Table of Content

    28 February 2023, Volume 4 Issue 1
    Invited Review
    Computational design and directed evolution strategies for optimizing protein stability
    Qingyun RUAN, Xin HUANG, Zijun MENG, Shu QUAN
    2023, 4(1):  5-29.  doi:10.12211/2096-8280.2022-038
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    Most natural proteins tend to be marginally stable, which allows them to gain flexibility for biological functions. However, marginal stability is often associated with protein misfolding and aggregation under stress conditions, presenting a challenge for protein research and applications such as proteins as biocatalysts and therapeutic agents. In addition, protein instability has been increasingly recognized as one of the major factors causing human diseases. For example, the formation of toxic protein aggregates is the hallmark of many neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Therefore, optimizing protein folding and maintaining protein homeostasis in cells are long-standing goals for the scientific community. Confronting these challenges, various methods have been developed to stabilize proteins. In this review, we classify and summarize various techniques for engineering protein stability, with a focus on strategies for optimizing protein sequences or cellular folding environments. We first outline the principles of protein folding, and describe factors that affect protein stability. Then, we describe two main approaches for protein stability engineering, namely, computational design and directed evolution. Computational design can be further classified into structure-based, phylogeny-based, folding energy calculation-based and artificial intelligence-assisted methods. We present the principles of several methods under each category, and also introduce easily accessible web-based tools. For directed evolution approaches, we focus on library-based, high-throughput screening or selection techniques, including cellular or cell-free display and stability biosensors, which link protein stability to easily detectable phenotypes. We not only introduce the applications of these techniques in protein sequence optimization, but also highlight their roles in identifying novel folding factors, including molecular chaperones, chemical chaperones, and inhibitors of protein aggregation. Moreover, we demonstrate the applications of protein stability engineering in biomedicine and pharmacotherapeutics, including identifying small molecules to stabilize disease-related, aggregation-prone proteins, obtaining conformation-fixed and stable antigens for vaccine development, and targeting protein stability as a means to control protein homeostasis. Finally, we look forward to the trends and prospects of protein stabilization technologies, and believe that protein stability engineering will lead to a better understanding of protein folding processes to facilitate the development of precision medicine. {L-End}

    Recent progress in computational tools for designing editing sequences used in microbial genetic manipulations
    Yi YANG, Yufeng MAO, Chunhe YANG, Meng WANG, Xiaoping LIAO, Hongwu MA
    2023, 4(1):  30-46.  doi:10.12211/2096-8280.2022-055
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    Genetic manipulations such as homologous recombination and CRISPR are basic technologies of synthetic biology aiming to design and construct artificial life. One key factor affecting the efficiency and accuracy of microbial genetic manipulations is the editing sequences (ES), namely the assisting sequences used for precisely locating and editing a target sequence in a genome, such as a primer, a homologous arm or a sgRNA sequence. For different genetic manipulation technologies, diverse manipulation types and multiple ESs are required for different stages of manipulation processes, especially for the high-throughput design with recently developed biofoundries to enable automatic strain modifications. Therefore, it is becoming essential to use computational tools for precise, fast, high-throughput and whole workflow ES design. This article reviews tools for ES design at different stages using various microbial genetic manipulation technologies. The tools are classified into four categories based on the types of ESs and their application scenario: primer design, DNA assembly design, sgRNA design and whole workflow ES design. We first give a brief introduction to the tools used for basic primer design with an emphasis on the widely used open-source tool Primer3. Then we have an extensive discussion on the tools used in the design of ESs for DNA assembly using technologies like Gibson and Golden Gate assembly which are required for linking the editing sequences and/or the inserted sequences together as one big fragment to be transformed into target strains. Various tools for designing sgRNA used in the latest CRISPR technologies for genome and base editing are also evaluated and compared in detail. Moreover, we argue that one-stop ES design tools which integrate various design methods to cover the whole genetic manipulation workflow would be very important in addressing challenges for the high throughput design raised by automatic strain construction biofoundries to enable highly precise and efficient genome editing for different sequence manipulations at any location and in any organism. These ES design tools will seamlessly link the genotype "Design" step and the strain "Build" step in the "design-build-test-learn (DBTL)" working cycle of synthetic biology to facilitate the creation of artificial organisms. {L-End}

