Genome engineering using the CRISPR/Cas system

Recently, an epoch-making genome engineering technology using clustered regularly at interspaced short palindromic repeats(CRISPR) and CRISPR associated(Cas) nucleases, was developed. Previous technologies for genome manipulation require the time-consuming design and construction of genome-engineered nucleases for each target and have, therefore, not been widely used in mouse research where standard techniques based on homologous recombination are commonly used. The CRISPR/Cas system only requires the design of sequences complementary to a target locus, making this technology fast and straightforward. In addition, CRISPR/Cas can be used to generate mice carrying mutations in multiple genes in a single step, an achievement not possible using other methods. Here, we review the uses of this technology in genetic analysis and manipulation, including achievements made possible to date and the prospects for future therapeutic applications.

Research on Copyright Protection Method of Material Genome Engineering Data Based on Zero-Watermarking

In order to effectively solve the problem of copyright protection of materials genome engineering data,this paper proposes a method for copyright protection of materials genome engineering data based on zero-watermarking technology.First,the important attribute values are selected from the materials genome engineering database;then,use the method of remainder to group the selected attribute values and extract eigenvalues;then,the eigenvalues sequence is obtained by the majority election method;finally,XOR the sequence with the actual copyright information to obtain the watermarking information and store it in the third-party authentication center.When a copyright dispute requires copyright authentication for the database to be detected.First,the zero-watermarking construction algorithm is used to obtain an eigenvalues sequence;then,this sequence is XORed with the watermarking information stored in the third-party authentication center to obtain copyright information to-be-detected.Finally,the ownership is determined by calculating the similarity between copyright information to-be-detected and copyright information that has practical significance.The experimental result shows that the zero-watermarking method proposed in this paper can effectively resist various common attacks,and can well achieve the copyright protection of material genome engineering database.

Genome engineering of the human gut microbiome

The human gut microbiome,a complex ecosystem,significantly influences host health,impacting crucial aspects such as metabolism and immunity.To enhance our comprehension and control of the molecular mechanisms orchestrating the intricate interplay between gut commensal bacteria and human health,the exploration of genome engineering for gut microbes is a promising frontier.Nevertheless,the complexities and diversities inherent in the gut microbiome pose substantial challenges to the development of effective genome engineering tools for human gut microbes.In this comprehensive review,we provide an overview of the current progress and challenges in genome engineering of human gut commensal bacteria,whether executed in vitro or in situ.A specific focus is directed towards the advancements and prospects in cargo DNA delivery and high-throughput techniques.Additionally,we elucidate the immense potential of genome engineering methods to enhance our understanding of the human gut microbiome and engineer the microorganisms to enhance human health.

Toward precise CRISPR DNA fragment editing and predictable 3D genome engineering

Ever since gene targeting or specific modification of genome sequences in mice was achieved in the early 1980s,the reverse genetic approach of precise editing of any genomic locus has greatly accelerated biomedical research and biotechnology development.In particular,the recent development of the CRISPR/Cas9 system has greatly expedited genetic dissection of 3D genomes.CRISPR gene-editing outcomes result from targeted genome cleavage by ectopic bacterial Cas9 nuclease followed by presumed random ligations via the host double-strand break repair machineries.Recent studies revealed,however,that the CRISPR genomeediting system is precise and predictable because of cohesive Cas9 cleavage of targeting DNA.Here,we synthesize the current understanding of CRISPR DNA fragment-editing mechanisms and recent progress in predictable outcomes from precise genetic engineering of 3D genomes.Specifically,we first briefly describe historical genetic studies leading to CRISPR and 3D genome engineering.We then summarize different types of chromosomal rearrangements by DNA fragment editing.Finally,we review significant progress from precise ID gene editing toward predictable 3D genome engineering and synthetic biology.The exciting and rapid advances in this emerging field provide new opportunities and challenges to understand or digest 3D genomes.

TALENs:Customizable Molecular DNA Scissors for Genome Engineering of Plants

Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases（TALENs）,consisting of an engineered specific（TALE） DNA binding domain and a Fok I cleavage domain,are newly developed versatile reagents for genome engineering in different organisms.Because of the simplicity of the DNA recognition code and their modular assembly,TALENs can act as customizable molecular DNA scissors inducing double-strand breaks（DSBs） at given genomic location.Thus,they provide a valuable approach to targeted genome modifications such as mutations, insertions,replacements or chromosome rearrangements.In this article,we review the development of TALENs,and summarize the principles and tools for TALEN-mediated gene targeting in plant cells,as well as current and potential strategies for use in plant research and crop improvement.

From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas

In the last years,tremendous progress has been achieved in the field of gene editing in plants.By the induction of single site-specific double-strand breaks(DSBs),the knockout of genes by non-homologous end joining has become routine in many plant species.Recently,the efficiency of inducing pre-planned mutations by homologous recombination has also been improved considerably.However,very little effort has been undertaken until now to achieve more complex changes in plant genomes by the simultaneous induction of several DSBs.Several reports have been published on the efficient induction of deletions.However,the induction of intrachromosomal inversions and interchromosomal recombination by the use of CRISPR/Cas has only recently been reported.In this review,we want to sum up these results and put them into context with regards to what is known about natural chromosome rearrangements in plants.Moreover,we review the recent progress in CRISPR/Cas-based mammalian chromosomal rearrangements,which might be inspiring for plant biologists.In the long run,the controlled restructuring of plant genomes should enable us to link or break linkage of traits at will,thus defining a new area of plant breeding.

