1.Advances of Cas9/sgRNA delivery system for gene editing.
Xuan HU ; Song WANG ; Lu YU ; Xiaopeng ZHANG ; Wei CHEN
Chinese Journal of Biotechnology 2021;37(11):3880-3889
In the application of CRISPR genome editing, direct cellular delivery of non-replicable Cas9/sgRNA may reduce unwanted gene targeting and integrational mutagenesis, thus offering greater specificity and safety. Cas9/sgRNA delivery system holds great potential for treating genetic diseases. This review summarizes the advances of Cas9/sgRNA delivery systems and its therapeutic applications, providing new understandings and inspirations for vector design and future clinical applications.
CRISPR-Cas Systems/genetics*
;
Gene Editing
;
RNA, Guide/genetics*
2.Identification of neoantigens derived from alternative splicing and RNA modification
Genomics & Informatics 2019;17(3):e23-
The acquisition of somatic mutations is the most common event in cancer. Neoantigens expressed from genes with mutations acquired during carcinogenesis can be tumor-specific. Since the immune system recognizes tumor-specific peptides, they are potential targets for personalized neoantigen-based immunotherapy. However, the discovery of druggable neoantigens remains challenging, suggesting that a deeper understanding of the mechanism of neoantigen generation and better strategies to identify them will be required to realize the promise of neoantigen-based immunotherapy. Alternative splicing and RNA editing events are emerging mechanisms leading to neoantigen production. In this review, we outline recent work involving the large-scale screening of neoantigens produced by alternative splicing and RNA editing. We also describe strategies to predict and validate neoantigens from RNA sequencing data.
Alternative Splicing
;
Carcinogenesis
;
Humans
;
Immune System
;
Immunotherapy
;
Mass Screening
;
Peptides
;
RNA Editing
;
RNA
;
Sequence Analysis, RNA
3.Bioinformatics Approaches for the Identification and Annotation of RNA Editing Sites.
Journal of Genetic Medicine 2013;10(1):27-32
Post-transcriptional nucleotide sequence modification of transcripts by RNA editing is an important molecular mechanism in the regulation of protein function and is associated with a variety of human disease phenotypes. Identification of RNA editing sites is the basic step for studying RNA editing. Databases and bioinformatics resources are used to annotate and evaluate as well as identify RNA editing sites. No method is free of limitations. Correctly establishing an analytic pipeline and strategic application of both experimental and bioinformatics methods constitute the first step in investigating RNA editing. This review summarizes modern bioinformatics approaches and related resources for RNA editing research.
Base Sequence
;
Computational Biology
;
Humans
;
Phenotype
;
Resin Cements
;
RNA
;
RNA Editing
4.Identification of Diverse Adenosine-to-Inosine RNA Editing Subtypes in Colorectal Cancer.
Si Hyun LEE ; Hwang Phill KIM ; Jun Kyu KANG ; Sang Hyun SONG ; Sae Won HAN ; Tae You KIM
Cancer Research and Treatment 2017;49(4):1077-1087
PURPOSE: RNA editing generates protein diversity by altering RNA sequences in coding regions without changing the overall DNA sequence. Adenosine-to-inosine (A-to-I) RNA editing events have recently been reported in some types of cancer, but they are rare in human colorectal cancer (CRC). Therefore, this study was conducted to identify diverse RNA editing in CRC. MATERIALS AND METHODS: We compared transcriptome data of 39 CRC samples and paired adjacent tissues from The Cancer Genome Atlas database to identify RNA editing patterns in CRC, focusing on canonical A-to-I RNA edits in coding sequence regions. We investigated nonsynonymous RNA editing patterns by comparing tumor and normal tissue transcriptome data. RESULTS: The number of RNA edits varied from 12 to 42 per sample. We also observed that hypoand hyper-RNA editing patterns were distinguishable within the samples. We found 10 recurrent nonsynonymous RNA editing candidates in nine genes (PDLIM, NEIL1, SRP9, GLI1, APMAP, IGFBP7, ZNF358, COPA, and ZNF587B) and validated some by Sanger sequencing and the inosine chemical erasing assay. We further showed that editing at these positions was performed by the adenosine deaminase acting on RNA 1 enzyme. Most of these genes are hypoedited in CRC, but editing of GLI1 was increased in cancer tissues compared with normal tissues. CONCLUSION: Our results show that nonsynonymous RNA editing patterns can be used to identify CRC patients and could serve as novel biomarkers for CRC.
Adenosine Deaminase
;
Base Sequence
;
Biomarkers
;
Clinical Coding
;
Colorectal Neoplasms*
;
Genome
;
Humans
;
Inosine
;
RNA Editing*
;
RNA*
;
Transcriptome
5.Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA.
Qihua PAN ; Junzhi LUO ; Yuewen JIANG ; Zhi WANG ; Ke LU ; Tiansheng CHEN
Journal of Zhejiang University. Science. B 2022;23(1):74-83
Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase(tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.
Animals
;
CRISPR-Cas Systems
;
Cell Line
;
Gene Editing
;
Oryzias/genetics*
;
RNA, Guide/genetics*
;
RNA, Transfer/genetics*
6.A Statistical Analysis of SNPs, In-Dels, and Their Flanking Sequences in Human Genomic Regions.