    Mining, engineering and functional expansion of CRISPR/Cas systems
    Ke LIU, Guihong LIN, Kun LIU, Wei ZHOU, Fengqing WANG, Dongzhi WEI
    2023, 4(1):  47-66.  doi:10.12211/2096-8280.2021-022
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    The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) were derived from an acquired immune system in microbes. Since their functions on gene editing have been reported, they have been rapidly used to enhance our ability to edit, regulate, annotate, detect, and image DNA and RNA fragments of various organisms, which consequently have faciliated fundamental research in life science, medicine, bioengineering and so on, and drived the development of synthetic biology and other disciplines. However, CRISPR/Cas systems also have some inherent drawbacks, such as off-target effect, constraint of protospacer-adjacent motif (PAM) on the target, and controllability of the gene editing activity, which substantially compromise their applications in highly precise and controllable gene editing. In order to overcome these challenges, two important strategies have been employed to develop enhanced CRISPR/Cas systems and expand the CRISPR toolbox, including modifying the Cas proteins by protein engineering and mining novel CRISPR/Cas systems with bioinformatics. In the review, focusing on the most widely used the type II CRISPR/Cas systems, we mainly introduce the basic structures and functions of three representative systems, including CRISPR/Cas9, CRISPR/Cas12a and CRISPR/Cas13a, as well as recent progress in their structural modifications and functional expansion. Thereinto, engineering strategies for CRISPR/Cas systems have been systematically commented, which include modification methods for Cas proteins and the way to expand the function of CRISPR/Cas systems by coupling specfic proteins with Cas. In addition, we also review some novel CRISPR/Cas systems with important characteristics and potential applications that have been discovered in recently years, such as CRISPR/CasФ and CRISPR/Cas12k. These engineering modifications and mining work have greatly addressed the inherent problems of the CRISPR/Cas systems, and effectively expanded their functions and applicability, which will further promote the applications of the CRISPR/Cas systems in many fields. {L-End}

    Optimization and development of CRISPR/Cas9 systems for genome editing
    Xiaolong TENG, Shuobo SHI
    2023, 4(1):  67-85.  doi:10.12211/2096-8280.2022-047
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    As an emerging technology developed within recent years, CRISPR/Cas9 exhibits fast, efficient, and precise gene editing and regulation capabilities in various organisms and tissues, and these advantages make it widely used in research with fundamental sciences and applied technologies as well such as synthetic biology. This review first briefly introduces the discovery history, classification, and mechanism of CRISPR/Cas9. The system of CRISPR/Cas9 usually contains a single guide RNA (gRNA) molecule for targeting a specific sequence, and a Cas9 endonuclease for catalyzing a double-strand break (DSB) in the sequence (target DNA strands). The recognition and cleavage of target DNA strictly require the presence of a protospacer adjacent motif (PAM) in the target sequence. The DSB(s) can be repaired by various DNA repair mechanisms, which allow various gene editing such as gene integration, gene replacement, and gene knockout. Due to limitations of CRISPR/Cas9, such as PAM dependence and high off-target rate, researchers have developed various fused or engineered Cas9 proteins and gRNAs that play significant roles in fulfilling various purposes. These Cas9 variants are modified for improving the performance of PAM, in particular its specificity and fidelity. Moreover, the DSBs generated by Cas9 are considered toxic to the cells, and the use of Cas9 nickase (nCas9) or catalytically deficient Cas9 (dCas9) in CRISPR has also been developed without generating DSBs. Meanwhile, different effector proteins can be fused with Cas9/dCas9/nCas9 to bring about new functions and applications in gene expression regulation, epigenome editing, and single base editing. Moreover, we introduce the current studies and applications of multiple gRNA expression strategies based on the multiplex advantages of the CRISPR/Cas9 system. In general, CRISPR/Cas9 systems have gradually become standardized and revolutionized genome editing systems for almost all possible genetic manipulations. Finally, we highlight perspectives on several applications of the versatile CRISPR/Cas9 toolbox as a genome editing tool, and discuss the safety and risk control issues when it is used in gene therapy. {L-End}

    Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems
    Liya LIANG, Rongming LIU
    2023, 4(1):  86-101.  doi:10.12211/2096-8280.2022-040
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    With different types of nucleases, genome editing technologies have opened up the possibility for targeting and modifying specific gene sequences, which show potential applications in basic and applied aspects of biotechnology research. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are artificial proteins generated by fusing a specific DNA-binding domain with a restriction enzyme FokI DNA-cleavage domain, which arise from their ability to customize the DNA-binding domain for recognition of targeting sequences and cleaving them by the FokI domain. However, the design and construction of such a system are time consuming, laborious and costly. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) protein is a unique acquired immune system of bacteria or archaea. Since researchers constructed the CRISPR/Cas system for gene editing, its high efficiency has revolutionized a variety of fields such as life sciences, bioengineering, biomedicine, food, and agricultural sciences. However, the CRISPR/Cas system still has some challenges, such as off-target effect and limited PAM site recognition range, which limit its further applications. In order to solve these problems, molecular engineering of Cas proteins has become an important strategy for developing and optimizing CRISPR/Cas systems. In this study, with CRISPR/Cas9 and CRISPR/Cas12a selected as representative examples for DNA-targeting Class II CRISPR/Cas systems, we focus on the optimization and modification methods, and progress of Cas9 and Cas12a proteins achieved within recent years, such as Cas protein engineering for improved on-target specificity and expanded PAM scopes, developing new functions using CRISPR/Cas systems as gene targeting tools, and introducing exogenous protein domains to regulate CRISPR/Cas functions. These studies have generated a series of high-specificity and high-precision CRISPR/Cas systems, which have greatly expanded their functions and scopes, and made important contributions to the wide-range applications of CRISPR/Cas systems. {L-End}

    Advances in optogenetics for biomedical research
    Yuanhuan YU, Yang ZHOU, Xinyi WANG, Deqiang KONG, Haifeng YE
    2023, 4(1):  102-140.  doi:10.12211/2096-8280.2022-030
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    Synthetic biology enables rational design of regulatory molecules and circuits to reprogram cellular behaviors, and its applications to human cells could lead to powerful gene- and cell-based therapies, which are well recognized as central pillars of next-generation medicines. However, the safety of these therapies remains to be assessed, and controllability is a critical issue affecting their safety and limiting their clinical applications. In recent years, optogenetic technologies have been widely used in biomedical applications, which provides new insights for treating intractable diseases due to their distinguishing features of non-invasiveness, reversibility, and spatiotemporal resolution. Light is an ideal inducer to control gene expression, enabling precise and spatiotemporal manipulation of gene expression and cell behaviors by illuminating with light of appropriate intensity and wavelength as a triggering signal to achieve pinpoint spatiotemporal control of cellular activities. With the development of optogenetic toolkits, optogenetics has recently been developed for therapeutic applications. In this review, we summarize various optogenetic tools responsive to different wavelengths and their applications for precise treatment of neurological diseases, tumors, cardiovascular diseases, diabetes, enteric diseases as well as for the optogenetic control of gene transcription, gene editing, gene recombination and organelle movement. At the same time, we introduce recent research progress in portable bioelectronic medicine and artificial intelligence-assisted diagnosis and treatment systems, which are based on optogenetic techniques and the intelligent electronic devices. The rapid development of optogenetics has enormously extended the scope of traditional bioelectronic medicine, and the remote-controllability, reversibility, and negligible toxicity of optical control systems provide a solid foundation for the application of optogenetics in biomedicine. The success of these approaches would have an impact on precision medicine in the future practice. Finally, we also discuss the shortcomings of existing optogenetic tools and the challenges that would be faced in the future clinical applications as well as the prospects of their development. {L-End}

    Technologies for precise spatiotemporal control of post-transcriptional RNA metabolism
    Renmei LIU, Leshi LI, Xiaoyan YANG, Xianjun CHEN, Yi YANG
    2023, 4(1):  141-164.  doi:10.12211/2096-8280.2022-050
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    RNA exhibits complex dynamics and functions at specific times and locations inside cells, which include changes in their expression, degradation, translocation, splicing and other chemical modifications. The precise regulation of RNA metabolism is crucial for the studies of gene and RNA functions, the analysis of cellular activities, as well as the development of treatments for diseases. In order to deeply understand the temporal and spatial distribution and functional mechanism of RNA, scientists are always pursuing technologies that can precisely control the activity of RNA molecules in live cells. There are several gene editing- or transcriptional regulation-based methodologies that can regulate RNA synthesis in live cells. However, technologies for controlling the post-transcriptional metabolic behaviors of RNA are highly desirable, but they are less attained. Traditional methodologies for regulating RNA metabolism, e.g., regulatory RNA or RNA-binding proteins-based synthetic RNA effectors, suffer from low spatiotemporal resolution, making them difficult to dynamically regulate the post-transcriptional RNA metabolism in real time. Optogenetics has been used for precise spatiotemporal control of RNA metabolism in live cells due to its unique advantages of high spatiotemporal resolution and non-invasiveness. At present, photochemical modifications of nucleotides and genetically encoded photosensitive factors-based optogenetic tools have been applied for spatiotemporal control of various RNA metabolism at transcriptional or post-transcriptional levels, including transcription, translocation, translation and degradation. This article introduces recent progress in regulation of RNA metabolism, in particular the optogenetic control of post-transcriptional RNA metabolism, including technologies based on photochemical modified nucleotides, light-induced protein heterodimerization combined with RNA tethering, light-induced interactions between RNA-binding proteins and their cognate RNA motifs. Finally, we highlight prospects on technologies for precise spatiotemporal control of post-transcriptional RNA metabolism. {L-End}

