Donor Plasmid Research Articles

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull

Genome editing followed by reproductive cloning was previously used to produce two hornless dairy bulls. We crossed one genome-edited dairy bull, homozygous for the dominant PC Celtic POLLED allele, with horned cows (pp) and obtained six heterozygous (PCp) polled calves. The calves had no horns and were otherwise healthy and phenotypically unremarkable. We conducted whole-genome sequencing of all animals using an Illumina HiSeq4000 to achieve ~20× coverage. Bioinformatics analyses revealed the bull was a compound heterozygote, carrying one naturally occurring PC Celtic POLLED allele and an allele containing an additional introgression of the homology-directed repair donor plasmid along with the PC Celtic allele. These alleles segregated in the offspring of this bull, and inheritance of either allele produced polled calves. No other unintended genomic alterations were observed. These data can be used to inform conversations in the scientific community, with regulatory authorities and with the public around ‘intentional genomic alterations’ and future regulatory actions regarding genome-edited animals.

A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum.

The heliobacteria are members of the bacterial order Clostridiales and form the only group of phototrophs in the phylum Firmicutes Several physiological and metabolic characteristics make them an interesting subject of investigation, including their minimalist photosynthetic system, nitrogen fixation abilities, and ability to reduce toxic metals. While the species Heliobacterium modesticaldum is an excellent candidate as a model system for the family Heliobacteriaceae, since an annotated genome and transcriptomes are available, studies in this organism have been hampered by the lack of genetic tools. We adapted techniques for genetic manipulation of related clostridial species for use with H. modesticaldum Five heliobacterial DNA methyltransferase genes were expressed in an Escherichia coli strain engineered as a conjugative plasmid donor for broad-host-range plasmids. Premethylation of the shuttle vectors before conjugation into H. modesticaldum is absolutely required for production of transconjugant colonies. The introduced shuttle vectors are maintained stably and can be recovered using a modified minipreparation procedure developed to inhibit endogenous DNase activity. Furthermore, we describe the formulation of various growth media, including a defined medium for metabolic studies and isolation of auxotrophic mutants.IMPORTANCE Heliobacteria are anoxygenic phototrophic bacteria with the simplest known photosynthetic apparatus. They are unique in using bacteriochlorophyll g as their main pigment and lacking a peripheral antenna system. Until now, research on this organism has been hampered by the lack of a genetic transformation system. Without such a system, gene knockouts, site-directed mutations, and gene expression studies cannot be performed to help us further understand or manipulate the organism. Here we report the genetic transformation of a heliobacterium, which should enable future genetic studies in this unique phototrophic organism.

Salmonella persisters promote the spread of antibiotic resistance plasmids in the gut.

SummaryThe emergence of antibiotic resistant bacteria by mutations or by acquisition of genetic material like resistance plasmids represents a major public health issue 1,2 (Extended Data Fig. 1a). Persisters are bacterial subpopulations surviving antibiotics by reversibly adapting their physiology 3–10. They promote the emergence of antibiotic resistant mutants 11. We asked if persisters can also promote the spread of resistance plasmids. In contrast to mutations, resistance plasmid transfer requires the co-occurrence of two different bacterial strains: a donor and a recipient (Extended Data Fig. 1a). For our experiments, we chose the facultative intracellular entero-pathogen Salmonella enterica serovar Typhimurium (S.Tm) and E. coli, a common microbiota member 12. S.Tm forms persisters surviving antibiotic therapy in several host tissues. We show that tissue-associated, S.Tm persisters account for long-lived reservoirs of plasmid donors or recipients. Persistent S.Tm reservoir formation requires Salmonella Pathogenicity Island (SPI) -1/2 in the gut-associated tissues or SPI-2 at systemic sites. Re-seeding of these bacteria into the gut lumen allows co-occurrence of donors with gut-resident recipients, thereby favouring plasmid transfer between various Enterobacteriaceae. We observe up to 99% transconjugants within 2-3 days after re-seeding. Mathematical modeling shows that rare re-seeding events may suffice for a high frequency of conjugation. Vaccination reduces tolerant reservoir formation after oral Salmonella infection and subsequent plasmid transfer. We conclude that even without selection for plasmid-encoded resistance genes, small persistent pathogen reservoirs can foster the spread of promiscuous resistance plasmids in the gut.