Cascade-Cas3 facilitates high-accuracy genome engineering in Pseudomona s using phage-encoded homologous recombination

Phage-encoded homologous recombination(PEHR)is an efficient tool for bacterial genome editing.We previously developed and utilized a Pseudomonas-specific PEHR system.However,when using the PEHR system for Pseu-domonas genome editing,false positives can be a problem.In this study,we combined a compact Cascade-Cas3 system from P.aeruginosa(PaeCas3c)with a Pseudomonas-specific PEHR system,and the results of our recom-bineering assay showed that this compact Cascade-Cas3 system can significantly improve PEHR recombineering accuracy.

Materials genome engineering accelerates the research and development of organic and perovskite photovoltaics

The emerging photovoltaic(PV)technologies,such as organic and perovskite PVs,have the characteristics of complex compositions and processing,resulting in a large multidimensional parameter space for the development and optimization of the technologies.Traditional manual methods are time-consuming and laborintensive in screening and optimizing material properties.Materials genome engineering(MGE)advances an innovative approach that combines efficient experimentation,big database and artificial intelligence(AI)algorithms to accelerate materials research and development.High-throughput(HT)research platforms perform multidimensional experimental tasks rapidly,providing a large amount of reliable and consistent data for the creation of materials databases.Therefore,the development of novel experimental methods combining HT and AI can accelerate materials design and application,which is beneficial for establishing material-processing-property relationships and overcoming bottlenecks in the development of emerging PV technologies.This review introduces the key technologies involved in MGE and overviews the accelerating role of MGE in the field of organic and perovskite PVs.

A double-edged sword: CRISPR-Cas9 is emerging as a revolutionary technique for genome editing

In May 2015, professor Xiao Yang authored a review on the development of CRISPR-Cas9 techniques in the journal of Military Medical Research. This review provided a valuable overview of this major scientific advance. It has been four years since the first publication of the CRISPR-Cas9 breakthrough. The use of this technique has expanded into various scientific areas and is being developed into a systematic technical platform that may contribute to many bioengineering fields involving DNA sequence editing.

A Comparative Analysis of CRISPR Screening Technologies

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.

Exploiting viral vectors to deliver genome editing reagents in plants

Genome editing holds great promise for the molecular breeding of plants,yet its application is hindered by the shortage of simple and effective means of delivering genome editing reagents into plants.Conventional plant transformation-based methods for delivery of genome editing reagents into plants often involve prolonged tissue culture,a labor-intensive and technically challenging process for many elite crop cultivars.In this review,we describe various virus-based methods that have been employed to deliver genome editing reagents,including components of the CRISPR/Cas machinery and donor DNA for precision editing in plants.We update the progress in these methods with recent successful examples of genome editing achieved through virus-based delivery in different plant species,highlight the advantages and limitations of these delivery approaches,and discuss the remaining challenges.

Advancement of chimeric antigen receptor-natural killer cells targeting hepatocellular carcinoma

With the advance of genome engineering technology,chimeric antigen receptors(CARs)-based immunotherapy has become an emerging therapeutic strategy for tumors.Although initially designed for T cells in tumor immunotherapy,CARs have been exploited to modify the function of natural killer(NK)cells against a variety of tumors,including hepatocellular carcinoma(HCC).CAR-NK cells have the potential to sufficiently kill tumor antigen-expressing HCC cells,independent of major histocompatibility complex matching or prior priming.In this review,we summarize the recent advances in genetic engineering of CAR-NK cells against HCC and discuss the current challenges and prospects of CAR-NK cells as a revolutionary cellular immunotherapy against HCC.

Genes and genetics in eye diseases: a genomic medicine approach for investigating hereditary and inflammatory ocular disorders

Past 25 y have witnessed an exponential increase in knowledge and understanding of ocular diseases and their respective genetic underpinnings. As a result, scientists have mapped many genes and their variants that can influence vision and health of our eyes. Based on these findings, it is becoming clear that an early diagnosis employing genetic testing can help evaluate patients＇ conditions for instituting treatment plan（s） and follow-up care to avoid vision complications later. For example, knowing family history becomes crucial for inherited eye diseases as it can benefit members in family who may have similar eye diseases or predispositions. Therefore, gathering information from an elaborate examination along with complete assessment of past medical illness by ophthalmologists followed by consultation with geneticists can help create a roadmap for making diagnosis and treatment precise and beneficial. In this review, we present an update on ocular genomic medicine that we believe has tremendous potential towards unraveling genetic implications in ocular diseases and patients＇ susceptibilities. We also discuss translational aspects of genetic ophthalmology and genome engineering that may help advance molecular diagnostics and therapeutics.

