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Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 3.0

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (20 June 2024) | Viewed by 33953

Special Issue Editors

Dr. Timofey S. Rozhdestvensky

E-Mail Website
Guest Editor
Medical Faculty, Core Facility Transgenic Animal and Genetic Engineering Models (TRAM), University of Muenster, Von-Esmarch-Str. 56, D-48149 Muenster, Germany
Interests: genome editing; CRISPR-Cas9 technology; programmable DNA endonucleases; nervous system diseases; RNA biology; disease-associated RNAs; non-protein coding RNAs; etc.
Special Issues, Collections and Topics in MDPI journals
Dr. Alexander Kondrashov

E-Mail Website
Guest Editor
Dept of Stem Cell Biology, Division of Cancer & Stem Cells, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK
Interests: genome editing (particularly CRISPR/Cas9 and TALEN-based systems); genome editing in stem/cancer cells for disease modelling & drug testing; role of specific polymorphisms in the regulation of expression/function of genes associated with disease development; regulation of gene expression, with emphasis on understanding mechanisms of reprogramming during stress and pathology
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer A. Doudna “for the development of a method for genome editing”. Since the initial publication on the mechanism of CRISPR/Cas9 nuclease cleavage activity and its application for the direct targeting of genomic sequences “in vitro” in 2012, multiple parallel studies in different organisms (from bacteria to human cells) have proven its unparalleled efficiency as genetic “molecular scissors” for “in vivo” manipulations. Within a short period of time, CRISPR/Cas9-mediated genome editing has emerged as a state-of-the-art approach for precise gene modification in all model organisms. It has become a powerful technology for the generation of custom-designed gene variants and gaining molecular insights into different biochemical pathways or human diseases at the cellular or organism level. Moreover, CRISPR/Cas9-mediated genome editing is currently under intensive development for application in the field of human gene therapy and has recently been approved for its first clinical trials. However, despite the many advantages and great potential for future research and clinical applications, existing problems (off-target activity, the induction of genome rearrangements, cellular mosaicism in transgenes, etc.) still impede the full-scale use of the technology in biomedical research. Further efforts are needed to overcome these hurdles. This Special Issue aims to cover all areas of molecular-based research that use CRISPR/Cas9 and alternative CRISPR systems for genome editing and other applications in cellular or animal models. It welcomes original research, reviews and short communication articles of which CRISPR technology is the main topic or the tool for answering mechanistic molecular questions. This includes, but is not limited to, methodological advances and identified pitfalls in CRISPR-based technology, and its application to functional genomics for disease and human gene therapy, epigenomics, proteomics, RNA biology, systems biology, etc.

  • Molecular insights into different approaches and methods for CRISPR/Cas9 genome editing;
  • The generation of disease models;
  • The CRISPR/Cas9-mediated genome editing of ES cells;
  • The CRISPR/Cas9 system in cancer biology;
  • CRISPR/Cas9-mediated genome editing in model organisms (plants, fungi and animals);
  • The CRISPR/Cas9 system in epigenetic research;
  • The CRISPR/Cas9 system in gene therapy—applications and the challenges in the implementation of this technology;
  • The CRISPR/Cas9 system beyond genome editing (molecular markers, biosensors, etc.).

Dr. Timofey S. Rozhdestvensky
Dr. Alexander Kondrashov
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

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Published Papers (7 papers)