Methods for Correction of the Single-Nucleotide Substitution c.840C>T in Exon 7 of the SMN2 Gene.

The CRISPR/Cas technology has a great potential in the treatment of many hereditary diseases. One of the prospective models for the CRISPR/Cas-mediated therapy is spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene that encodes the SMN protein required for the survival of motor neurons. SMA patients' genomes contain either single or several copies of SMN2 gene, which is a paralog of SMN1. Exon 7 of SMN2 has the single-nucleotide substitution c.840C>T leading to the defective splicing and decrease in the amounts of the full-length SMN. The objective of this study was to create and test gene-editing systems for correction of the single-nucleotide substitution c.840C>T in exon 7 of the SMN2 gene in fibroblasts, induced pluripotent stem cells, and motor neuron progenitors derived from a SMA patient. For this purpose, we used plasmid vectors expressing CRISPR/Cas9 and CRISPR/Cpf1, plasmid donor, and 90-nt single-stranded oligonucleotide templates that were delivered to the target cells by electroporation. Although sgRNA_T2 and sgRNA_T3 guiding RNAs were more efficient than sgRNA_T1 in fibroblasts (p < 0.05), no significant differences in the editing efficiency of sgRNA_T1, sgRNA_T2, and sgRNA_T3 was observed in patient-specific induced pluripotent stem cells and motor neuron progenitors. The highest editing efficiency in induced pluripotent stem cells and motor neuron progenitors was demonstrated by the sgRNA_T1 and 90-nt single-stranded oligonucleotide donors.

Efficient and modular CRISPR-Cas9 vector system for Physcomitrella patens.

CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the moss Physcomitrella patens. However, CRISPR‐Cas9‐mediated genome editing in P. patens was shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing in P. patens, here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping.

Sequence-specific retrotransposition of 28S rDNA-specific LINE R2Ol in human cells.

R2 is a long interspersed element (LINE) found in a specific sequence of the 28S rDNA among a wide variety of animals. Recently, we observed that R2Ol isolated from medaka fish, Oryzias latipes, retrotransposes sequence specifically into the target sequence of zebrafish. Because the 28S target and flanking regions are widely conserved among vertebrates, we examined whether R2Ol can also integrate in a sequence-specific manner in human cells. Using adenovirus-mediated expression of R2Ol constructs, we confirmed an accurate insertion of R2Ol into the 28S target of human 293T cells. However, the R2Ol mutant devoid of endonuclease (EN) activity showed no retrotransposition ability, suggesting that the sequence-specific integration of R2Ol into 28S rDNA occurs via the cleavage activity of EN. By introducing both R2Ol helper virus and donor plasmid in human cells, we succeeded in retrotransposing an exogenous EGFP gene into the 28S target site by the trans-complementation system, which enabled simplification of specific gene knock-in in a time-efficient manner. We believe that R2Ol may provide an alternative targeted gene knock-in method for practical applications such as gene therapy in future.

ZB transposon and chicken vasa homologue (Cvh) promoter interact to increase transfection efficiency of primordial germ cells in vivo

