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CRISPR-Cas Systems and Genome Editing—2nd Edition

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

Deadline for manuscript submissions: closed (20 March 2025) | Viewed by 14498

Special Issue Editor

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
Interests: CRISPR/Cas; genome editing; yeast; intracellular proteolysis; transcription regulation; Bacillii; stress responses
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Almost everyone has heard of CRISPR/Cas systems. The use of CRISPR/Cas systems as RNA-programmable site-directed nucleases has given a powerful impulse to the further development of genome editing technology. Moreover, numerous modifications of CRISPR/Cas systems have enabled the development of principally new methods for genome engineering. New molecular tools have also become widespread in other areas of science, biotechnology and biomedicine. The emergence of such versatile, relatively easy-to-use and effective molecular tools has increased human hope for effective treatment of hereditary, bacterial, or viral diseases. However, CRISPR/Cas systems have a number of drawbacks that limit their application in therapeutic approaches. Thus, research to find new genome editors, modify existing genome editing technologies, and study the universe of defense mechanisms of prokaryotes may lead to the development of fundamentally new approaches to genome editing. This special issue aims to cover all areas of both basic research on CRISPR/Cas and other genome editing systems and their applications in basic science, biotechnology, and biomedicine. It welcomes original research, theoretical articles, reviews, and communications in which CRISPR/Cas or other genome editing technologies are a major topic or tool for mechanistic molecular research. This includes, but is not limited to, methodological advances in genome editing technologies and their applications to the study and treatment of hereditary, bacterial or viral human diseases, the development of new microbial producents, cultivated plant lines and fungi.

Please note that, for IJMS’s paper, theoretical studies should offer new insights into the understanding of experimental results or suggest new experimentally testable hypotheses.

Dr. Dmitry Karpov
Guest Editor

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Keywords

  • prokaryotic defense systems with genome editing potential (CRISPR/Cas, bacterial argonauts, etc.)
  • development or improvement of genome editing tools
  • genome editing technologies in the generation and study of cellular, multicellular or animal models of hereditary, bacterial, or viral human diseases
  • CRISPR/Cas systems in the therapy of human diseases
  • genome editing to probe biotechnological processes and improve microbial producents, cultivated plants, and fungi

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Published Papers (10 papers)