Therapeutic gene editing strategies using CRISPR-Cas9 for theβ-hemoglobinopathies

With advancements in gene editing technologies,our ability to make precise and efficient modifications to the genome is increasing at a remarkable rate,paving the way for scientists and clinicians to uniquely treat a multitude of previously irremediable diseases.CRISPR-Cas9,short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9,is a gene editing platform with the ability to alter the nucleotide sequence of the genome in living cells.This technology is increasing the number and pace at which new gene editing treatments for genetic disorders are moving toward the clinic.Theβ-hemoglobinopathies are a group of monogenic diseases,which despite their high prevalence and chronic debilitating nature,continue to have few therapeutic options available.In this review,we will discuss our existing comprehension of the genetics and current state of treatment forβ-hemoglobinopathies,consider potential genome editing therapeutic strategies,and provide an overview of the current state of clinical trials using CRISPR-Cas9 gene editing.

A Prediction Method of Fracture Toughness of Nickel-Based Superalloys

Fracture toughness plays a vital role in damage tolerance design of materials and assessment of structural integrity.To solve these problems of com-plexity,time-consuming,and low accuracy in obtaining the fracture toughness value of nickel-based superalloys through experiments.A combination prediction model is proposed based on the principle of materials genome engineering,the fracture toughness values of nickel-based superalloys at different temperatures,and different compositions can be predicted based on the existing experimental data.First,to solve the problem of insufficient feature extraction based on manual experience,the Deep Belief Network(DBN)is used to extract features,and an attention mechanism module is introduced.To achieve the purpose of strengthen-ing the important features,an attention weight is assigned to each feature accord-ing to the importance of the feature.Then,the feature vectors obtained by the DBN module based on the Attention mechanism(A-DBN)are spliced with the original features.Thus,the prediction accuracy of the model is improved by extracting high-order combined features and low-order linear features between input and output data.Finally,the spliced feature vectors are put into the Support Vector Regression(SVR)model to further improve the regression prediction abil-ity of the model.The results of the contrast experiment show that the model can effectively improve the prediction accuracy of the fracture toughness value of nickel-based superalloys.

CRISPR/Cas9 and Genome Editing in Drosophila

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.

Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system(CRISPR/Cas)has recently become the most powerful tool available for genome engineering in various organisms.With efficient and proper expression of multiple guide RNAs(gRNAs),the CRISPR/Cas system is particularly suitable for multiplex genome editing.During the past several years,different CRISPR/Cas expression strategies,such as two-component transcriptional unit,single transcriptional unit,and bidirectional promoter systems,have been developed to efficiently express gRNAs as well as Cas nucleases.Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes,endogenous RNases,and exogenous endoribonuclease(Csy4).Besides being constitutively and ubiquitously expressed,inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible,tissue-specific,and/or synthetic promoters for specific research purposes.Most recently,the emergence of CRISPR/Cas ribonucleoprotein delivery methods,such as engineered nanoparticles,further revolutionized transgene-free and multiplex genome editing.In this review,we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.

Study the lipidoid nanoparticle mediated genome editing protein delivery using 3D intestinal tissue model

Lipid nanoparticles are promising carriers for oral drug delivery.For bioactive cargos with intracellular targets,e.g.gene-editing proteins,it is essential for the cargo and carrier to remain complexed after crossing the epithelial layer of intestine in order for the delivery system to transport the cargos inside targeted cells.However,limited studies have been conducted to verify the integrity of cargo/carrier nanocomplexes and their capability in facilitating cargo delivery intracellularly after the nanocomplex crossing the epithelial barrier.Herein,we used a traditional 2D transwell system and a recently developed 3D tissue engineered intestine model and demonstrated the synthetic lipid nanoparticle(carrier)and protein(cargo)nanocomplexes are able to cross the epithelial layer and deliver the protein cargo inside the underneath cells.We found that the EC16-63 LNP efficiently encapsulated the GFP-Cre recombinase,penetrated the intestinal monolayer cells in both the 2D cell culture and 3D tissue models through temporarily interrupting the tight junctions between epithelial layer.After transporting across the intestinal epithelia,the EC16-63 and GFP-Cre recombinase nanocomplexes can enter the underneath cells to induce gene recombination.These results suggest that the in vitro 3D intestinal tissue model is useful for identifying effective lipid nanoparticles for potential oral drug delivery.

Genome reprogramming for synthetic biology

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.

The emerging role of recombineering in microbiology

Recombineering is a valuable technique for generating recombinant DNA in vivo,primarily in bacterial cells,and is based on homologous recombination using phage-encoded homologous recombinases,such as Red βγ from the lambda phage and RecET from the Rac prophage.The recombineering technique can efficiently mediate homol-ogous recombination using short homologous arms(∼50 bp)and is unlimited by the size of the DNA molecules or positions of restriction sites.In this review,we summarize characteristics of recombinases,mechanism of recombineering,and advances in recombineering for DNA manipulation in Escherichia coli and other bacteria.Furthermore,the broad applications of recombineering for mining new bioactive microbial natural products,and for viral mutagenesis,phage genome engineering,and understanding bacterial metabolism are also reviewed.