ABSTRACT 1. In order to increase the efficiency of generating transgenic chicken, this trial focused on two points: primordial germ cells (PGCs）transfection in vivo and a germline-specific promoter. 2. In order to transfect PGCs in vivo, two plasmids (pZB-CAG-GFP, pCMV-ZB）were co-injected into chicken embryos via the subgerminal cavity at Hamburger and Hamilton (HH) stage 2–3 or via blood vessel at HH stage 13–14. Results showed that the percentage of GFP+ embryos, viability and hatching rate of embryos injected at HH stage 13–14 were significantly higher than that at HH stage 2–3. 3. Two plasmid transposon systems were used for chicken embryo micro-injections. The donor plasmid, with a green fluorescent protein (GFP) reporter gene, was mediated by the ZB transposon. The helper plasmid was a transposase expression vector driven by the promoter of the chicken vasa homologue (Cvh) gene or Human cytomegalovirus (CMV) promoter. Results showed that 60.98% of gonads in Cvh group expressed GFP, which was 52.50% higher than seen in the CMV group. Only gonad tissue from the Cvh group showed any GFP signal, whereas both gonads and other tissues in the CMV group showed green fluorescence. 4. The data suggested that ZB transposon-mediated gene transfer was efficient for transfecting PGCs in vivo; the Cvh promoter drove the transposase gene specifically in the germline and increased the efficiency of germline transmission. Blood vessels injection at HH stage 13–14 may be a more efficient route for PGCs transfection in vivo.

Expression, purification and characterization of recombinant PLCζ protein in baculovirus-insect cell expression system

PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.

Feasible development of stable HEK293 clones by CRISPR/Cas9-mediated site-specific integration for biopharmaceuticals production.

To inspect the feasibility of recombinant stable HEK293 cell lines development for biopharmaceuticals production using CRISPR/Cas9-mediated site-specific integration. Using EGFP as a model protein, we first confirmed that the 'safe harbor' AAVS1 locus could be successfully targeted and the exogenous genes could be integrated through homology-directed repair induced by CRISPR/Cas9 technology. Then we constructed a donor plasmid harboring CTLA4Ig gene with an upstream CMV promoter and a downstream puromycin N-acetyltransferase gene to accelerate the efficient integration and selection of CTLA4Ig expression clones. After puromycin enrichment, the transfected pool was diluted for single clone selection, and 12 recombinant clones with CTLA4Ig expression were finally selected with a targeting efficiency of 25.8%. Productivity assay demonstrated that a frequency of 83.3% of selected clone were of consistent productivities, thus illustrating the high efficiency and success rate of this strategy. CRISPR/Cas9 mediated site-specific integration is an efficient and reliable tool to establishment recombinant stable HEK293 cell lines for both academic and industrial applications.

Efficient genome editing in Aspergillus niger with an improved recyclable CRISPR-HDR toolbox and its application in introducing multiple copies of heterologous genes

Aspergillus niger is an important industrial producer of enzymes due to its high capacity for producing exocellular secretory proteins. The CRISPR/Cas9 system has been developed as a genetic manipulation tool in A. niger. However, only the basic functions of the CRISPR/Cas9 system, such as codon optimization of Cas9 nucleases and promoter screening of guide RNA (gRNA) expression, have been developed in A. niger. The CRISPR/Cas9 system for manipulating large genomic fragments and multiple gene knock-ins still needs to be established. Here, we improved the CRISPR/Cas9 homologous direct repair (CRISPR-HDR) tool box based on donor DNAs (dDNAs) and plasmid harboring AMA1 and the pyrG marker, allowing recycling of pyrG and Cas9 components. Furthermore, we used the CRISPR-HDR tool box to knock out the 0 kb (protospacer only), 2 kb, 10 kb and even 50 kb gene fragments. This CRISPR-HDR tool box could also be used to simultaneously knock in multiple genes at the loci of two highly expressed extracellular secreted proteins, glucoamylase A (glaA) and alpha-amylase (amyA, two copies). In our study, two or three copies of glucose oxidase (goxC) were precisely knocked in at the loci of amyA and glaA, resulting in 4-fold increased enzyme activity (869.86 U/mL). This CRISPR-HDR tool box can be easily manipulated, and the AMA1-based plasmid can be easily removed under selective pressure of 5-fluoroorotic acid and uridine.

Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii

BackgroundClostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.ResultsWe have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source.ConclusionsThe developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.