Seung Wook SHIN ; Young Joo KIM ; Byung Dong KIM
Genomics & Informatics 2007;5(2):68-76
Due to the increasing interest in SNPs and mutational hot spots for disease traits, it is becoming more important to define and understand the relationship between SNPs and their flanking sequences. To study the effects of flanking sequences on SNPs, statistical approaches are necessary to assess bias in SNP data. In this study we mainly applied Markov chains for SNP sequences, particularly those located in intronic regions, and for analysis of in-del data. All of the pertaining sequences showed a significant tendency to generate particular SNP types. Most sequences flanking SNPs had lower complexities than average sequences, and some of them were associated with microsatellites. Moreover, many Alu repeats were found in the flanking sequences. We observed an elevated frequency of single-base-pair repeat-like sequences, mirror repeats, and palindromes in the SNP flanking sequence data. Alu repeats are hypothesized to be associated with C-to-T transition mutations or A-to-I RNA editing. In particular, the in-del data revealed an association between particular changes such as palindromes or mirror repeats. Results indicate that the mechanism of induction of in-del transitions is probably very different from that which is responsible for other SNPs. From a statistical perspective, frequent DNA lesions in some regions probably have effects on the occurrence of SNPs.
Bias (Epidemiology)
;
DNA
;
Humans*
;
Introns
;
Markov Chains
;
Microsatellite Repeats
;
Polymorphism, Single Nucleotide*
;
RNA Editing
7.Application of CRISPR-Cas9 genome editing for constructing animal models of human diseases.
Chinese Journal of Medical Genetics 2016;33(4):559-563
The CRISPR-Cas9 system is a new targeted nuclease for genome editing, which can directly introduce modifications at the targeted genomic locus. The system utilizes a short single guide RNA (sgRNA) to direct the endonuclease Cas9 in the genome. Upon targeting, Cas9 can generate DNA double-strand breaks (DSBs). As such DSBs are repaired by non-homologous end joining (NHEJ) or homology directed repair (HDR), therefore facilitates introduction of random or specific mutations, repair of endogenous mutations, or insertion of DNA elements. The system has been successfully used to generate gene targeted cell lines including those of human, animals and plants. This article reviews recent advances made in this rapidly evolving technique for the generation of animal models for human diseases.
Animals
;
Clustered Regularly Interspaced Short Palindromic Repeats
;
genetics
;
Disease Models, Animal
;
Humans
;
RNA Editing
;
genetics
8.Advances of CRISPR/Cas9 activation system.
Xiao DING ; Zhuanxia PAN ; Liuliu YANG ; Xiaoli LUO ; Nan JIANG ; Mengjie ZHU ; Cuicui WU ; Gang LAN ; Pengbo LI
Chinese Journal of Biotechnology 2022;38(8):2713-2724
Gene editing technology has been a hotspot in the field of biotechnology. CRISPR/Cas systems are efficient gene editing tools because of its specificity, simplicity and flexibility, these features enabled the rapid application of CRISPR/Cas systems in a variety of organisms. Moreover, the combination of transcriptional activator with dead Cas protein can achieve specific regulation of gene expression at the transcription level, which has made important contributions to the development of biotechnology in medical and agriculture. Overexpression of foreign genes is a common method to verify gene function and regulation. However, due to the limitation of vector capacity, it is difficult to achieve overexpression of multiple genes. CRISPR/Cas9 activation system can regulate the expression of multiple genes under the guidance of different guide RNAs to verify gene functions at the regulatory level. This review summarizes the composition of the CRISPR/Cas9 activation system and different activation strategies, and summarizes solutions for excessive activation. It may facilitate the application of CRISPR/Cas9 activation system in genetic improvement of cotton and herbicide resistance research.
Biotechnology
;
CRISPR-Cas Systems/genetics*
;
Gene Editing
;
Phenotype
;
RNA, Guide, Kinetoplastida/metabolism*
9.Optimization of CRISPR/Cas9-based multiplex base editing in Corynebacterium glutamicum.
Hui LU ; Qi ZHANG ; Sili YU ; Yu WANG ; Ming KANG ; Shuangyan HAN ; Ye LIU ; Meng WANG
Chinese Journal of Biotechnology 2022;38(2):780-795
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.
CRISPR-Cas Systems/genetics*
;
Corynebacterium glutamicum/metabolism*
;
Gene Editing
;
Plasmids
;
RNA, Guide/metabolism*
10.Construction of a stable TrxR1 knockout HCT-116 cell line using CRISPR/Cas9 gene editing system.
Zhiyin ZHOU ; Xiaomei LÜ ; Li ZHU ; Ji ZHOU ; Huidan HUANG ; Chao ZHANG ; Xiaoping LIU
Chinese Journal of Biotechnology 2022;38(3):1074-1085
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.
CRISPR-Cas Systems/genetics*
;
Gene Editing
;
Gene Knockout Techniques
;
HCT116 Cells
;
Humans
;
RNA, Guide/metabolism*