Tools for large-scale genetic manipulation of yeast genome / 生物工程学报

Large-scale genetic manipulation of the genome refers to the genetic modification of large fragments of DNA using knockout, integration and translocation. Compared to small-scale gene editing, large-scale genetic manipulation of the genome allows for the simultaneous modification of more genetic information, which is important for understanding the complex mechanisms such as multigene interactions. At the same time, large-scale genetic manipulation of the genome allows for larger-scale design and reconstruction of the genome, and even the creation of entirely new genomes, with great potential in reconstructing complex functions. Yeast is an important eukaryotic model organism that is widely used because of its safety and easiness of manipulation. This paper systematically summarizes the toolkit for large-scale genetic manipulation of the yeast genome, including recombinase-mediated large-scale manipulation, nuclease-mediated large-scale manipulation, de novo synthesis of large DNA fragments and other large-scale manipulation tools, and introduces their basic working principles and typical application cases. Finally, the challenges and developments in large-scale genetic manipulation are presented.

Multiplex gene editing and regulation techniques based on CRISPR/Cas system / 生物工程学报

The CRISPR/Cas systems comprising the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an acquired immune system unique to archaea or bacteria. Since its development as a gene editing tool, it has rapidly become a popular research direction in the field of synthetic biology due to its advantages of high efficiency, precision, and versatility. This technique has since revolutionized the research of many fields including life sciences, bioengineering technology, food science, and crop breeding. Currently, the single gene editing and regulation techniques based on CRISPR/Cas systems have been increasingly improved, but challenges still exist in the multiplex gene editing and regulation. This review focuses on the development and application of multiplex gene editing and regulation techniques based on the CRISPR/Cas systems, and summarizes the techniques for multiplex gene editing or regulation within a single cell or within a cell population. This includes the multiplex gene editing techniques developed based on the CRISPR/Cas systems with double-strand breaks; or with single-strand breaks; or with multiple gene regulation techniques, etc. These works have enriched the tools for the multiplex gene editing and regulation and contributed to the application of CRISPR/Cas systems in the multiple fields.

Preliminary report of preclinical trial of multi-genome engineering pig-to-macaque heart, liver and kidney transplantation / 器官移植

Objective To investigate the application prospect of the most extensive genome engineering pig internationally in preclinical xenotransplantation. Methods Porcine endogenous retrovirus (PERV) knockout combined with 3 major heterologous antigen gene knockouts and 9 humanized genes for inhibition of complement activation, regulation of coagulation disorders, anti-inflammatory and anti-phagocytosis were transferred into a pig (PERV-KO/3-KO/9-TG) as a donor, and the heart, liver and kidney were obtained and transplanted to 3 Rhesus macaque recipients respectively to establish a preclinical research model of pig-to-Rhesus macaque xenotransplantation. The functional status of xenografts after blood flow reconstruction was observed and the survival of recipients was summarized. The hemodynamics of xenografts were monitored. The change of hematological indexes of each recipient was compared. The histopathological manifestation of xenografts was observed. Results After the blood flow was reconstructed, all xenografts showed ruddy color, soft texture and good perfusion. The transplant heart, liver and kidney showed full arterial and venous blood flow and good perfusion at 1 d after operation. The postoperative survival time of heart, liver, and kidney transplant recipients was 7, 26, and 1 d, respectively. The levels of creatine kinase, creatine kinase isoenzyme, and lactate dehydrogenase increased in heart transplant recipient at 1 d after operation, and gradually recovered to near normal levels at 6 d after operation. All indexes increased sharply at 7 d after operation. The level of aspartate aminotransferase increased in liver transplant recipients at 2 d after operation, and the alanine aminotransferase basically returned to normal at 10 d after operation, but the total bilirubin continued to increase. Both aspartate aminotransferase and alanine aminotransferase increased at 12 d after operation, and reached a peak at 15 d after operation. The kidney transplant recipient developed mild proteinuria at 1 d after operation, and died of sudden severe arrhythmia. Histopathology showed that the tissue structure of cardiac and renal xenografts was close to normal, and liver xenografts presented with patchy necrosis, the liver tissue structure was disordered, accompanied by inflammatory damage, interstitial hemorrhage and thrombotic microangiopathy. Conclusions PERV-KO/3-KO/9-TG pig shows advantages in overcoming hyperacute rejection, mitigating humoral rejection and coagulation dysregulation. However, whether it can be used as potential donor for clinical xenotransplantation needs further evaluation.