Renal medullary tonicity regulates RNF183 expression in the collecting ducts via NFAT5

Nuclear factor of activated T-cells 5 (NFAT5) directly binds to the promoter of the RING finger protein 183 (RNF183) gene and induces its transcription under hypertonic conditions in mouse inner-medullary collecting duct (mIMCD-3) cells. However, there is no specific anti-RNF183 antibody for immunostaining; therefore, it is unclear whether NFAT5 regulates RNF183 expression in vivo and where RNF183 is localized in the kidney. This study investigated NFAT5-regulated in vivo RNF183 expression and localization using CRISPR/Cas9-mediated RNF183-green fluorescent protein (RNF183-GFP) knock-in mice. GFP with linker sequences was introduced upstream of an RNF183 open reading frame in exon 3 by homologous recombination through a donor plasmid. Immunofluorescence staining using GFP antibody revealed that GFP signals gradually increase from the outer medulla down to the inner medulla and colocalize with aquaporin-2. Furosemide treatment dramatically decreased RNF183 expression in the renal medulla, consistent with the decrease in NFAT5 protein and target gene mRNA expression. Furosemide treatment of mIMCD-3 cells did not affect mRNA expression and RNF183 promoter activities. These results indicated that RNF183 is predominantly expressed in the renal medullary collecting ducts, and that decreased renal medullary tonicity by furosemide treatment decreases RNF183 expression by NFAT5 downregulation.

Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing.

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.

CRISPR-SONIC: targeted somatic oncogene knock-in enables rapid in vivo cancer modeling

CRISPR/Cas9 has revolutionized cancer mouse models. Although loss-of-function genetics by CRISPR/Cas9 is well-established, generating gain-of-function alleles in somatic cancer models is still challenging because of the low efficiency of gene knock-in. Here we developed CRISPR-based Somatic Oncogene kNock-In for Cancer Modeling (CRISPR-SONIC), a method for rapid in vivo cancer modeling using homology-independent repair to integrate oncogenes at a targeted genomic locus. Using a dual guide RNA strategy, we integrated a plasmid donor in the 3′-UTR of mouse β-actin, allowing co-expression of reporter genes or oncogenes from the β-actin promoter. We showed that knock-in of oncogenic Ras and loss of p53 efficiently induced intrahepatic cholangiocarcinoma in mice. Further, our strategy can generate bioluminescent liver cancer to facilitate tumor imaging. This method simplifies in vivo gain-of-function genetics by facilitating targeted integration of oncogenes.

Antimicrobial Resistant Salmonella enterica Typhimurium Colonizing Chickens: The Impact of Plasmids, Genotype, Bacterial Communities, and Antibiotic Administration on Resistance

The rise in Salmonella resistance to cephalosporins and fluoroquinolones has become a significant threat to public health. At issue, is whether agricultural use of antimicrobials is driving emergent antibiotic resistance in Salmonella and the degree to which large antimicrobial resistance gene reservoirs, present in animal manures, drive the emergence of resistance. Two in vivo studies were performed to address these questions. In the first study, chickens were administered Salmonella and commensals, including an Escherichia coli strain with a mobile, ceftiofur-resistance plasmid, in order to determine how antibiotic administration impacted resistance in E. coli and Salmonella. All antibiotic treatments increased streptomycin resistance in E. coli. However, only ceftiofur administration increased resistance in Salmonella and specifically to extended-spectrum -lactams and cephalosporins (ESBL). There was no significant increase in emergence of ESBL-resistant Salmonella in chickens administered a ceftiofur-resistance plasmid donor. In the second study, chickens were administered two different isolates of S. enterica Typhimurium and a resistant cecal microbiome to serve as a gene donor. Birds were subsequently administered chlortetracycline or streptomycin. Antimicrobial administration significantly altered aminoglycoside and tetracycline resistance in the Enterobacteriaceae population. However, there was no significant increase in antimicrobial resistant Salmonella. Administration of a resistant microbiome had no significant impact on prevalence of resistance in Enterobacteriaceae populations, including Salmonella. Evident, from both studies, was that these treatments had minimal effect on increasing the prevalence of resistance in Salmonella, suggesting that other factors such as vertical transmission may be more important in dissemination of antimicrobial resistant Salmonella in chickens.

Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9-mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off-target effects. The genome-modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop many chicken models and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

SummaryWe describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.

De novo synthesis of the complete genome of coxsackievirus A10 based on Golden Gate cloning combined with promoter reconstruction

In the present study, a donor plasmid derived from pUC19 and two recipient plasmids, which had been modified from the donor plasmid and contained the red fluorescence protein gene mCherry as a reporter gene downstream of the hybrid tac promoter with the −35 region deletion mutation, were constructed. The complete genome sequence of coxsackievirus A10 downstream of the T7 promoter was divided into 7 fragments and synthesized by overlap extension PCR and the DNAworks program. Using the Golden Gate cloning strategy, the 7 fragments were then cloned into the donor plasmid and transferred to the recipient plasmid upstream of the deletion mutation tac promoter in a defined order and orientation without any deletions or insertions at the junction sites. Because the −35 region of the tac promoter was introduced into the 3′ end of the last fragment during construction, the hybrid promoter was reconstructed to promote expression of mCherry, which facilitated the selection of colonies with the complete genome of coxsackievirus A10 to generate an infectious cDNA clone via reverse genetic engineering.

CRISPR/Cas9-mediated genome editing of splicing mutation causing congenital hearing loss

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has ushered in a new era of gene therapy. In this study, we aimed to demonstrate precise CRISPR/Cas9-mediated genome editing of the splicing mutation c.919-2A > G in intron 7 of the SLC26A4 gene, which is the second most common causative gene of congenital hearing loss. We designed candidate single-guide RNAs (sgRNAs) aimed to direct the targeting of Staphylococcus aureus Cas9 to either exon 7 or exon 8 of SLC26A4. Several of the designed sgRNAs showed targeting activity, with average indel efficiencies ranging from approximately 14% to 25%. The usage of dual sgRNAs delivered both into Neuro2a cells and primary mouse embryonic fibroblasts resulted in the successful removal of large genomic fragments within the target locus. We subsequently evaluated genome editing in the presence of artificial donor templates to induce precise target modification via homology-directed repair. Using this approach, two different donor plasmids successfully introduced silent mutations within the c.919-2A region of Slc26a4 without evident off-target activities. Overall, these results indicate that CRISPR/Cas9-mediated correction of mutations in the Slc26a4 gene is a feasible therapeutic option for restoration of hearing loss.

Targeting antimicrobial resistance genes in clinical isolates from healthcare-associated infections using CRISPR-Cas9

Of the numerous approaches to counteracting the spread of antimicrobial resistance (AMR), one that is receiving increased attention is the use of CRISPR-Cas-based gene editing technology to remove resistance-conferring sequences from bacteria. Proof-of-principle studies have shown that CRISPR-Cas is able to successfully remove AMR genes from monocultures or very simple bacterial communities in the laboratory, but these constructs and their delivery must be adapted to be used in real-world medical settings. One area where such a technology may be implemented is tackling healthcare-associated infections. Development of a CRISPR-Cas9 cassette that is able to target particular resistance genes would be a powerful tool in addressing these infections. CRISPR-Cas9 will therefore be implemented to target AMR genes in clinical isolates of vancomycin-resistant Enterococcus faecium, and extended-spectrum β-lactamase (CTX-M) or carbapenemase (Oxa-48)-producing Klebsiella pneumoniae and Escherichia coli, by utilising a broad host range conjugative plasmid to deliver this construct via conjugation. Preliminary work using E. coli MG1655 as plasmid donor have shown that all strains can receive the plasmid, and there is significant variation in conjugation frequency between E. coli recipients.