    Advances and applications of droplet-based microfluidics in evolution and screening of engineered microbial strains
    Ran TU, Shixin LI, Haoni LI, Meng WANG
    2023, 4(1):  165-184.  doi:10.12211/2096-8280.2021-105
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    Microbial strains are perquisites for biomanufacting through microbial culture and fermentation. However, most strains usually need to be engineered to improve their performances for industrial applications. Therefore how to efficiently screen and isolate robust strains is a critical step of strain engineering. As an advanced high-throughput screening technology, droplet-based microfluidics developed with micro-chips can generate highly independent and uniform micro- or nano-liter droplets, in which single cells can be encapsulated, inoculated, detected, and analyzed for strain engineering. It is especially useful in the evolution of microbial strains for producing extracellular products. In this review, we first introduce the basic components of the droplet-based microfluidic system and the main steps involved in the strain screening. We then summarize key factors for the application of the droplet-based microfluidic technology in strain engineering, such as the signal sources of droplet detection, the difficulties of handling droplet screening, and the scopes of droplet sorting instruments. Based on the instruments used for the droplet sorting, we group the application cases into two types either via fluorescence-activated droplet sorting (FADS) using microfluidic equipment or via fluorescence-activated cell sorting (FACS) using flow cytometry instrument. While FADS using single-layer water-in-oil droplet can be further classified into cellular signature, fluorescent reporter protein, and substrate-based reaction according to the signal sources, FACS can be divided into double-layers water-in-oil-in-water (W/O/W) droplet or microgel droplet according to the droplet property. Finally, we outline challenges and prospects for the droplet microfluidic technology, and provide some guidelines for its applications in synthetic biology. Compared with traditional screening methods such as shaking flask or microplate with a throughput of hundreds to thousands of samples per day in milli- or micro-liter volume, the droplet-based microfluidic technology can achieve millions of samples per day in pico- or nano-liter volume, resulting in an increase of thousand-folds in screening speed and cost-saving for million-folds. By integrating with an automated station, the droplet-based microfluidic technology can be further improved for its screening efficiencies and application potentials in microbial synthetic biology. {L-End}

    Research progress in the construction of nucleic acid and protein biomolecular sensor arrays and their applications for rapid detection
    Weiwei NI, Lingjia ZHOU, Hao WANG, Fei LI, Jinsong HAN
    2023, 4(1):  185-203.  doi:10.12211/2096-8280.2022-035
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    The advent of sensor arrays is inspired by the superior performance of biological olfactory and taste systems on detection, recognition, tracking, and localization. By simulating the sensing mechanism of cross-response with the olfactory and taste systems, a sensor array can be constructed. Meanwhile, a series of algorithms are used to detect and distinguish targets based on fingerprints generated by the analyte to the array. However, the fabrication of sensing elements still faces many challenges, such as difficult design, complicated synthesis, and low signal collecting efficiency. Nucleic acids and proteins have advantages in biocompatibility, flexible programmability, easy functionalization, and superior molecular recognition properties. At present, a variety of sensor arrays based on nucleic acid and protein sensing elements have been constructed through rational design and controllable preparation methods. In this review, the multi-target detection applications of these sensor arrays are highlighted, and their application prospects and challenges are addressed. {L-End}