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Research

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26 pages, 4704 KiB  
Article
CRISPR/Cas9 Ribonucleoprotein Delivery Enhanced by Lipo-Xenopeptide Carriers and Homology-Directed Repair Modulators: Insights from Reporter Cell Lines
by Xianjin Luo, Eric Weidinger, Tobias Burghardt, Miriam Höhn and Ernst Wagner
Int. J. Mol. Sci. 2025, 26(9), 4361; https://doi.org/10.3390/ijms26094361 (registering DOI) - 3 May 2025
Abstract
CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to [...] Read more.
CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy. Full article
(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing—2nd Edition)
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21 pages, 6509 KiB  
Article
Generation of the Krt24-CreERT2 Mouse Line Targeting Outer Bulge Hair Follicle Cells
by Jiao Wang, Yifei Qiu, Yansheng Zhu, Xuejiao Ren, Xiaoqi Zhou, Xia Wang, Huiyang Song, Jianhao Li, Chengming Gao, Gangqiao Zhou and Pengbo Cao
Int. J. Mol. Sci. 2025, 26(7), 3165; https://doi.org/10.3390/ijms26073165 - 29 Mar 2025
Viewed by 340
Abstract
Outer bulge (OB) hair follicle stem cells (HFSCs) play a crucial role in maintaining hair follicle structural stability and regulating the hair follicle cycle. Previous studies demonstrated that keratin 24 (Krt24) exhibits spatiotemporally restricted expression in OB HFSCs. Here, we report [...] Read more.
Outer bulge (OB) hair follicle stem cells (HFSCs) play a crucial role in maintaining hair follicle structural stability and regulating the hair follicle cycle. Previous studies demonstrated that keratin 24 (Krt24) exhibits spatiotemporally restricted expression in OB HFSCs. Here, we report the generation of the Krt24-CreERT2 mouse line. When crossed with Rosa26LSL-tdTomato or Rosa26LSL-DTR reporter lines, offspring exhibited specific labeling (Krt24-CreERT2;Rosa26LSL-tdTomato) or ablation (Krt24-CreERT2;Rosa26LSL-DTR) of Krt24+ cells. In Krt24-CreERT2;Rosa26LSL-tdTomato mice, phase-specific tamoxifen (TAM) administration demonstrated spatiotemporal fidelity of Cre activity to endogenous Krt24 expression patterns. Lineage tracing revealed that tdTomato-labeled Krt24+ cells differentiated into the outer root sheath (ORS) during the anagen phase and persisted when hair follicles reentered telogen. Ablation of Krt24+ cells via diphtheria toxin (DT) administration significantly delayed anagen initiation. Mice under continuous depletion of Krt24+ HFSCs experienced substantial mortality after ionizing irradiation. Notably, ionizing radiation triggered a marked expansion of tdTomato-labeled Krt24+ cells, accompanied by maintained hair follicle homeostasis. Taken together, this study established a Krt24-CreERT2 mouse line targeting OB HFSCs, which are essential for hair follicle development and damage repair. Full article
(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing—2nd Edition)
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13 pages, 884 KiB  
Article
Insight into crRNA Processing in Streptococcus mutans P42S and Application of SmutCas9 in Genome Editing
by Cas Mosterd and Sylvain Moineau
Int. J. Mol. Sci. 2025, 26(5), 2005; https://doi.org/10.3390/ijms26052005 - 25 Feb 2025
Viewed by 471
Abstract
CRISPR-Cas is an adaptive immune system found in bacteria and archaea that provides resistance against invading nucleic acids. Elements of this natural system have been harnessed to develop several genome editing tools, including CRISPR-Cas9. This technology relies on the ability of the nuclease [...] Read more.
CRISPR-Cas is an adaptive immune system found in bacteria and archaea that provides resistance against invading nucleic acids. Elements of this natural system have been harnessed to develop several genome editing tools, including CRISPR-Cas9. This technology relies on the ability of the nuclease Cas9 to cut DNA at specific locations directed by a guide RNA. In addition, the nuclease activity of Cas9 requires the presence of a short nucleotide motif (5′-NGG-3′ for Cas9 from Streptococcus pyogenes) called PAM, flanking the targeted region. As the reliance on this PAM is typically strict, diverse Cas9 variants recognising different PAM motifs have been studied to target a broader range of genomic sites. In this study, we assessed the potential of Cas9 from Streptococcus mutans strain P42S (SmutCas9) in gene editing. SmutCas9 recognises the rarely targeted 5′-NAA-3′ and 5′-NGAA-3′ PAMs. To test its efficacy, two genes of the virulent lactococcal phage p2 were edited, thereby demonstrating the potential of SmutCas9 for gene editing purposes, particularly in AT-rich genomes. Sequencing of total RNA also revealed the RNA components of this system, allowing further molecular characterisation of the type II-A CRISPR-Cas system of S. mutans. Full article
(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing—2nd Edition)
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23 pages, 5972 KiB  
Article
Mutations in Filamin C Associated with Both Alleles Do Not Affect the Functioning of Mice Cardiac Muscles
by Leonid A. Ilchuk, Ksenia K. Kochegarova, Iuliia P. Baikova, Polina D. Safonova, Alexandra V. Bruter, Marina V. Kubekina, Yulia D. Okulova, Tatiana E. Minkovskaya, Nadezhda A. Kuznetsova, Daria M. Dolmatova, Anna Yu. Ryabinina, Andrey A. Mozhaev, Vsevolod V. Belousov, Boris P. Ershov, Peter S. Timashev, Maxim A. Filatov and Yulia Yu. Silaeva