A biotecnologia envolvida no sistema CRISPR / Biotechnology involved in the CRISPR system

Introdução: o sistema CRISPR trata-se de uma ferramenta molecular, ordenada por um RNA guia e a enzima Cas9 capaz de corrigir a expressão de uma gama de genes alvo. Objetivo: apresentar uma revisão da literatura acerca do sistema CRISPR e sua contribuição para a biotecnologia. Metodologia: trata-se de uma revisão bibliográfica realizada com base em periódicos nacionais e internacionais com vista à reunir as melhores informações acerca do Sistema CRISPR. Resultados: após compilação dos dados, verificou-se que o sistema CRISPR tem se tornado um artifício promissor da biologia molecular, haja vista sua versatilidade de uso em diferentes sistemas e sua capacidade de destruir múltiplos genes invasores. Conclusão: nesse sentido, constata-se que a partir do sistema CRISPR podem surgir novas alternativas para o tratamento terapêutico de diversas doenças ligadas ao genoma.

Introduction: the CRISPR system is a molecular tool, ordered by a guiding RNA and a Cas9 enzyme able of correcting the expression of target genes. Objective: to present a review of the literature on the CRISPR system and its contribution to biotechnology Methodology: this is a bibliographic review based on national and international journals to gather the best information about the CRISPR System. Results: after compiling the data, it was verified that the CRISPR system has become a promising artifice for molecular biology, given its versatility of use in different systems and its ability to destroy multiple invading genes. Conclusion: in this sense, it can be seen that from the CRISPR system new alternatives can arise for the therapeutic treatment of various diseases linked to the genome

Genome Editing: A Comparative Study on the Efficiency of CRISPR/Cas9 Nuclease Versus Nickase Using HIV as a Model System

@#Introduction: CRISPR/Cas9 nuclease has gained popularity as a genome editing tool due to its straight-forward mechanism. However, there are concerns that CRISPR nuclease would cause off-target and toxicity. The CRISPR/ Cas9 D10A nickase was designed to enhance genome editing. Nevertheless, this raised the question of whether the efficiency of nickase is compromised compared to CRISPR/Cas9 nuclease. Targeting HIV genes, we investigated if CRISPR nuclease performed better than the nickase in efficacy and safety. Methods: CRISPR nucleases and nickases were designed to target Gag, Pol, Rev, Vif, Tat and LTR. HIV latently infected cell line, ACH-2, was transfected with the nucleases and nickases. Changes to viral load after CRISPR treatment was measured using p24 ELISA. Safety of nuclease and nickase was monitored using GFP expression with fluorescence microscopy and flow cytometry. Targeting two sites within the same gene, and targeting multiple genes concurrently were also studied to determine efficacy of CRISPR in reducing viral load. Results: A 44.9 to 68.1% and a 34.4 to 49.7% decrease in viral load was seen in CRISPR nuclease and nickase respectively. Microscopy and flow cytometry results showed that the nickase system was slightly toxic with a 0.31 to 0.7-fold cell death. There was a 34% decrease in viral load when two sites were targeted within a gene, and the largest decrease was seen when all the nucleases were combined, giving a 75.4% decrease in viral load at day 5. Conclusion: The knowledge gained from this study will be employed to improve genome editing in other disease models.

Methods for the genetic manipulation of marine bacteria

Genetic manipulation of bacteria is a procedure necessary to obtain new strains that express peculiar and defined genetic determinants or to introduce genetic variants responsible for phenotypic modifications. This procedure can be applied to explore the biotechnological potential associated with environmental bacteria and to utilize the functional properties of specific genes when inserted into an appropriate host. In the past years, marine bacteria have received increasing attention because they represent a fascinating reservoir of genetic and functional diversity that can be utilized to fuel the bioeconomy sector. However, there is an urgent need for an in-depth investigation and improvement of the genetic manipulation tools applicable to marine strains because of the paucity of knowledge regarding this. This review aims to describe the genetic manipulation methods hitherto used in marine bacteria, thus highlighting the limiting factors of the different techniques available today to increase manipulation efficiency. In particular, we focus on methods of natural and artificial transformations (especially electroporation) and conjugation because they have been successfully applied to several marine strains. Finally, we emphasize that, to avoid failure, future work should be carried out to establish tailored methodologies for marine bacteria.

Re-engineering the mitochondrial genomes in mammalian cells / 대한해부학회지

Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. They have long been recognized as the generators of energy for the cell and also have been known to associate with several metabolic pathways that are crucial for cellular function. Mitochondria have their own genome, mitochondrial DNA (mtDNA), that is completely separated and independent from the much larger nuclear genome, and even have their own system for making proteins from the genes in this mtDNA genome. The human mtDNA is a small (~16.5 kb) circular DNA and defects in this genome can cause a wide range of inherited human diseases. Despite of the significant advances in discovering the mtDNA defects, however, there are currently no effective therapies for these clinically devastating diseases due to the lack of technology for introducing specific modifications into the mitochondrial genomes and for generating accurate mtDNA disease models. The ability to engineer the mitochondrial genomes would provide a powerful tool to create mutants with which many crucial experiments can be performed in the basic mammalian mitochondrial genetic studies as well as in the treatment of human mtDNA diseases. In this review we summarize the current approaches associated with the correction of mtDNA mutations in cells and describe our own efforts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation.