CRISPR Genome Editing: Principle, Method, Tool and Application

A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Cell Methods".

Deadline for manuscript submissions: closed (31 October 2022) | Viewed by 19664

Special Issue Editors


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Guest Editor
Department of Biology, East Carolina University, Greenville, NC 27858, USA
Interests: genome editing; small regulatory RNAs; molecular genetics; plant biotechnology; abiotic stress; gene expression and regulation
Special Issues, Collections and Topics in MDPI journals

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Guest Editor
Ficus Biotechnology, Ostim Teknopark, No: 1/1/76, Yenimahalle, Ankara 06378, Turkey
Interests: genomics; transcriptomics; miRNA; CRISPR

Special Issue Information

Dear Colleagues,

Genome editing is becoming a powerful, robust, and versatile tool for gene functional study and for gene therapy, which can lead to improvements in both plants and animals. This field has developed rapidly in recent years, as evidenced in the new Cas that have been identified and the new bioinformatics tools that have been developed. Genome editing is widely used in many fields at present, not only for gene functional study but also for gene therapy and crop improvement. In this Special Issue, both reviews and research papers, as well as method and bioinformatics papers, are welcomed.

Prof. Dr. Baohong Zhang
Dr. Turgay Unver
Guest Editors

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Keywords

  • genome editing
  • CRISPR/Cas9
  • gene functional study
  • gene knockout
  • gene regulation
  • gene therapy
  • epigenetic
  • plant improvement
  • biotechnology

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Published Papers (7 papers)

