Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively e...Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.展开更多
Whole genome engineering is now feasible with the aid of genome editing and synthesis tools. Synthesizing a genome from scratch allows modifications of the genomic structure and function to an extent that was hitherto...Whole genome engineering is now feasible with the aid of genome editing and synthesis tools. Synthesizing a genome from scratch allows modifications of the genomic structure and function to an extent that was hitherto not possible, which will finally lead to new insights into the basic principles of life and enable valuable applications. With several recent genome synthesis projects as examples, the technical details to synthesize a genome and applications of synthetic genome are addressed in this perspective. A series of ongoing or future synthetic genomics projects, including the different genomes to be synthesized in GPwrite, synthetic minimal genome, massively recoded genome, chimeric genome and synthetic genome with expanded genetic alphabet, are also discussed here with a special focus on theoretical and technical impediments in the design and synthesis process. Synthetic genomics will become a commonplace to engineer pathways and genomes according to arbitrary sets of design principles with the development of high-efficient, low-cost genome synthesis and assembly technologies.展开更多
Transcription activator-like effectors （TALEs） from Xanthomonas sp. have been used as customizable DNA- binding modules for genome-engineering applications, Ralstonia solanacearum TALE-like proteins （RTLs） exhibit...Transcription activator-like effectors （TALEs） from Xanthomonas sp. have been used as customizable DNA- binding modules for genome-engineering applications, Ralstonia solanacearum TALE-like proteins （RTLs） exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues （RVDs）, which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts spe- cifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.展开更多
This paper offers a general review and comparative analysis of various types of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies. It evaluates the strengths and weaknesses of these techn...This paper offers a general review and comparative analysis of various types of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies. It evaluates the strengths and weaknesses of these technologies to identify the optimal approach for conducting genetic screens. Through an extensive literature review, this paper examines CRISPR nuclease, CRISPR activation (CRISPRa), and CRISPR interference (CRISPRi) screens. This study concludes that CRISPRa and CRISPRi are more advantageous due to their use of deactivated Cas9 proteins that only over-express or deactivate genes rather than irreversibly breaking genes like CRISPRn. Notably, CRISPRa is unique in its ability to over-express genes, while the other two technologies deactivate genes. Future studies may focus on inducing multiple mutations simultaneously—both gain-of-function and gene knockout—to carry out a more complete screen that can test the combinatorial effect of genes. Likewise, targeting both exons and introns can offer a more thorough understanding of a specific phenotype.展开更多
The tools available for genome engineering have significantly improved over the last 5 years, allowing scientist to make precise edits to the genome. Along with the development of these new genome editing tools has co...The tools available for genome engineering have significantly improved over the last 5 years, allowing scientist to make precise edits to the genome. Along with the development of these new genome editing tools has come advancements in technologies used to deliver them. In mammals genome engineering tools are typically delivered into in vitro fertilized single cell embryos which are subsequently cultured and then implanted into a recipient animal.In avian species this is not possible, so other methods have been developed for genome engineering in birds. The most common involves in vitro culturing of primordial germ cells(PGCs), which are cells that migrate through the embryonic circulatory system to the developing gonad and colonize the gonad, eventually differentiating into the gonadocytes which produce either sperm or ova. While in culture the PGCs can be modified to carry novel transgenes or gene edits, the population can be screened and enriched, and then transferred into a recipient embryo. The largest drawback of PGC culture is that culture methods do not transfer well across avian species, thus there are reliable culture methods for only a few species including the chicken. Two newer technologies that appear to be more easily adapted in a wider range of avian species are direct injection and sperm transfection assisted gene editing(STAGE).The direct injection method involves injecting genome engineering tools into the circulatory system of the developing embryo just prior to the developmental time point when the PGCs are migrating to the gonads. The genome engineering tools are complexed with transfection reagents, allowing for in vivo transfection of the PGCs. STAGE utilizes sperm transfection to deliver genome engineering tools directly to the newly fertilized embryo. Preliminary evidence indicates that both methodologies have the potential to be adapted for use in birds species other than the chicken, however further work is needed in this area.展开更多
The ability to go from a digitized DNA sequence to a predictable biological function is central to synthetic biology. Genome engineering tools facilitate rewriting and implementation of engineered DNA sequences. Recen...The ability to go from a digitized DNA sequence to a predictable biological function is central to synthetic biology. Genome engineering tools facilitate rewriting and implementation of engineered DNA sequences. Recent development of new programmable tools to reengineer genomes has spurred myriad advances in synthetic biology. Tools such as clustered regularly interspace short palindromic repeats enable RNA-guided rational redesign of organisms and implementation of synthetic gene systems. New directed evolution methods generate organisms with radically restructured genomes. These restructured organisms have useful new phenotypes for biotechnology, such as bacteriophage resistance and increased genetic stability. Advanced DNA synthesis and assembly methods have also enabled the construction of fully synthetic organisms, such as J. Craig Venter Institute （JCVI）-syn 3.0. Here we summarize the recent advances in programmable genome engineering tools.展开更多
基金supported by the UK Medical Research Council and the European Research Council (DARCGENs, No. 249869)
文摘Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Traditional techniques for generating genetic mutations in most organisms have relied on selection from large pools of randomly induced mutations for those of particular interest, or time-consuming gene targeting by homologous recombination. Drosophila melanogaster has always been at the forefront of genetic analysis, and application of these new genome editing techniques to this organism will revolutionise our approach to performing analysis of gene function in the future. We discuss the recent techniques that apply the CRISPR/Cas9 system to Drosophila, highlight potential uses for this technology and speculate upon the future of genome engineering in this model organism.
