Development of a tau-V337M mouse model using CRISPR/Cas9 system and enhanced ssODN-mediated recombination / 生物工程学报

The generation of a tau-V337M point mutation mouse model using gene editing technology can provide an animal model with fast disease progression and more severe symptoms, which facilitate the study of pathogenesis and treatment of Alzheimer's disease (AD). In this study, single guide RNAs (sgRNA) and single-stranded oligonucleotides (ssODN) were designed and synthesized in vitro. The mixture of sgRNA, Cas9 protein and ssODN was microinjected into the zygotes of C57BL/6J mice. After DNA cutting and recombination, the site homologous to human 337 valine (GTG) in exon 11 was mutated into methionine (ATG). In order to improve the efficiency of recombination, a Rad51 protein was added. The female mice mated with the nonvasectomy male mice were used as the surrogates. Subsequently, the 2-cell stage gene edited embryos were transferred into the unilateral oviduct, and the F0 tau-V337M mutation mice were obtained. Higher mutation efficiency could be obtained by adding Rad51 protein. The F0 tau-V337M point mutation mice can pass the mutation on to the F1 generation mice. In conclusion, this study successfully established the first tau-V337M mutation mouse by using Cas9, ssODN and Rad51. These results provide a new method for developing AD mice model which can be used in further research on the pathogenesis and treatment of AD.

Precision gene editing technologies based on CRISPR/Cas9: a review / 生物工程学报

Gene editing technology is a genetic operation technology that can modify the DNA sequence at the genomic level. The precision gene editing technology based on CRISPR/Cas9 system is a gene editing technology that is easy to operate and widely used. Unlike the traditional CRISPR/Cas9 system, the precision gene editing technology can perform site-directed mutation of genes without DNA template. This review summarizes the recent development of precision gene editing technology based on CRISPR/Cas9, and prospects the challenges and opportunities of this technology.

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.

Advances in the RNA-targeting CRISPR-Cas systems / 生物工程学报

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) system is an adaptive immune system of bacteria and archaea against phages, plasmids and other exogenous genetic materials. The system uses a special RNA (CRISPR RNA, crRNA) guided endonuclease to cut the exogenous genetic materials complementary to crRNA, thus blocking the infection of exogenous nucleic acid. According to the composition of the effector complex, CRISPR-Cas system can be divided into two categories: class 1 (including type Ⅰ, Ⅳ, and Ⅲ) and class 2 (including type Ⅱ, Ⅴ, and Ⅵ). Several CRISPR-Cas systems have been found to have very strong ability to specifically target RNA editing, such as type Ⅵ CRISPR-Cas13 system and type Ⅲ CRISPR-Cas7-11 system. Recently, several systems have been widely used in the field of RNA editing, making them a powerful tool for gene editing. Understanding the composition, structure, molecular mechanism and potential application of RNA-targeting CRISPR-Cas systems will facilitate the mechanistic research of this system and provide new ideas for developing gene editing tools.

Application of CRISPR-Cas9 gene editing technology in crop breeding / 生物工程学报

The CRISPR-Cas9 system is composed of a clustered regularly interspaced short palindromic repeat (CRISPR) and its associated proteins, which are widely present in bacteria and archaea, serving as a specific immune protection against viral and phage secondary infections. CRISPR-Cas9 technology is the third generation of targeted genome editing technologies following zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). The CRISPR-Cas9 technology is now widely used in various fields. Firstly, this article introduces the generation, working mechanism and advantages of CRISPR-Cas9 technology; secondly, it reviews the applications of CRISPR-Cas9 technology in gene knockout, gene knock-in, gene regulation and genome in breeding and domestication of important food crops such as rice, wheat, maize, soybean and potato. Finally, the article summarizes the current problems and challenges encountered by CRISPR-Cas9 technology and prospects future development and application of CRISPR-Cas9 technology.

CRISPR-based molecular diagnostics: a review / 生物工程学报

Rapid and accurate detection technologies are crucial for disease prevention and control. In particular, the COVID-19 pandemic has posed a great threat to our society, highlighting the importance of rapid and highly sensitive detection techniques. In recent years, CRISPR/Cas-based gene editing technique has brought revolutionary advances in biotechnology. Due to its fast, accurate, sensitive, and cost-effective characteristics, the CRISPR-based nucleic acid detection technology is revolutionizing molecular diagnosis. CRISPR-based diagnostics has been applied in many fields, such as detection of infectious diseases, genetic diseases, cancer mutation, and food safety. This review summarized the advances in CRISPR-based nucleic acid detection systems and its applications. Perspectives on intelligent diagnostics with CRISPR-based nucleic acid detection and artificial intelligence were also provided.

