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.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
7.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
9.Advances in the RNA-targeting CRISPR-Cas systems.
Chinese Journal of Biotechnology 2023;39(4):1363-1373
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.
CRISPR-Cas Systems/genetics*
;
RNA/genetics*
;
Bacteria/genetics*
;
Gene Editing
;
Archaea
10.Development of a CRISPR/Cpf1 gene editing system in silkworm Bombyx mori.
Zhanqi DONG ; Qi QIN ; Xinling ZHANG ; Kejie LI ; Peng CHEN ; Minhui PAN
Chinese Journal of Biotechnology 2021;37(12):4342-4350
The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type (V type) CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5'T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1 (AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3A-346 knockout vectors. The efficiency for editing the target genes BmATAD3A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.
Animals
;
Bombyx/metabolism*
;
CRISPR-Cas Systems/genetics*
;
Endonucleases/genetics*
;
Gene Editing
;
RNA, Guide/genetics*