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Human dignity and gene editing: Using human dignity as an argument against modifying the human genome and germline is a logical fallacy.
A common argument against gene editing the human germline is the notion of dignity as linked to the human genome. But this based on a logical fallacy and should rather prompt us to consider gene editing for therapeutic purposes in order to preserve human dignity. [Image: see text]
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Optimised genome editing for precise DNA insertion and substitution using prime editors in zebrafish.
CRISPR/Cas9-mediated genome editing has rapidly become a popular tool for studying gene functions and generating genetically modified organisms. However, using this system, stochastic integration of random insertions and deletions restricts precise genome manipulation. Advanced CRISPR/Cas9 technologies using Prime Editors (PEs), Cas9 proteins fused with reverse transcriptase, enable programmed integration of short DNA modifications into the genome. However, its application in precise genome editing in animal models is challenging. Here, we utilise a nickase- and a nuclease-based PE to perform programmed short DNA substitutions and insertions at various loci in the zebrafish genome. Whereas nickase-based PE2 mediated a higher ratio of precise prime edits to the total edits, nuclease-based PEn was more efficient for short DNA modifications, achieving up to 27.3% precise insertion. To further evaluate our approach, we inserted a nuclear localisation signal into a reporter transgene to incorporate longer fragments by prime editing. These gene modifications were transmitted to the next generation. We show that PE-mediated prime editing can efficiently manipulate genome information in zebrafish without using exogenous donor DNA.
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Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.
Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc.
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Potential benefits of gene editing for the future of poultry farming.
The chicken is an exemplar of efficient intensive animal agriculture and provides two valuable food products, chicken meat and eggs. Only aquaculture is better, by efficiency, but poultry is still top, by mass of animal protein produced as food in the global context. However this efficiency and intensive production comes with a number of challenges. Though the genetics of selective breeding have led to dramatic improvements in yield, efficiency and product quality, traits that relate to disease and welfare outcomes have not been so tractable. These two issues are major impacts to the industry in terms of production and in terms of public perception. Both transgenic technology and genome editing have clear potential for impact in these two important areas. The reproductive biology of birds requires techniques very specific to birds to achieve heritable (germline) edited traits. These are quite involved and, even though they are now well-defined and reliable, there is room for improvement and advances can be expected in the future. Currently the key targets for this technology are modifying chicken genes involved in virus-receptor interactions and cellular response involved in infection. For the egg industry the technology is being applied to the issue of sex-selection for layer hens (and the removal of males), removal of allergens from egg white and the tailoring of eggs system to enhance the yield of influenza vaccine doses. Regulation and trading of the animals generated, and resulting food products, will significantly impact the value and future development of genome editing for poultry.
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Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system.
The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.
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A protocol for the detection of precision genome editing in human cells using One-pot DTECT.
Prime editing is a highly versatile CRISPR-based genome editing technology that allows for the precise installation of desired genetic variants. This protocol describes how to use One-pot DTECT to assess prime editing efficiency in human cells. Key steps include conducting prime editing, extracting genomic DNA, performing AcuI-tagging PCR, capturing genetic signatures, and detecting captured signatures through qualitative, quantitative, and visual methods. One-pot DTECT enables same-day detection of targeted genetic signatures introduced by precision genome editing technologies using off-the-shelf reagents. For complete details on the use and execution of this protocol, please refer to Baudrier et al.1.
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In recognition of the Spanish origin of CRISPR/Cas9. Implications for the treatment of familial heart disease.
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Shock greets claim of CRISPR-edited babies.
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New Developments in CRISPR/Cas-based Functional Genomics and their Implications for Research Using Zebrafish.
INTRODUCTION: Genome editing using CRISPR/Cas9 has advanced very rapidly in its scope, versatility and ease of use. Zebrafish (Danio rerio) has been one of the vertebrate model species where CRISPR/Cas9 has been applied very extensively for many different purposes and with great success. In particular, disease modeling in zebrafish is useful for testing specific gene variants for pathogenicity in a preclinical setting. Here we describe multiple advances in diverse species and systems that can improve genome editing in zebrafish. OBJECTIVE: To achieve temporal and spatial precision of genome editing, many new technologies can be applied in zebrafish such as artificial transcription factors, drug-inducible or optogenetically-driven expression of Cas9, or chemically-inducible activation of Cas9. Moreover, chemically- or optogenetically- inducible reconstitution of dead Cas9 (catalytically inactive, dCas9) can enable spatiotemporal control of gene regulation. In addition to controlling where and when genome editing occurs, using oligonucleotides allows for the introduction (knock-in) of precise modifications of the genome. CONCLUSION: We review recent trends to improve the precision and efficiency of oligo-based point mutation knock-ins and discuss how these improvements can apply to work in zebrafish. Similarly to how chemical mutagenesis enabled the first genetic screens in zebrafish, multiplexed sgRNA libraries and Cas9 can enable the next revolutionary transition in how genetic screens are performed in this species. We discuss the first examples and prospects of approaches using sgRNAs as specific and effective mutagens. Moreover, we have reviewed methods aimed at measuring the phenotypes of single cells after their mutagenic perturbation with vectors encoding individual sgRNAs. These methods can range from different cell-based reporters to single-cell RNA sequencing and can serve as great tools for high-throughput genetic screens.
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Toehold-Based CRISPR-dCas9 Transcriptional Activation Platform for Spatiotemporally Controllable Gene Therapy in Tumor and Diabetic Mouse Models.
The CRISPR-Cas system has been extensively employed as a genome editing tool with the dCas9-based transcriptional activation system emerging as a particularly promising approach for gene editing in the treatment of diseases at the gene level. Nevertheless, the challenge of achieving effective spatiotemporal control of the transcriptional activation system of dCas9 has thus far restricted its broader application. In this study, we present an miRNA-responsive CRISPR-dCas9 transcriptional activation (mCTA) system. This system is capable of responding specifically to exogenous and endogenous miRNAs in mammalian cells and enables the specific imaging of miRNAs during neural development or in the deep tissues of mice. Furthermore, the replacement of downstream functional genes with DTA has been demonstrated to result in the effective apoptosis of tumor cells and inhibition of xenografted tumor growth in mice. Finally, in a diabetic mouse model, the m122CTA system was shown to reduce the blood glucose in diabetic mice via the activation of PDX-1 gene. Our work provides an effective platform for miRNA imaging and gene therapy via spatiotemporal control of gene regulation.
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