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Research

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31 pages, 6625 KiB  
Article
Human RNA Polymerase II Segregates from Genes and Nascent RNA and Transcribes in the Presence of DNA-Bound dCas9
by João Pessoa and Célia Carvalho
Int. J. Mol. Sci. 2024, 25(15), 8411; https://doi.org/10.3390/ijms25158411 - 1 Aug 2024
Viewed by 1031
Abstract
RNA polymerase II (Pol II) dysfunction is frequently implied in human disease. Understanding its functional mechanism is essential for designing innovative therapeutic strategies. To visualize its supra-molecular interactions with genes and nascent RNA, we generated a human cell line carrying ~335 consecutive copies [...] Read more.
RNA polymerase II (Pol II) dysfunction is frequently implied in human disease. Understanding its functional mechanism is essential for designing innovative therapeutic strategies. To visualize its supra-molecular interactions with genes and nascent RNA, we generated a human cell line carrying ~335 consecutive copies of a recombinant β-globin gene. Confocal microscopy showed that Pol II was not homogeneously concentrated around these identical gene copies. Moreover, Pol II signals partially overlapped with the genes and their nascent RNA, revealing extensive compartmentalization. Using a cell line carrying a single copy of the β-globin gene, we also tested if the binding of catalytically dead CRISPR-associated system 9 (dCas9) to different gene regions affected Pol II transcriptional activity. We assessed Pol II localization and nascent RNA levels using chromatin immunoprecipitation and droplet digital reverse transcription PCR, respectively. Some enrichment of transcriptionally paused Pol II accumulated in the promoter region was detected in a strand-specific way of gRNA binding, and there was no decrease in nascent RNA levels. Pol II preserved its transcriptional activity in the presence of DNA-bound dCas9. Our findings contribute further insight into the complex mechanism of mRNA transcription in human cells. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 3.0)
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18 pages, 2149 KiB  
Article
Deep Characterization and Comparison of Different Retrovirus-like Particles Preloaded with CRISPR/Cas9 RNPs
by Max Wichmann, Cecile L. Maire, Niklas Nuppenau, Moataz Habiballa, Almut Uhde, Katharina Kolbe, Tanja Schröder, Katrin Lamszus, Boris Fehse and Dawid Głów
Int. J. Mol. Sci. 2023, 24(14), 11399; https://doi.org/10.3390/ijms241411399 - 13 Jul 2023
Cited by 5 | Viewed by 3048
Abstract
The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced [...] Read more.
The CRISPR/Cas system has a broad range of possible medical applications, but its clinical translation has been hampered, particularly by the lack of safe and efficient vector systems mediating the short-term expression of its components. Recently, different virus-like particles (VLPs) have been introduced as promising vectors for the delivery of CRISPR/Cas genome editing components. Here, we characterized and directly compared three different types of retrovirus-based (R) VLPs, two derived from the γ-retrovirus murine leukemia virus (gRVLPs and “enhanced” egRVLPs) and one from the lentivirus human immunodeficiency virus, HIV (LVLPs). First, we unified and optimized the production of the different RVLPs. To ensure maximal comparability of the produced RVLPs, we adapted several assays, including nanoparticle tracking analysis (NTA), multi-parametric imaging flow cytometry (IFC), and Cas9-ELISA, to analyze their morphology, surface composition, size, and concentration. Next, we comparatively tested the three RVLPs targeting different genes in 293T model cells. Using identical gRNAs, we found egRVLPs to mediate the most efficient editing. Functional analyses indicated better cargo (i.e., Cas9) transfer and/or release as the underlying reason for their superior performance. Finally, we compared on- and off-target activities of the three RVLPs in human-induced pluripotent stem cells (hiPSC) exploiting the clinically relevant C-C motif chemokine receptor 5 (CCR5) as the target. Again, egRVLPs facilitated the highest, almost 100% knockout rates, importantly with minimal off-target activity. In conclusion, in direct comparison, egRVLPs were the most efficient RVLPs. Moreover, we established methods for in-depth characterization of VLPs, facilitating their validation and thus more predictable and safe application. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 3.0)
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24 pages, 76572 KiB  
Article
A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells
by Sara Cuevas-Ocaña, Jin Ye Yang, Magomet Aushev, George Schlossmacher, Christine E. Bear, Nicholas R. F. Hannan, Neil D. Perkins, Janet Rossant, Amy P. Wong and Michael A. Gray
Int. J. Mol. Sci. 2023, 24(12), 10266; https://doi.org/10.3390/ijms241210266 - 17 Jun 2023
Cited by 4 | Viewed by 3938
Abstract
Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of [...] Read more.
Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3–6 weeks in order to understand genetic determinants of disease and precision medicine. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 3.0)
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Review

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20 pages, 2165 KiB  
Review
CRISPR/Cas9-Mediated Genome Editing in Cancer Therapy
by Shuai Ding, Jinfeng Liu, Xin Han and Mengfan Tang
Int. J. Mol. Sci. 2023, 24(22), 16325; https://doi.org/10.3390/ijms242216325 - 15 Nov 2023
Cited by 6 | Viewed by 5166
Abstract
The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, an RNA-based adaptive immune system found in bacteria and archaea, has catalyzed the development and application of a new generation of gene editing tools. Numerous studies have shown that this system can [...] Read more.
The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, an RNA-based adaptive immune system found in bacteria and archaea, has catalyzed the development and application of a new generation of gene editing tools. Numerous studies have shown that this system can precisely target a wide range of human genes, including those associated with diseases such as cancer. In cancer research, the intricate genetic mutations in tumors have promoted extensive utilization of the CRISPR/Cas9 system due to its efficient and accurate gene editing capabilities. This includes improvements in Chimeric Antigen Receptor (CAR)-T-cell therapy, the establishment of tumor models, and gene and drug target screening. Such progress has propelled the investigation of cancer molecular mechanisms and the advancement of precision medicine. However, the therapeutic potential of genome editing remains underexplored, and lingering challenges could elevate the risk of additional genetic mutations. Here, we elucidate the fundamental principles of CRISPR/Cas9 gene editing and its practical applications in tumor research. We also briefly discuss the primary challenges faced by CRISPR technology and existing solutions, intending to enhance the efficacy of this gene editing therapy and shed light on the underlying mechanisms of tumors. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 3.0)
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40 pages, 1770 KiB  
Review
CRISPR/Cas9 Landscape: Current State and Future Perspectives
by Marina Tyumentseva, Aleksandr Tyumentsev and Vasiliy Akimkin
Int. J. Mol. Sci. 2023, 24(22), 16077; https://doi.org/10.3390/ijms242216077 - 8 Nov 2023
Cited by 15 | Viewed by 14640
Abstract
CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a unique genome editing tool that can be easily used in a wide range of applications, including functional genomics, transcriptomics, epigenetics, biotechnology, plant engineering, livestock breeding, gene therapy, diagnostics, and so on. This review is [...] Read more.
CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a unique genome editing tool that can be easily used in a wide range of applications, including functional genomics, transcriptomics, epigenetics, biotechnology, plant engineering, livestock breeding, gene therapy, diagnostics, and so on. This review is focused on the current CRISPR/Cas9 landscape, e.g., on Cas9 variants with improved properties, on Cas9-derived and fusion proteins, on Cas9 delivery methods, on pre-existing immunity against CRISPR/Cas9 proteins, anti-CRISPR proteins, and their possible roles in CRISPR/Cas9 function improvement. Moreover, this review presents a detailed outline of CRISPR/Cas9-based diagnostics and therapeutic approaches. Finally, the review addresses the future expansion of genome editors’ toolbox with Cas9 orthologs and other CRISPR/Cas proteins. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 3.0)
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17 pages, 2189 KiB  
Review
CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination
by Tahmina Tabassum, Giovanni Pietrogrande, Michael Healy and Ernst J. Wolvetang
Int. J. Mol. Sci. 2023, 24(19), 14701; https://doi.org/10.3390/ijms241914701 - 28 Sep 2023
Cited by 3 | Viewed by 3004
Abstract
DNA repair in mammalian cells involves the coordinated action of a range of complex cellular repair machinery. Our understanding of these DNA repair processes has advanced to the extent that they can be leveraged to improve the efficacy and precision of Cas9-assisted genome [...] Read more.
DNA repair in mammalian cells involves the coordinated action of a range of complex cellular repair machinery. Our understanding of these DNA repair processes has advanced to the extent that they can be leveraged to improve the efficacy and precision of Cas9-assisted genome editing tools. Here, we review how the fusion of CRISPR-Cas9 to functional domains of proteins that directly or indirectly impact the DNA repair process can enhance genome editing. Such studies have allowed the development of diverse technologies that promote efficient gene knock-in for safer genome engineering practices. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 3.0)
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Other

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11 pages, 1502 KiB  
Brief Report
CRISPR-Cas9-Mediated Cytosine Base Editing Screen for the Functional Assessment of CALR Intron Variants in Japanese Encephalitis Virus Replication
by Youcai Xiong, Xiaoning Xi, Yue Xiang, Sheng Li, Hailong Liu, Yinyu Su, Ruigao He, Chong Xiong, Bingrong Xu, Xinyi Wang, Liangliang Fu, Changzhi Zhao, Xiaosong Han, Xinyun Li, Shengsong Xie and Jinxue Ruan
Int. J. Mol. Sci. 2023, 24(17), 13331; https://doi.org/10.3390/ijms241713331 - 28 Aug 2023
Cited by 1 | Viewed by 1751
Abstract
The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of [...] Read more.
The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes viral encephalitis in humans, pigs and other mammals across Asia and the Western Pacific. Genetic screening tools such as CRISPR screening, DNA sequencing and RNA interference have greatly improved our understanding of JEV replication and its potential antiviral approaches. However, information on exon and intron mutations associated with JEV replication is still scanty. CRISPR-Cas9-mediated cytosine base editing can efficiently generate C: G-to-T: A conversion in the genome of living cells. One intriguing application of base editing is to screen pivotal variants for gene function that is yet to be achieved in pigs. Here, we illustrate that CRISPR-Cas9-mediated cytosine base editor, known as AncBE4max, can be used for the functional analysis of calreticulin (CALR) variants. We conducted a CRISPR-Cas9-mediated cytosine base editing screen using 457 single guide RNAs (sgRNAs) against all exons and introns of CALR to identify loss-of-function variants involved in JEV replication. We unexpectedly uncovered that two enriched sgRNAs targeted the same site in intron-2 of the CALR gene. We found that mutating four consecutive G bases in the intron-2 of the CALR gene to four A bases significantly inhibited JEV replication. Thus, we established a CRISPR-Cas9-mediated cytosine-base-editing point mutation screening technique in pigs. Our results suggest that CRISPR-mediated base editing is a powerful tool for identifying the antiviral functions of variants in the coding and noncoding regions of the CALR gene. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 3.0)
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