    Advances with applications of Raman spectroscopy in single-cell phenotype sorting and analysis
    Xixian WANG, Qing SUN, Zhidian DIAO, Jian XU, Bo MA
    2023, 4(1):  204-224.  doi:10.12211/2096-8280.2022-043
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    In synthetic biology, methodological innovations in sequencing, editing and synthesis of genes and whole genomes have resulted in unprecedented development in "design and manufacturing of genotypes". On the other hand, "testing of cellular phenotypes and functions" has increasingly become one of major bottlenecks. Single-cell technologies have tremendous impacts and potentials in rapid testing of cellular phenotypes and functions. However, such single-cell methods should allow non-invasive live-cell probing, be label-free, provide landscape-like phenotype sorting, distinguish complex functions, operate with high speed, sufficient throughput and low-cost, and finally, be able to integrate with downstream omics analysis. Raman spectroscopy has all the above features, and can provide information on the chemical composition and molecular structure of single cells, making it an efficient single-cell phenotyping technology. In this review, we first introduce the concept of Ramanome and Ramanome-based phenotyping technologies, including detecting and quantifying products, measuring profiles of substrates and metabolites, discriminating cell types or states, and characterizing stress response and modeling environmental changes. We then summarize the development of existing Raman-activated cell sorting (RACS) platforms in phenotyping and sorting of cell factories such as including spontaneous Raman, resonance Raman, and coherent Raman, the modes for acquiring Raman signals including static modes on dry slice and in liquid as well, flow modes by trap-free and trap-and-release manners, and principles for target cells sorting including Ejection by pulsed laser, dragging by optical tweezer, and sorting by microfluidics operation and droplets. We also highlight the applications of different RACS platforms, including the sorting of carotenoid-producing yeast and cyanobacteria cells, astaxanthin (AXT)-hyperproducing microalgae cells, triacylglycerol (TAG)-producing yeast cells, etc. Finally, challenges with single cell Raman spectroscopy (SCRS) in the phenotyping and sorting of synthetic cells and their perspectives are outlined and discussed. We propose that SCRS will bridge phenotypes and genotypes in science and technologies through coupling with downstream high-throughput cell sorting and omics profiling. This bridge will lead to novel and creative solutions to high-throughput, landscape-like testing and screening of synthetic cells. Moreover, it will fulfill the promise of Raman spectroscopy-enabled single-cell "phenome-genome" as a new type of biological big-data, and accelerate the pace of "data-driven" synthetic biology. {L-End}

    Research Article
    Mutator-driven continuous genome evolution of Saccharomyces cerevisiae
    Yingjia PAN, Siyang XIA, Chang DONG, Jin CAI, Jiazhang LIAN
    2023, 4(1):  225-240.  doi:10.12211/2096-8280.2022-051
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    Saccharomyces cerevisiae, one of the most commonly used cell factories for industrial biotechnology, is widely employed for mass production of bio-based chemicals and value-added compounds. Due to complicated cellular metabolism and regulatory network of biological systems, genome evolution is generally required for engineering with complicated phenotypes, which are coordinated and regulated by multiple genes. To achieve rapid evolution of S. cerevisiae genome, this study employed Clustered Regularly Interspaced Short Palindromic Repeats Interference (CRISPRi) to regulate the expression of genes closely related to chromosome replication and maintenance, such as MSH2, TSA1, RAD27, and CLB5, known as mutator genes. By designing guide RNAs (gRNAs) with differential repression efficiency, four mutators: MSH2 mutator mainly for point mutations and small InDels (MM), TAS1 mutator mainly for point mutations, small InDels, and structural variants (TM), RAD27 mutator mainly for small InDels and structural variants (RM), and CLB5 mutator mainly for structural variants and aneuploidy chromosomes (CM) were constructed to control genome mutation rates and types (e.g. point mutation, small InDels, structural variants, and aneuploid chromosomes). These mutators were used for continuous evolution of a series of industrially relevant phenotypes, such as isobutanol tolerance, xylose utilization, and β-carotene biosynthesis. Interestingly, TM was more efficient for evolving isobutanol tolerance, but TM and CM were preferred for evolving β-carotene overproduction, and all mutators were verified to have comparable performance for evolving xylose utilization with yeast strains. We also discovered that the effectiveness of mutators was dependent on the phenotype to be evolved. To address challenges in the evolution of phenotypes without pre-determined knowledge on mutation rates and types, a mixed mutator (MTRC) was constructed to rapidly evaluate the mutator-phenotype relationship. Finally, a combinatorial mutator (MTRC*2) were constructed to explore synergistic interactions among various mutators for the continuous genome evolution of S. cerevisiae. The established mutators can not only be used for constructing robust yeast cell factories, but also be further developed as a generally applicable genome evolution tool.