Int. J. Mol. Sci. 2025, 26(4), 1409; https://doi.org/10.3390/ijms26041409 - 7 Feb 2025
Viewed by 807
Abstract
Filamin C (FLNC) is a structural protein of muscle fibers. Mutations in the FLNC gene are known to cause myopathies and cardiomyopathies in humans. Here we report the generation by a CRISPR/Cas9 editing system injected into zygote pronuclei of two mouse strains carrying [...] Read more.
Filamin C (FLNC) is a structural protein of muscle fibers. Mutations in the FLNC gene are known to cause myopathies and cardiomyopathies in humans. Here we report the generation by a CRISPR/Cas9 editing system injected into zygote pronuclei of two mouse strains carrying filamin C mutations—one of them (AGA) has a deletion of three nucleotides at position c.7418_7420, causing E>>D substitution and N deletion at positions 2472 and 2473, respectively. The other strain carries a deletion of GA nucleotides at position c.7419_7420, leading to a frameshift and a premature stop codon. Homozygous animals (FlncAGA/AGA and FlncGA/GA) were embryonically lethal. We determined that FlncGA/GA embryos died prior to the E12.5 stage and illustrated delayed development after the E9.5 stage. We performed histological analysis of heart tissue and skeletal muscles of heterozygous strains carrying mutations in different combinations (FlncGA/wt, FlncAGA/wt, and FlncGA/AGA). By performing physiological tests (grip strength and endurance tests), we have shown that heterozygous animals of both strains (FlncGA/wt, FlncAGA/wt) are functionally indistinguishable from wild-type animals. Interestingly, compound heterozygous mice (FlncGA/AGA) are viable, develop normally, reach puberty and it was verified by ECG and Eco-CG that their cardiac muscle is functionally normal. Intriguingly, FlncGA/AGA mice demonstrated better results in the grip strength physiological test in comparison to WT animals. We also propose a structural model that explains the complementary interaction of two mutant variants of filamin C. Full article
(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing—2nd Edition)
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21 pages, 2940 KiB  
Article
Construction of a TAT-Cas9-EGFP Site-Specific Integration Eukaryotic Cell Line Using Efficient PEG10 Modification
by Shiyu Qi, Yibo Wang, Zhimei Liu, Sujun Wu, Yue Zhao, Yan Li, Shoulong Deng, Kun Yu and Zhengxing Lian
Int. J. Mol. Sci. 2025, 26(3), 1331; https://doi.org/10.3390/ijms26031331 - 4 Feb 2025
Viewed by 767
Abstract
The CRISPR/Cas9 system enables precise and efficient modification of eukaryotic genomes. Among its various applications, homology-directed repair (HDR) mediated knock-in (KI) is crucial for creating human disease models, gene therapy, and agricultural genetic enhancements. Despite its potential, HDR-mediated knock-in efficiency remains relatively low. [...] Read more.
The CRISPR/Cas9 system enables precise and efficient modification of eukaryotic genomes. Among its various applications, homology-directed repair (HDR) mediated knock-in (KI) is crucial for creating human disease models, gene therapy, and agricultural genetic enhancements. Despite its potential, HDR-mediated knock-in efficiency remains relatively low. This study investigated the impact of 5′ end PEG10 modification on site-specific integration of the target gene. The HEK293 cell line is considered a highly attractive expression system for the production of recombinant proteins, with the construction of site-specific integration cell lines at the AAVS1 locus enabling stable protein expression. This study investigated the impact of the 5′ end PEG10 modification on the site-specific integration of the target gene at the AAVS1 locus in the 293T cell line. Utilizing this 5′ end PEG10 modification resulted in a 1.9-fold increase in knock-in efficiency for a 1.8 kb target fragment, improving efficiency from 26% to 49%. An optimized system was utilized to successfully establish a high-expression, site-specific integration 293T cell line for TAT-Cas9-EGFP, providing a reliable resource of seed cells for subsequent protein production. Full article
(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing—2nd Edition)
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17 pages, 3623 KiB  
Article
Two Novel Mouse Models of Duchenne Muscular Dystrophy with Similar Dmd Exon 51 Frameshift Mutations and Varied Phenotype Severity
by Iuliia P. Baikova, Leonid A. Ilchuk, Polina D. Safonova, Ekaterina A. Varlamova, Yulia D. Okulova, Marina V. Kubekina, Anna V. Tvorogova, Daria M. Dolmatova, Zanda V. Bakaeva, Evgenia N. Kislukhina, Natalia V. Lizunova, Alexandra V. Bruter and Yulia Yu. Silaeva
Int. J. Mol. Sci. 2025, 26(1), 158; https://doi.org/10.3390/ijms26010158 - 27 Dec 2024
Viewed by 1343
Abstract
Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder caused by an array of mutations in the dystrophin gene, with the most commonly mutated regions being exons 48–55. One of the several existing approaches to treat DMD is gene therapy, based on [...] Read more.
Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder caused by an array of mutations in the dystrophin gene, with the most commonly mutated regions being exons 48–55. One of the several existing approaches to treat DMD is gene therapy, based on alternative splicing and mutant exon skipping. Testing of such therapy requires animal models that carry mutations homologous to those found in human patients. Here, we report the generation of two genetically modified mouse lines, named “insT” and “insG”, with distinct mutations at the same position in exon 51 that lead to a frameshift, presumably causing protein truncation. Hemizygous males of both lines exhibit classical signs of muscular dystrophy in all muscle tissues except for the cardiac tissue. However, pathological changes are more pronounced in one of the lines. Membrane localization of the protein is reduced to the point of absence in one of the lines. Moreover, an increase in full-length isoform mRNA was detected in diaphragms of insG line mice. Although further work is needed to qualify these mutations as sole origins of dissimilarity, both genetically modified mouse lines are suitable models of DMD and can be used to test gene therapy based on alternative splicing. Full article
(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing—2nd Edition)
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30 pages, 3897 KiB  
Article
Efficient Genome Editing Using ‘NanoMEDIC’ AsCas12a-VLPs Produced with Pol II-Transcribed crRNA
by Sofiia E. Borovikova, Mikhail V. Shepelev, Dmitriy V. Mazurov and Natalia A. Kruglova
Int. J. Mol. Sci. 2024, 25(23), 12768; https://doi.org/10.3390/ijms252312768 - 27 Nov 2024
Viewed by 1422
Abstract
Virus-like particles (VLPs) are an attractive vehicle for the delivery of Cas nuclease and guide RNA ribonucleoprotein complexes (RNPs). Most VLPs are produced by packaging SpCas9 and its sgRNA, which is expressed from the RNA polymerase III (Pol III)-transcribed U6 promoter. VLPs assemble [...] Read more.
Virus-like particles (VLPs) are an attractive vehicle for the delivery of Cas nuclease and guide RNA ribonucleoprotein complexes (RNPs). Most VLPs are produced by packaging SpCas9 and its sgRNA, which is expressed from the RNA polymerase III (Pol III)-transcribed U6 promoter. VLPs assemble in the cytoplasm, but U6-driven sgRNA is localized in the nucleus, which hinders the efficient formation and packaging of RNPs into VLPs. In this study, using the nuclease packaging mechanism of ‘NanoMEDIC’ VLPs, we produced VLPs with AsCas12a and exploited its ability to process pre-crRNA. This allowed us to direct crRNA in the cytoplasm as part of a Pol II-driven transcript where AsCas12a excised mature crRNA, thus boosting RNP incorporation into VLPs. CMV-driven crRNA increased Venus and CCR5 transgene knockout levels in 293 cells from 30% to 50–90% and raised the level of endogenous CXCR4 knockout in Jurkat T cells from 1% to 20%. Changing a single crRNA to an array of three or six identical crRNAs improved CXCR4 knockout rates by up to 60–70%. Compared to SpCas9-VLPs, the editing efficiencies of AsCas12a-VLPs were higher, regardless of promoter usage. Thus, we showed that AsCas12a and CMV-driven crRNA could be efficiently packaged into VLPs and mediate high levels of gene editing. AsCas12a-VLPs are a new and promising tool for the delivery of RNPs into mammalian cells that will allow efficient target genome editing and may be useful for gene therapy applications. Full article
(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing—2nd Edition)
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14 pages, 4168 KiB  
Article
Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines
by Zhimei Liu, Yue Zhao, Sujun Wu, Shiyu Qi, Yefeng Qiu and Zhengxing Lian
Int. J. Mol. Sci. 2024, 25(20), 11320; https://doi.org/10.3390/ijms252011320 - 21 Oct 2024
Viewed by 1407
Abstract
The technology for obtaining the high-efficiency expression of target proteins through site-specific recombination has made progress. However, using the CRISPR/Cas9 system for site-specific integration of long fragments and the expression of active proteins remains a challenge. This study optimized the linear DNA circularization [...] Read more.
The technology for obtaining the high-efficiency expression of target proteins through site-specific recombination has made progress. However, using the CRISPR/Cas9 system for site-specific integration of long fragments and the expression of active proteins remains a challenge. This study optimized the linear DNA circularization system, eliminated the prokaryotic plasmid backbone on the traditional foreign gene vector, and generated a homologous arm-free circular donor template with a single guide RNA target site (sgRNA TS). This strategy significantly increased the co-transfection efficiency of the 1.6 kb template and Cas9 plasmid by 1.15-fold, and the average knock-in (KI) efficiency of the 4.7 kb long-fragment template for the two target gene sites increased by 1.3-fold. Subsequently, we used rhBCHE as a reporter gene to efficiently integrate the 5.4 kb fragment containing the gene of interest (GOI) into specific sites in the HEK293T cell line to detect the expression of the circular template at different target sites. Overall, this study further verifies that the length of the circular donor is more conducive to non-homologous integration, and more importantly, we provide a simple and optimized strategy for the construction of long-fragment site integration cell lines. Full article
(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing—2nd Edition)
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Review