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Research

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14 pages, 2729 KiB  
Article
CRISPR/Cas9-Mediated Mutagenesis of Sex-Specific Doublesex Splicing Variants Leads to Sterility in Spodoptera frugiperda, a Global Invasive Pest
by Junwen Gu, Jingyi Wang, Honglun Bi, Xuehai Li, Austin Merchant, Porui Zhang, Qi Zhang and Xuguo Zhou
Cells 2022, 11(22), 3557; https://doi.org/10.3390/cells11223557 - 10 Nov 2022
Cited by 5 | Viewed by 1722
Abstract
Spodoptera frugiperda (J. E. Smith), an emerging invasive pest worldwide, has posed a serious agricultural threat to the newly invaded areas. Although somatic sex differentiation is fundamentally conserved among insects, the sex determination cascade in S. frugiperda is largely unknown. In this study, [...] Read more.
Spodoptera frugiperda (J. E. Smith), an emerging invasive pest worldwide, has posed a serious agricultural threat to the newly invaded areas. Although somatic sex differentiation is fundamentally conserved among insects, the sex determination cascade in S. frugiperda is largely unknown. In this study, we cloned and functionally characterized Doublesex (dsx), a “molecular switch” modulating sexual dimorphism in S. frugiperda using male- and female-specific isoforms. Given that Lepidoptera is recalcitrant to RNAi, CRISPR/Cas9-mediated mutagenesis was employed to construct S. frugiperda mutants. Specifically, we designed target sites on exons 2, 4, and 5 to eliminate the common, female-specific, and male-specific regions of S. frugiperda dsx (Sfdsx), respectively. As expected, abnormal development of both the external and internal genitalia was observed during the pupal and adult stages. Interestingly, knocking out sex-specific dsx variants in S. frugiperda led to significantly reduced fecundity and fertility in adults of corresponding sex. Our combined results not only confirm the conserved function of dsx in S. frugiperda sex differentiation but also provide empirical evidence for dsx as a potential target for the Sterile Insect Technique (SIT) to combat this globally invasive pest in a sustainable and environmentally friendly way. Full article
(This article belongs to the Special Issue CRISPR Genome Editing: Principle, Method, Tool and Application)
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13 pages, 7389 KiB  
Article
Masculinizer and Doublesex as Key Factors Regulate Sexual Dimorphism in Ostrinia furnacalis
by Honglun Bi, Xiaowei Li, Xia Xu, Yaohui Wang, Shutang Zhou and Yongping Huang
Cells 2022, 11(14), 2161; https://doi.org/10.3390/cells11142161 - 11 Jul 2022
Cited by 7 | Viewed by 2357
Abstract
Sex determination is an important and traditional biological process. In Lepidoptera, Masculinizer (Masc) and doublesex (dsx) are the essential genes for sex determination and play critical roles in sexual differentiation and development. The functions of Masc and dsx have [...] Read more.
Sex determination is an important and traditional biological process. In Lepidoptera, Masculinizer (Masc) and doublesex (dsx) are the essential genes for sex determination and play critical roles in sexual differentiation and development. The functions of Masc and dsx have been characterized in several model insect species. However, the molecular mechanism and sex determination functions of Masc and dsx in Ostrinia furnacalis, an agricultural pest, are still unknown. Here, we successfully used the CRISPR/Cas9 genome editing system to knock out OfMasc and Ofdsx. Mutation of OfMasc induced male external genital defects and sterility. Disruptions of the Ofdsx common region caused sex-specific defects in the external genitals and adult sterility. In addition, we found that OfMasc and Ofdsx can regulate the pigmentation genes that control wing pigmentation patterns. These results demonstrate that OfMasc and Ofdsx play key roles in the sex determination of O. furnacalis, and suggest novel genetic control approaches for the management of pests, including O. furnacalis. Full article
(This article belongs to the Special Issue CRISPR Genome Editing: Principle, Method, Tool and Application)
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15 pages, 5512 KiB  
Article
A Novel Cre/lox71-Based System for Inducible Expression of Recombinant Proteins and Genome Editing
by Maxim Karagyaur, Daniyar Dyikanov, Pyotr Tyurin-Kuzmin, Stalik Dzhauari, Mariya Skryabina, Maksim Vigovskiy, Alexandra Primak, Natalia Kalinina and Vsevolod Tkachuk
Cells 2022, 11(14), 2141; https://doi.org/10.3390/cells11142141 - 7 Jul 2022
Cited by 1 | Viewed by 2764
Abstract
In this study, we developed a novel Cre/lox71-based system for the controlled transient expression of target genes. We used the bacteriophage P1 Cre recombinase, which harbors a short, highly specific DNA-binding site and does not have endogenous binding sites within mouse or human [...] Read more.
In this study, we developed a novel Cre/lox71-based system for the controlled transient expression of target genes. We used the bacteriophage P1 Cre recombinase, which harbors a short, highly specific DNA-binding site and does not have endogenous binding sites within mouse or human genomes. Fusing the catalytically inactive form of Cre recombinase and the VP64 transactivation domain (VP16 tetramer), we constructed the artificial transcription factor Cre-VP64. This transcription factor binds to the lox71 sites within the promoter region of the target gene and, therefore, upregulates its expression. We tested the Cre-VP64/lox71 system for the controlled expression of several genes, including growth factors and the genome editor CRISPR/Cas9, and obtained superior efficiency in the regulation of transgene expression, achieving a high expression level upon induction together with low basal activity. This system or its modified forms can be suggested as a novel effective tool for the transitory controlled expression of target genes for functional genomic studies, as well as for gene therapy approaches. Full article
(This article belongs to the Special Issue CRISPR Genome Editing: Principle, Method, Tool and Application)
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16 pages, 21998 KiB  
Article
Reverting TP53 Mutation in Breast Cancer Cells: Prime Editing Workflow and Technical Considerations
by Asmaa Y. Abuhamad, Nurul Nadia Mohamad Zamberi, Ling Sheen, Safaa M. Naes, Siti Nur Hasanah Mohd Yusuf, Asilah Ahmad Tajudin, M. Aiman Mohtar, Amir Syahir Amir Hamzah and Saiful Effendi Syafruddin
Cells 2022, 11(10), 1612; https://doi.org/10.3390/cells11101612 - 11 May 2022
Cited by 9 | Viewed by 3737
Abstract
Breast cancer is the leading cause of cancer-related deaths in women. The aggressive breast cancer subtype is commonly linked to the genetic alterations in the TP53 tumor suppressor gene, predominantly the missense mutations. Robust experimental models are needed to gain better insights into [...] Read more.
Breast cancer is the leading cause of cancer-related deaths in women. The aggressive breast cancer subtype is commonly linked to the genetic alterations in the TP53 tumor suppressor gene, predominantly the missense mutations. Robust experimental models are needed to gain better insights into these mutations’ molecular properties and implications in tumorigenesis. The generation of such models harboring the alterations is feasible with the CRISPR-based gene editing technology. Moreover, the development of new CRISPR applications, particularly DNA base and prime editing, has considerably improved the precision and versatility of gene editing. Here, we employed the prime editing tool to revert a TP53 missense C > T mutation (L194F) in a T47D luminal A breast cancer cell line. In parallel, this prime editing tool was also utilized to introduce the L194F mutation in HEK293T cells. To assess the prime editing efficiency in both cell lines, we first performed Sanger sequencing in the prime-edited cells pool and single cell-derived clones. However, the Sanger sequencing approach did not detect any base substitution in these cell lines. Next, by employing the more sensitive amplicon target sequencing, we managed to identify the expected substitution in these T47D and HEK293T cells, albeit the editing efficiency was low. In light of these findings, we discussed the technical aspects and provided suggestions for improve the prime editing workflow and efficiency for future experiments. Full article
(This article belongs to the Special Issue CRISPR Genome Editing: Principle, Method, Tool and Application)
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15 pages, 67051 KiB  
Article
5′-Nucleotidase Plays a Key Role in Uric Acid Metabolism of Bombyx mori
by Linmeng Tang, Dehong Yang, Yaohui Wang, Xu Yang, Kai Chen, Xingyu Luo, Jun Xu, Yujia Liu, Zheng Tang, Qianqian Zhang, Zhiwei Liu and Yongping Huang
Cells 2021, 10(9), 2243; https://doi.org/10.3390/cells10092243 - 30 Aug 2021
Cited by 11 | Viewed by 2751
Abstract
Uric acid (UA) is the end-product in the human purine metabolism pathway. The UA that accumulates in silkworm tissues is excreted as a nitrogen waste product. Here, we first validated that Bombyx mori has a homolog of the human gene that encodes the [...] Read more.
Uric acid (UA) is the end-product in the human purine metabolism pathway. The UA that accumulates in silkworm tissues is excreted as a nitrogen waste product. Here, we first validated that Bombyx mori has a homolog of the human gene that encodes the 5′-nucleotidase (5′N) involved in purine metabolism. The B. mori gene, Bm5′N, is located upstream of other genes involved in UA metabolism in the silkworm. Disruption of Bm5′N via the CRISPR/Cas9 system resulted in decreased UA levels in the silkworm epidermis and caused a translucent skin phenotype. When Bm5′N mutant silkworms were fed with the uric acid precursor inosine, the UA levels in the epidermis increased significantly. Furthermore, the metabolomic and transcriptomic analyses of Bm5′N mutants indicated that loss of the Bm5′N affected purine metabolism and the ABC transport pathway. Taken together, these results suggest that the UA pathway is conserved between the silkworm and humans and that the Bm5′N gene plays a crucial role in the uric acid metabolism of the silkworm. Thus, the silkworm may be a suitable model for the study of UA metabolism pathways relevant to human disease. Full article
(This article belongs to the Special Issue CRISPR Genome Editing: Principle, Method, Tool and Application)
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Review