基金supported by the National Natural Science Foundation of China (31725002)by Bureau of International Cooperation, Chinese Academy of Sciences (172644KYSB20170042)+3 种基金by the Key Research Program of the Chinese Academy of Sciences (KFZD-SW215)the financial support from Youth Innovation Promotion Association of the Chinese Academy of Sciences (2018396)China Postdoctoral Science Foundation (2017M620393)Technical Innovation Special Fund of Hubei Province (2018ACA149)
文摘Whole genome engineering is now feasible with the aid of genome editing and synthesis tools. Synthesizing a genome from scratch allows modifications of the genomic structure and function to an extent that was hitherto not possible, which will finally lead to new insights into the basic principles of life and enable valuable applications. With several recent genome synthesis projects as examples, the technical details to synthesize a genome and applications of synthetic genome are addressed in this perspective. A series of ongoing or future synthetic genomics projects, including the different genomes to be synthesized in GPwrite, synthetic minimal genome, massively recoded genome, chimeric genome and synthetic genome with expanded genetic alphabet, are also discussed here with a special focus on theoretical and technical impediments in the design and synthesis process. Synthetic genomics will become a commonplace to engineer pathways and genomes according to arbitrary sets of design principles with the development of high-efficient, low-cost genome synthesis and assembly technologies.
文摘Transcription activator-like effectors （TALEs） from Xanthomonas sp. have been used as customizable DNA- binding modules for genome-engineering applications, Ralstonia solanacearum TALE-like proteins （RTLs） exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues （RVDs）, which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts spe- cifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp.
文摘This paper offers a general review and comparative analysis of various types of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies. It evaluates the strengths and weaknesses of these technologies to identify the optimal approach for conducting genetic screens. Through an extensive literature review, this paper examines CRISPR nuclease, CRISPR activation (CRISPRa), and CRISPR interference (CRISPRi) screens. This study concludes that CRISPRa and CRISPRi are more advantageous due to their use of deactivated Cas9 proteins that only over-express or deactivate genes rather than irreversibly breaking genes like CRISPRn. Notably, CRISPRa is unique in its ability to over-express genes, while the other two technologies deactivate genes. Future studies may focus on inducing multiple mutations simultaneously—both gain-of-function and gene knockout—to carry out a more complete screen that can test the combinatorial effect of genes. Likewise, targeting both exons and introns can offer a more thorough understanding of a specific phenotype.
文摘The tools available for genome engineering have significantly improved over the last 5 years, allowing scientist to make precise edits to the genome. Along with the development of these new genome editing tools has come advancements in technologies used to deliver them. In mammals genome engineering tools are typically delivered into in vitro fertilized single cell embryos which are subsequently cultured and then implanted into a recipient animal.In avian species this is not possible, so other methods have been developed for genome engineering in birds. The most common involves in vitro culturing of primordial germ cells(PGCs), which are cells that migrate through the embryonic circulatory system to the developing gonad and colonize the gonad, eventually differentiating into the gonadocytes which produce either sperm or ova. While in culture the PGCs can be modified to carry novel transgenes or gene edits, the population can be screened and enriched, and then transferred into a recipient embryo. The largest drawback of PGC culture is that culture methods do not transfer well across avian species, thus there are reliable culture methods for only a few species including the chicken. Two newer technologies that appear to be more easily adapted in a wider range of avian species are direct injection and sperm transfection assisted gene editing(STAGE).The direct injection method involves injecting genome engineering tools into the circulatory system of the developing embryo just prior to the developmental time point when the PGCs are migrating to the gonads. The genome engineering tools are complexed with transfection reagents, allowing for in vivo transfection of the PGCs. STAGE utilizes sperm transfection to deliver genome engineering tools directly to the newly fertilized embryo. Preliminary evidence indicates that both methodologies have the potential to be adapted for use in birds species other than the chicken, however further work is needed in this area.
文摘The ability to go from a digitized DNA sequence to a predictable biological function is central to synthetic biology. Genome engineering tools facilitate rewriting and implementation of engineered DNA sequences. Recent development of new programmable tools to reengineer genomes has spurred myriad advances in synthetic biology. Tools such as clustered regularly interspace short palindromic repeats enable RNA-guided rational redesign of organisms and implementation of synthetic gene systems. New directed evolution methods generate organisms with radically restructured genomes. These restructured organisms have useful new phenotypes for biotechnology, such as bacteriophage resistance and increased genetic stability. Advanced DNA synthesis and assembly methods have also enabled the construction of fully synthetic organisms, such as J. Craig Venter Institute （JCVI）-syn 3.0. Here we summarize the recent advances in programmable genome engineering tools.
