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Recent metabolomics and gene editing approaches for synthesis of microbial secondary metabolites for drug discovery and development.
Microbial secondary metabolites (SMs) have been identified as an important natural source of drugs for several metabolic and neurological diseases. Along with biomedical applications, SMs are also used in the food and biochemical industries. SMs include natural products such as pigments, alkaloids, toxins, antimicrobials obtained from cultured microorganisms, while other non-cultivable microorganisms have also acted as a rich source of SMs. But, the isolation of SMs from these sources is a very tedious task. Metabolomics provides complete identification and structural information about the entire cellular metabolome under specific conditions using highly sophisticated instrumentation. Further, gene editing techniques such as cloning and gene refactoring, including advanced CRISPR-Cas, can be used for engineering microbes that have the potential to produce natural SMs that were not produced in native microbial strain. The present review describes integrated metabolomics and gene editing approaches for the synthesis of novel microbial SMs and their potential application towards drug discovery and development.
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Genome editing in the green alga Chlamydomonas: past, present practice and future prospects.
The green alga Chlamydomonas is an important and versatile model organism for research topics ranging from photosynthesis and metabolism, cilia, and basal bodies to cellular communication and the cellular cycle and is of significant interest for green bioengineering processes. The genome in this unicellular green alga is contained in 17 haploid chromosomes and codes for 16 883 protein coding genes. Functional genomics, as well as biotechnological applications, rely on the ability to remove, add, and change these genes in a controlled and efficient manner. In this review, the history of gene editing in Chlamydomonas is put in the context of the wider developments in genetics to demonstrate how many of the key developments to engineer these algae follow the global trends and the availability of technology. Building on this background, an overview of the state of the art in Chlamydomonas engineering is given, focusing primarily on the practical aspects while giving examples of recent applications. Commonly encountered Chlamydomonas-specific challenges, recent developments, and community resources are presented, and finally, a comprehensive discussion on the emergence and evolution of CRISPR/Cas-based precision gene editing is given. An outline of possible future paths for gene editing based on current global trends in genetic engineering and tools for gene editing is presented.
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Genetic Modification of Mice Using Prime Editing.
Genetically modifying mice traditionally involved complex methods of designing and validating targeting constructs, embryonic stem cell electroporation and selection, blastocyst injection, and breeding chimeras for germline transmission. Such arduous steps were best carried out by specialized gene targeting cores in academia or through expensive commercial vendors. Further, the time from initiation to completion of a project often took at least 1 year and, in some cases, much longer (or never), with no guarantees of success. The RNA-programmable CRISPR system of gene editing has greatly streamlined the generation of gene modifications (e.g., small substitutions, insertions, and deletions) in the mouse with high rates of success. Several editing platforms exist for gene/genome targeting in mice and other animal models previously difficult or impossible to alter. Here, we provide a simplified method of generating genetically modified mice using the prime editing platform. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Design, cloning, and synthesis of engineered pegRNA (epegRNA) Basic Protocol 2: Microinjection of PE2 components into mouse zygote Basic Protocol 3: Genotyping founder mice and breeding for germline transmission.
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Applications of CRISPR-Cas9 in gynecological cancer research.
Gynecological cancers pose a significant threat to women's health worldwide, with cervical cancer, ovarian cancer, and endometrial cancer having high incidences. Current gynecological cancer treatment methods mainly include surgery, chemotherapy, radiotherapy, and chemoradiotherapy. The CRISPR-Cas9 gene editing technology as a new therapeutic method has shown tremendous effect in the treatment of other cancers, promoting research on its potential therapeutic effect in gynecological cancer. In this article, we reviewed the current research status of CRISPR-Cas9 technology in gynecological cancer, focusing on the importance of studying the mechanism of CRISPR-Cas9 in gynecological cancer treatment, thereby laying a foundation for further research on its clinical application.
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Use of an exogenous DNA-free system to generate MSTN-KO calves by CRISPR/Cas9 and SCNT.
This study aimed to obtain myostatin (MSTN)-knockout calves, while avoiding the risk of exogenous DNA integration during gene editing. To achieve this, we combined CRISPR/Cas9 ribonucleoprotein gene editing with somatic cell nuclear transfer (SCNT) technology. In the first experiment (E1), we compared the gene editing efficiency of four gRNAs targeting different coding regions of the MSTN gene using plasmid-based CRISPR/Cas9 in bovine fetal fibroblasts (BFF-E1 cells). The highest bioinformatically-predicted editing rate (BPE) was obtained with gRNA2 (96 %), which was subsequently used for further experiments. Next, embryos were produced by SCNT using BFF-E1-edited cells as nuclear donors. Sanger sequencing of the embryos showed biallelic MSTN editing. In the second experiment (E2), plasmid-based editing was replaced with CAS9 protein and trac:crRNA oligoribonucleotides. Editing efficiency was assessed on one edited bovine fetal fibroblast line (BFF-E2-maleed) and two edited bovine mesenchymal stem lines (MSC-E2-maleed and MSC-E2-femed) derived from price-winning animals. BPEs were 58.8 %, 31 % and 59 % in fibroblast and MSC cells, and 64 %, 73.3 %, and 66.6 % in SCNT embryos derived from BFF-E2-maleed, MSC-E2-maleed and MSC-E2-femed, respectively. Transfer of 35 MSC-E2-femed embryos to recipient cows, resulted in the birth of one MSTN-edited calf with a heterozygous genotype. A second-generation clone was subsequently produced, using a fibroblast sample as nuclear donor. In conclusion, we established an efficient protocol for generating high rates of edited blastocysts with a desirable genetic background, resulting in the birth of two MSTN-knockout calves. This study provides a foundation for gene editing to improve productive or biomedical traits.