Principle and development of single base editing technology and its application in livestock breeding / 生物工程学报

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) is widely used in the field of livestock breeding. However, its low efficiency, untargeted cutting and low safety have greatly hampered its use for introducing single base mutations in livestock breeding. Single base editing, as a new gene editing tool, can directly replace bases without introducing double strand breaks. Single base editing shows high efficiency and strong specificity, and provides a simpler and more effective method for precise gene modification in livestock breeding. This paper introduces the principle and development of single base editing technology and its application in livestock breeding.

An efficient marker-free genome editing method for Aspergillus niger / 生物工程学报

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.

Recent advances in CRISPR-related transposable elements / 生物工程学报

A new wave of research has been inspired by the CRISPR-Cas system with respect to their application in genome editing. The CRISPR-Cas system can not only be applied in gene knockout and insertion, but also be used in base editing, transcriptional regulation and recombination of gene clusters. However, the low efficiency of homology-directed repair (HDR) limits its application. Unlike the CRISPR-Cas system, mobile genetic elements (MGE) can insert DNA fragments into cell chromosomes without the aid of HDR. Recently, it is reported that CRISPR-related transposable elements can guide targeted DNA insertion. Their transposition mechanisms and reprogramming abilities have brought novel opportunities to the development of this field. This review summarized the research progress and application development of natural CRISPR-related transposable elements in recent years, as well as the applications of fused dCas9-transposase. It proposed the application prospects and potential challenges of CRISPR-related transposable elements in the future, which provided a reference for the development direction of gene editing tools.

CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice / 亚洲男科学杂志(英文版)

Gene expression analyses suggest that more than 1000-2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.

Propagation and phenotypic analysis of mutant rabbits with MSTN homozygous mutation / 生物工程学报

Myostatin gene (MSTN) encodes a negative regulator for controlling skeletal muscle growth in animals. In this study, MSTN-/- homozygous mutants with "double muscle" phenotypic traits and stable inheritance were bred on the basis of MSTN gene editing rabbits, with the aim to establish a method for breeding homozygous progeny from primary MSTN biallelic mutant rabbits. MSTN-/- primary mutant rabbits were generated by CRISPR/Cas9 gene editing technology. The primary mutant rabbits were mated with wild type rabbits to produce F1 rabbits, whereas the F2 generation homozygous rabbits were bred by half-sibling mating or backcrossing with F1 generation rabbits of the same mutant strain. Sequence analysis of PCR products and its T vector cloning were used to screen homozygous rabbits. The MSTN mutant rabbits with 14-19 week-old were weighed and the difference of gluteus maximus tissue sections and muscle fiber cross-sectional area were calculated and analyzed. Five primary rabbits with MSTN gene mutation were obtained, among which three were used for homozygous breeding. A total of 15 homozygous rabbits (5 types of mutants) were obtained (M2-a: 3; M2-b: 2; M3-a: 2; M7-a: 6; M7-b: 2). The body weight of MSTN-/- homozygous mutant rabbits aged 14-19 weeks were significantly higher than that of MSTN+/+ wild-type rabbits of the same age ((2 718±120) g vs. (1 969±53) g, P < 0.01, a 38.0% increase). The mean cross sections of gluteus maximus muscle fiber in homozygous mutant rabbits were not only significantly higher than that of wild type rabbits ((3 512.2±439.2) μm2 vs. (1 274.8±327.3) μm2, P < 0.01), but also significantly higher than that of MSTN+/- hemizygous rabbits ((3 512.2±439.2) μm2 vs. (2 610.4±604.4) μm2, P < 0.05). In summary, five homozygous mutants rabbits of MSTN-/- gene were successfully bred, which showed a clear lean phenotype. The results showed that the primary breeds were non-chimeric mutant rabbits, and the mutant traits could be inherited from the offspring. MSTN-/- homozygous mutant rabbits of F2 generation could be obtained from F1 hemizygous rabbits by inbreeding or backcrossing. The progenies of the primary biallelic mutant rabbits were separated into two single-allelic mutants, both of which showed a "double-muscle" phenotype. Thus, this study has made progress in breeding high-quality livestock breeds with gene editing technology.

Thermostable CRISPR/Cas9 genome editing system and its application in construction of cell factories with thermophilic bacteria: a review / 生物工程学报

The diverse thermophilic strains of Thermoanaerobacter, serving as unique platforms with a broad range of application in biofuels and chemicals, have received wide attention from scholars and practitioners. Although biochemical experiments and genome sequences have been reported for a variety of Thermoanaerobacter strains, an efficient genetic manipulation system remains to be established for revealing the biosynthetic pathways of Thermoanaerobacter. In line with this demand, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems for editing, regulating and targeting genomes have been well developed in thermophiles. Here, we reviewed and discussed the current status, associated challenges, and future perspectives of the construction of thermostable CRISPR/Cas9 genome editing systems for some representative Thermoanaerobacter species. The establishment, optimization, and application of thermostable CRISPR/Cas genome editing systems would potentially provide a foundation for further genetic modification of thermophilic bacteria.