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19 pages, 966 KiB  
Review
Engineering Abiotic Stress Tolerance in Crop Plants through CRISPR Genome Editing
by Mehboob-ur Rahman, Sana Zulfiqar, Muhammad Ahmad Raza, Niaz Ahmad and Baohong Zhang
Cells 2022, 11(22), 3590; https://doi.org/10.3390/cells11223590 - 13 Nov 2022
Cited by 12 | Viewed by 3245
Abstract
Environmental abiotic stresses challenge food security by depressing crop yields often exceeding 50% of their annual production. Different methods, including conventional as well as genomic-assisted breeding, mutagenesis, and genetic engineering have been utilized to enhance stress resilience in several crop species. Plant breeding [...] Read more.
Environmental abiotic stresses challenge food security by depressing crop yields often exceeding 50% of their annual production. Different methods, including conventional as well as genomic-assisted breeding, mutagenesis, and genetic engineering have been utilized to enhance stress resilience in several crop species. Plant breeding has been partly successful in developing crop varieties against abiotic stresses owning to the complex genetics of the traits as well as the narrow genetic base in the germplasm. Irrespective of the fact that genetic engineering can transfer gene(s) from any organism(s), transgenic crops have become controversial mainly due to the potential risk of transgene-outcrossing. Consequently, the cultivation of transgenic crops is banned in certain countries, particularly in European countries. In this scenario, the discovery of the CRISPR tool provides a platform for producing transgene-free genetically edited plants—similar to the mutagenized crops that are not extensively regulated such as genetically modified organisms (GMOs). Thus, the genome-edited plants without a transgene would likely go into the field without any restriction. Here, we focused on the deployment of CRISPR for the successful development of abiotic stress-tolerant crop plants for sustaining crop productivity under changing environments. Full article
(This article belongs to the Special Issue CRISPR Genome Editing: Principle, Method, Tool and Application)
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15 pages, 1450 KiB  
Review
The Power of Gene Technologies: 1001 Ways to Create a Cell Model
by Maxim Karagyaur, Alexandra Primak, Anastasia Efimenko, Mariya Skryabina and Vsevolod Tkachuk
Cells 2022, 11(20), 3235; https://doi.org/10.3390/cells11203235 - 14 Oct 2022
Cited by 5 | Viewed by 1691
Abstract
Modern society faces many biomedical challenges that require urgent solutions. Two of the most important include the elucidation of mechanisms of socially significant diseases and the development of prospective drug treatments for these diseases. Experimental cell models are a convenient tool for addressing [...] Read more.
Modern society faces many biomedical challenges that require urgent solutions. Two of the most important include the elucidation of mechanisms of socially significant diseases and the development of prospective drug treatments for these diseases. Experimental cell models are a convenient tool for addressing many of these problems. The power of cell models is further enhanced when combined with gene technologies, which allows the examination of even more subtle changes within the structure of the genome and permits testing of proteins in a native environment. The list and possibilities of these recently emerging technologies are truly colossal, which requires a rethink of a number of approaches for obtaining experimental cell models. In this review, we analyze the possibilities and limitations of promising gene technologies for obtaining cell models, and also give recommendations on the development and creation of relevant models. In our opinion, this review will be useful for novice cell biologists, as it provides some reference points in the rapidly growing universe of gene and cell technologies. Full article
(This article belongs to the Special Issue CRISPR Genome Editing: Principle, Method, Tool and Application)
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