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CRISPR/Cas9-mediated gene editing of GmJAGGED1 increased yield in the low-latitude soybean variety Huachun 6.
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Mutational rescue of the activity of high-fidelity Cas9 enzymes.
Programmable DNA endonucleases derived from bacterial genetic defense systems, exemplified by CRISPR-Cas9, have made it significantly easier to perform genomic modifications in living cells. However, unprogrammed, off-target modifications can have serious consequences, as they often disrupt the function or regulation of non-targeted genes and compromise the safety of therapeutic gene editing applications. High-fidelity mutants of Cas9 have been established to enable more accurate gene editing, but these are typically less efficient. Here, we merge the strengths of high-fidelity Cas9 and hyperactive Cas9 variants to provide an enzyme, which we dub HyperDriveCas9, that yields the desirable properties of both parents. HyperDriveCas9 functions efficiently in mammalian cells and introduces insertion and deletion mutations into targeted genomic regions while maintaining a favorable off-target profile. HyperDriveCas9 is a precise and efficient tool for gene editing applications in science and medicine.
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Optimizing gene editing in pigs: The role of electroporation and lipofection.
The production of genetically modified pigs is becoming increasingly important in both the agricultural and biomedical fields. Optimization of these processes is a key objective to improve the precision, scalability and viability of genetically modified animals for research and commercial applications. Among the available techniques, electroporation and lipofection have emerged as promising alternatives to traditional methods such as microinjection and somatic cell nuclear transfer (SCNT) due to their simplicity, cost-effectiveness, and potential for high-throughput applications. These methods allow the direct delivery of CRISPR/Cas components into zygotes and embryos, reducing the technical expertise required and bypassing some of the challenges associated with cloning. This review examines the application, efficacy, and outcomes of electroporation and lipofection as gene editing techniques in porcine gametes and embryos. We provide a comprehensive synthesis of recent advances, compare their efficacy, and discuss their potential in agricultural and biomedical research. The principles and mechanisms of both methods are reviewed, highlighting their advantages, such as cost-effectiveness and ease of implementation, over traditional approaches such as microinjection. In addition, we address their limitations, including variability in efficiency, and discuss recent protocol optimizations aimed at improving reproducibility and applicability. By analyzing these developments, this review provides valuable insights into the evolving role of electroporation and lipofection in porcine genetic modification strategies.
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Gene Replacement by Intron Targeting with CRISPR-Cas9.
The CRISPR-Cas9 system has become the most widely adopted genome editing platform and is used in an expanding number of organisms, mainly by creating targeted knockouts through non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). It would also be highly desirable to be able to use homology-directed repair (HDR) to perform precise gene editing, for example, by replacing a small section of DNA to substitute one amino acid for another in a given gene product. However, this remains a serious challenge in plants. Here, we describe a recently developed intron-mediated site-specific gene replacement method acting through the NHEJ pathway in which Cas9 simultaneously introduces DSBs in adjacent introns and the donor template. This approach is of general use for replacing targeted gene fragments at specific genomic sites in plants.
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Comparison of Multiple Strategies for Precision Transgene Knock-In in Gallus gallus Genome via Microhomology-Mediated End Joining.
Precision exogenous gene knock-in is an attractive field for transgenic Gallus gallus (chicken) generation. In this article, we constructed multiple Precise Integration into Target Chromosome (PITCh) plasmid systems mediated by microhomology-mediated end-joining (MMEJ) for large-fragment integration in DF-1 cells and further assess the possibility of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a genomic safe harbor for chickens. We designed three targeted sgRNAs for the all-in-one plasmid at the 3'UTR of GAPDH near the stop codon. The donor-plasmid-carrying microhomology arms correspond to sgRNA and EGFP fragments in the forward and reverse directions. MMEJ-mediated EGFP insertion can be efficiently expressed in DF-1 cells. Moreover, the differences between the forward and reverse fragments indicated that promoter interference does affect the transfection efficiency of plasmids and cell proliferation. The comparison of the 20 bp and 40 bp microhomology arms declared that the short one has higher knock-in efficiency. Even though all three different transgene insertion sites in GAPDH could be used to integrate the foreign gene, we noticed that the G2-20R-EGFP cell reduced the expression of GAPDH, and the G3-20R-EGFP cell exhibited significant growth retardation. Taken together, G1, located at the 3'UTR of GAPDH on the outer side of the last base of the terminator, can be a candidate genomic safe harbor (GSH) loci for the chicken genome. In addition, deleted-in-azoospermia-like (DAZL) and actin beta (ACTB) site-specific gene knock-in indicated that MMEJ has broad applicability and high-precision knock-in efficiency for genetically engineered chickens.
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