas9.

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system from Streptococcus pyogenes has recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing of Bacillus subtilis The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication for Escherichia coli, the temperature-sensitive replication origin of plasmid pE194(ts) for B. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries the cas9 gene under the control of the B. subtilis mannose-inducible promoter PmanP and a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5' end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in the B. subtilis chromosome and by repair of the trpC2 mutation of B. subtilis 168. In prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing of B. subtilis overcomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

Genome engineering tools for building cellular models of disease.

With the recent development of methods for genome editing of human pluripotent stem cells, study of the genetic basis of human diseases has been rapidly advancing. Genome-edited differentiated stem cells have provided new and more accurate insights into genomic underpinnings of diseases for which there have not been adequate treatments, and moving toward clinical application of genome editing holds great promise for acceleration of therapeutic translation. Here, we review recent advances in genome-editing technologies and their application to human biology in disease modeling and beyond.

Gene transcription repression in Clostridium beijerinckii using CRISPR-dCas9.

CRISPR-Cas9 has been explored as a powerful tool for genome engineering for many organisms. Meanwhile, dCas9 which lacks endonuclease activity but can still bind to target loci has been engineered for efficient gene transcription repression. Clostridium beijerinckii, an industrially significant species capable of biosolvent production, is generally difficult to metabolically engineer. Recently, we reported our work in developing customized CRISPR-Cas9 system for genome engineering in C. beijerinckii. However, in many cases, gene expression repression (rather than actual DNA mutation) is more desirable for various biotechnological applications. Here, we further demonstrated gene transcription repression in C. beijerinckii using CRISPR-dCas9. A small RNA promoter was employed to drive the expression of the single chimeric guide RNA targeting on the promoter region of amylase gene, while a constitutive thiolase promoter was used to drive Streptococcus pyogenes dCas9 expression. The growth assay on starch agar plates showed qualitatively significant repression of amylase activity in C. beijerinckii transformant with CRISPR-dCas9 compared to the control strain. Further amylase activity quantification demonstrated consistent repression (65-97% through the fermentation process) on the activity in the transformant with CRISPR-dCas9 versus in the control. Our results provided essential references for engineering CRISPR-dCas9 as an effective tool for tunable gene transcription repression in diverse microorganisms. Biotechnol. Bioeng. 2016;113: 2739-2743. © 2016 Wiley Periodicals, Inc.