Application of gene editing technology in Escherichia coli / 生物工程学报

Gene editing technology can be used to modify the genome of Escherichia coli for the investigation of gene functions, or to change the metabolic pathways for the efficient production of high-value products in engineered strains with genetic stability. A variety of gene editing technologies have been applied in prokaryotes, such as λ-Red homologous recombination and CRISPR/Cas9. As a traditional gene editing technique, λ-Red recombination is widely used. However, it has a few shortcomings, such as the limited integration efficiency by the integrated fragment size, the cumbersome gene editing process, and the FRT scar in the genome after recombination. CRISPR/Cas9 is widely used for genome editing at specific sites, which requires specific DNA segments according to the editing site. As the understanding of the two technologies deepens, a variety of composite gene editing techniques have been developed, such as the application of λ-Red homologous recombination in combination with homing endonucleaseⅠ-SceⅠ or CRISPR/Cas9. In this review, we summarized the basic principles of common gene editing techniques and composite gene editing techniques, as well as their applications in Escherichia coli, which can provide a basis for the selection of gene editing methods in prokaryotes.

Construction of a stable TrxR1 knockout HCT-116 cell line using CRISPR/Cas9 gene editing system / 生物工程学报

To investigate the cellular target selectivity of small molecules targeting thioredoxin reductase 1, we reported the construction and functional research of a stable TrxR1 gene (encode thioredoxin reductase 1) knockout HCT-116 cell line. We designed and selected TrxR1 knockout sites according to the TrxR1 gene sequence and CRISPR/Cas9 target designing principles. SgRNA oligos based on the selected TrxR1 knockout sites were obtained. Next, we constructed knockout plasmid by cloning the sgRNA into the pCasCMV-Puro-U6 vector. After transfection of the plasmid into HCT-116 cells, TrxR1 knockout HCT-116 cells were selected using puromycin resistance. The TrxR1 knockout efficiency was identified and verified by DNA sequencing, immunoblotting, TRFS-green fluorescent probe, and cellular TrxR1 enzyme activity detection. Finally, the correlation between TrxR1 expression and cellular effects of drugs specifically targeting TrxR1 was investigated by CCK-8 assay. The results demonstrated that the knockout plasmid expressing the sgRNA effectively knocked-out TrxR1 gene within HCT-116 cells, and no expression of TrxR1 protein could be observed in stable TrxR1 knockout HCT-116 (HCT116-TrxR1-KO) cells. The TrxR1-targeting inhibitor auranofin did not show any inhibitory activity against either cellular TrxR1 enzyme activity or cell proliferation. Based on these results, we conclude that a stable TrxR1 gene knockout HCT-116 cell line was obtained through CRISPR/Cas9 techniques, which may facilitate investigating the role of TrxR1 in various diseases.

Optimization of CRISPR/Cas9-based multiplex base editing in Corynebacterium glutamicum / 生物工程学报

As a new CRISPR/Cas-derived genome engineering technology, base editing combines the target specificity of CRISPR/Cas and the catalytic activity of nucleobase deaminase to install point mutations at target loci without generating DSBs, requiring exogenous template, or depending on homologous recombination. Recently, researchers have developed a variety of base editing tools in the important industrial strain Corynebacterium glutamicum, and achieved simultaneous editing of two and three genes. However, the multiplex base editing based on CRISPR/Cas9 is still limited by the complexity of multiple sgRNAs, interference of repeated sequence and difficulty of target loci replacement. In this study, multiplex base editing in C. glutamicum was optimized by the following strategies. Firstly, the multiple sgRNA expression cassettes based on individual promoters/terminators was optimized. The target loci can be introduced and replaced rapidly by using a template plasmid and Golden Gate method, which also avoids the interference of repeated sequence. Although the multiple sgRNAs structure is still complicated, the editing efficiency of this strategy is the highest. Then, the multiple gRNA expression cassettes based on Type Ⅱ CRISPR crRNA arrays and tRNA processing were developed. The two strategies only require one single promoter and terminator, and greatly simplify the structure of the expression cassette. Although the editing efficiency has decreased, both methods are still applicable. Taken together, this study provides a powerful addition to the genome editing toolbox of C. glutamicum and facilitates genetic modification of this strain.

