CRISPR-Cas基因编辑技术在微生物组工程中的应用

微生物组工程是对复杂微生物群落进行改造,在基因组、代谢组及群落生态结构等多个层次上实现微生物组的精准调控.成簇规律间隔短回文序列(CRISPR)及其相关蛋白(CRISPR-Cas)系统是近期发展迅速的高效基因编辑工具.本文回顾了微生物组工程领域CRISPR-Cas系统的重要研究工作,重点关注CRISPR-Cas系统在微生物组的基因编辑和生态调控方面的应用.针对CRISPR-Cas系统在微生物组工程领域的应用挑战,本文从外源DNA递送方式和基因表达调控元件两个方面总结了微生物组工程的关键辅助方法与发展趋势,展望了微生物组工程领域所面临的挑战与机遇.

转录因子HNF1A、HNF4A和FOXA2调节肝细胞蛋白质N-糖基化

Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.

Review: Plant Genome Editing Targeted by RNA-Guided DNA Endonuclease CRISPR/Cas9

This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequence, of biological significance and of the molecular mechanism for adaptive bacterial immunity. It describes recent works on structural and functional diversity of CRISPR/Cas systems, and on three-dimensional structure-based improvements of on-target specificity of CRISPR/Cas9 and Cpf1 endonucleases. The review ends with the application of CRISPR/Cas9 to targeted editing of plant genomes. Importantly, plant commodities modified by CRISPR-Cas9 have not been considered as genetically modified organisms (GMO) as long as foreign DNAs from plant pests were not introduced, according to the recent determination by the USDA.

Modulation of mitochondrial bioenergetics as a therapeutic strategy in Alzheimer＇s disease

Alzheimer’s disease （AD） is an increasingly pressing worldwide public-health, social, political and economic concern. Despite significant investment in multiple traditional therapeutic strategies that have achieved success in preclinical models addressing the pathological hallmarks of the disease, these efforts have not translated into any effective disease-modifying therapies. This could be because interventions are being tested too late in the disease process. While existing therapies provide symptomatic and clinical benefit, they do not fully address the molecular abnormalities that occur in AD neurons. The pathophysiology of AD is complex; mitochondrial bioenergetic deficits and brain hypometabolism coupled with increased mitochondrial oxidative stress are antecedent and potentially play a causal role in the disease pathogenesis. Dysfunctional mitochondria accumulate from the combination of impaired mitophagy, which can also induce injurious inflammatory responses, and inadequate neuronal mitochondrial biogenesis. Altering the metabolic capacity of the brain by modulating/potentiating its mitochondrial bioenergetics may be a strategy for disease prevention and treatment. We present insights into the mechanisms of mitochondrial dysfunction in AD brain as well as an overview of emerging treatments with the potential to prevent, delay or reverse the neurodegenerative process by targeting mitochondria.

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010 s,clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)has rapidly been developed into a robust,multifunctional genome editing tool with many uses.Following the discovery of the initial CRISPR/Cas-based system,the technology has been advanced to facilitate a multitude of different functions.These include development as a base editor,prime editor,epigenetic editor,and CRISPR interference(CRISPRi)and CRISPR activator(CRISPRa)gene regulators.It can also be used for chromatin and RNA targeting and imaging.Its applications have proved revolutionary across numerous biological fields,especially in biomedical and agricultural improvement.As a diagnostic tool,CRISPR has been developed to aid the detection and screening of both human and plant diseases,and has even been applied during the current coronavirus disease 2019(COVID-19)pandemic.CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases,including cancers,and has aided drug development.In terms of agricultural breeding,precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins,starch,oil,and other functional components for crop improvement.Adding to this,CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators.Looking to the future,increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology.This review provides an in-depth overview of current CRISPR development,including the advantages and disadvantages of the technology,recent applications,and future considerations.

Precise genome editing with base editors

Single-nucleotide variants account for about half of known pathogenic genetic variants in human.Genome editing strategies by reversing pathogenic point mutations with minimum side effects have great therapeutic potential and are now being actively pursued.The emerge of precise and effcient genome editing strategies such as base editing and prime editing provide powerful tools for nucleotide conversion without inducing double-stranded DNA breaks(DSBs),which have shown great potential for curing genetic disorders.A diverse toolkit of base editors has been devel-oped to improve the editing effciency and accuracy in different context of application.Here,we summarized the evolving of base editors(BEs),their limitations and future perspective of base editing-based therapeutic strategies.