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20 pages, 1134 KiB  
Review
Expanding Horizons of CRISPR/Cas Technology: Clinical Advancements, Therapeutic Applications, and Challenges in Gene Therapy
by Ahmad Bairqdar, Polina E. Karitskaya and Grigory A. Stepanov
Int. J. Mol. Sci. 2024, 25(24), 13321; https://doi.org/10.3390/ijms252413321 - 12 Dec 2024
Cited by 2 | Viewed by 3197
Abstract
CRISPR–Cas technology has transformed the field of gene editing, opening new possibilities for treatment of various genetic disorders. Recent years have seen a surge in clinical trials using CRISPR–Cas-based therapies. This review examines the current landscape of CRISPR–Cas implementation in clinical trials, with [...] Read more.
CRISPR–Cas technology has transformed the field of gene editing, opening new possibilities for treatment of various genetic disorders. Recent years have seen a surge in clinical trials using CRISPR–Cas-based therapies. This review examines the current landscape of CRISPR–Cas implementation in clinical trials, with data from key registries, including the Australian New Zealand Clinical Trials Registry, the Chinese Clinical Trial Register, and ClinicalTrials.gov. Emphasis is placed on the mechanism of action of tested therapies, the delivery method, and the most recent findings of each clinical trial. Full article
(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing—2nd Edition)
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38 pages, 2307 KiB  
Review
Control of HSV-1 Infection: Directions for the Development of CRISPR/Cas-Based Therapeutics and Diagnostics
by Anastasiia O. Sosnovtseva, Natalia A. Demidova, Regina R. Klimova, Maxim A. Kovalev, Alla A. Kushch, Elizaveta S. Starodubova, Anastasia A. Latanova and Dmitry S. Karpov
Int. J. Mol. Sci. 2024, 25(22), 12346; https://doi.org/10.3390/ijms252212346 - 17 Nov 2024
Viewed by 3135
Abstract
It is estimated that nearly all individuals have been infected with herpesviruses, with herpes simplex virus type 1 (HSV-1) representing the most prevalent virus. In most cases, HSV-1 causes non-life-threatening skin damage in adults. However, in patients with compromised immune systems, it can [...] Read more.
It is estimated that nearly all individuals have been infected with herpesviruses, with herpes simplex virus type 1 (HSV-1) representing the most prevalent virus. In most cases, HSV-1 causes non-life-threatening skin damage in adults. However, in patients with compromised immune systems, it can cause serious diseases, including death. The situation is further complicated by the emergence of strains that are resistant to both traditional and novel antiviral drugs. It is, therefore, imperative that new methods of combating HSV-1 and other herpesviruses be developed without delay. CRISPR/Cas systems may prove an effective means of controlling herpesvirus infections. This review presents the current understanding of the underlying molecular mechanisms of HSV-1 infection and discusses four potential applications of CRISPR/Cas systems in the fight against HSV-1 infections. These include the search for viral and cellular genes that may serve as effective targets, the optimization of anti-HSV-1 activity of CRISPR/Cas systems in vivo, the development of CRISPR/Cas-based HSV-1 diagnostics, and the validation of HSV-1 drug resistance mutations. Full article
(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing—2nd Edition)
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