A CRISPR/dCpf1-based transcriptional repression system for Gluconobacter oxydans / 生物工程学报

Gluconobacter oxydans are widely used in industrial due to its ability of oxidizing carbohydrate rapidly. However, the limited gene manipulation methods and less of efficient gene editing tools impose restrictions on its application in industrial production. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been widely used in genome editing and transcriptional regulation which improves the efficiency of genome editing greatly. Here we constructed a CRISPR/dCpf1-mediated gene transcriptional repression system, the expression of a nuclease inactivation Cpf1 protein (dCpf1) in Gluconobacter oxydans together with a 19 nt direct repeats showed effective repression in gene transcription. This system in single gene repression had strong effect and the relative repression level had been increased to 97.9%. While it could be applied in multiplex gene repression which showed strong repression ability at the same time. Furthermore, this system was used in the metabolic pathway of L-sorbose and the regulatory of respiratory chain. The development of CRISPR transcriptional repression system effectively covered the shortage of current gene regulation methods in G. oxydans and provided an efficient gene manipulation tool for metabolic engineering modification in G. oxydans.

Plant prime editing technique: a new genome editing tool for plants / 生物工程学报

The CRISPR/Cas9 based prime editing (PE) technique enables all 12 types of base substitutions and precise small DNA deletions or insertions without generating DNA double-strand breaks. Prime editing has been successfully applied in plants and plays important roles in plant precision breeding. Although plant prime editing (PPE) can substantially expand the scope and capabilities of precise genome editing in plants, its editing efficiency still needs to be further improved. Here, we review the development of PPE technique, and introduce structural composition, advantages and limitations of PPE. Strategies to improve the PPE editing efficiency, including the Tm-directed PBS length design, the RT template length, the dual-pegRNA strategy, the PlantPegDesigner website, and the strategies for optimizing the target proteins of PPE, were highlighted. Finally, the prospects of future development and application of PPE were discussed.

Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing

Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.

CRISPR-Cas, el editor de genes / CRISPR, gene editor

El término CRISPR, por su acrónimo en inglés refiere a Clustered Regularly Interspaced Short Palindromic Repeats, es decir, repeticiones palindrómicas cortas, agrupadas y regularmente esparcidas, por sus características en el genoma, pertenece naturalmente al sistema de defensa de bacterias y arqueas. Este ha sido adaptado biotecnológicamente para la edición del ADN de células eucariotas, incluso de células humanas. El sistema CRISPR-Cas para editar genes consta, en forma generalizada, de dos componentes: una proteína nucleasa (Cas) y un ARN guía (sgRNA). La simplicidad del complejo lo hace una herramienta molecular reprogramable capaz de ser dirigida y de editar cualquier sitio en un genoma conocido. Su principal foco son las terapias para enfermedades hereditarias monogénicas y para el cáncer. Sin embargo, además de editor de genes, la tecnología CRISPR se utiliza para edición epigenética, regulación de la expresión génica y método de diagnóstico molecular. Este artículo tiene por objetivo presentar una revisión de las aplicaciones de la herramienta molecular CRISPR-Cas, particularmente en el campo biomédico, posibles tratamientos y diagnósticos, y los avances en investigación clínica, utilizando terapia génica con CRISPR/Cas más relevantes hasta la fecha. (AU)

CRISPR are Clustered Regularly Interspaced Short Palindromic Repeats, which naturally belong to the defense system of bacteria and archaea. It has been biotechnologically adapted for editing the DNA of eukaryotic cells, including human cells. The CRISPR-Cas system for editing genes generally consists of two components, a nuclease protein (Cas) and a guide RNA (sgRNA). The simplicity of the complex makes it a reprogrammable molecular tool capable of being targeted and editing any site in a known genome. Its main focus is therapies for monogenic inherited diseases and cancer. However, in addition to gene editor, CRISPR technology is used for epigenetic editing, regulation of gene expression, and molecular diagnostic methods. This article aims to present a review of the applications of the CRISPR-Cas molecular tool, particularly in the biomedical field, possible treatments and diagnoses, and the advances in clinical research, using the most relevant CRISPR-Cas gene therapy to date. (AU)

Application of CRISPR in evolution analysis, detecting and typing, virulence and antibiotic resistance regulation in food-borne pathogens / 生物工程学报

Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein gene system can limit the horizontal gene transfer, thereby effectively preventing the invasion of foreign gene elements such as bacteriophages. CRISPR arrays of different bacteria are diverse. Based on the differences in the CRISPR system, this review summarizes the application of CRISPR in food-borne pathogen evolution analysis, detection and typing, virulence and antibiotic resistance in recent years. We also address bacterial detection typing method developed based on the characteristics of CRISPR arrays and the association of CRISPR with virulence and drug resistance of food-borne pathogens. The shortcomings of CRISPR in evolution, detection and typing, virulence and resistance applications are analyzed. In addition, we suggest standardizing CRISPR typing methods, improving and expanding the CRISPR database of pathogenic bacteria, and further exploring the co-evolution relationship between phages and bacteria, to provide references for further exploration of CRISPR functions.