International Journal of Molecular Sciences

Editorial

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Open AccessEditorial

CRISPR-Cas Systems and Genome Editing: Beginning the Era of CRISPR/Cas Therapies for Humans

by Dmitry S. Karpov

Int. J. Mol. Sci. 2024, 25(10), 5292; https://doi.org/10.3390/ijms25105292 - 13 May 2024

Cited by 4 | Viewed by 3329

Abstract

Harnessing of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated genes) systems for detection, chemical modification, and sequence editing of nucleic acids dramatically changed many fields of fundamental science, biotechnology, and biomedicine [...] Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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Research

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12 pages, 2281 KiB

Open AccessArticle

An Efficient Expression and Purification Protocol for SpCas9 Nuclease and Evaluation of Different Delivery Methods of Ribonucleoprotein

by Konstantin Evmenov, Nikolay Pustogarov, Dmitri Panteleev, Artur Safin and Elena Alkalaeva

Int. J. Mol. Sci. 2024, 25(3), 1622; https://doi.org/10.3390/ijms25031622 - 28 Jan 2024

Cited by 4 | Viewed by 4536

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a revolutionary tool for precise genome editing across various cell types. Ribonucleoproteins (RNPs), encompassing the Cas9 protein and guide RNA (gRNA), have emerged as a promising technique due to their increased specificity and [...] Read more.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a revolutionary tool for precise genome editing across various cell types. Ribonucleoproteins (RNPs), encompassing the Cas9 protein and guide RNA (gRNA), have emerged as a promising technique due to their increased specificity and reduced off-target effects. This method eliminates the need for plasmid DNA introduction, thereby preventing potential integration of foreign DNA into the target cell genome. Given the requirement for large quantities of highly purified protein in various Cas9 studies, we present an efficient and simple method for the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein. This method leverages the Small Ubiquitin Like Modifier(SUMO) tag system, which includes metal-affinity chromatography followed by anion-exchange chromatography purification. Furthermore, we compare two methods of CRISPR-Cas9 system delivery into cells: transfection with plasmid DNA encoding the CRISPR-Cas9 system and RNP transfection with the Cas9-gRNA complex. We estimate the efficiency of genomic editing and protein lifespan post-transfection. Intriguingly, we found that RNP treatment of cells, even in the absence of a transfection system, is a relatively efficient method for RNP delivery into cell culture. This discovery is particularly promising as it can significantly reduce cytotoxicity, which is crucial for certain cell cultures such as induced pluripotent stem cells (iPSCs). Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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17 pages, 7396 KiB

Open AccessArticle

A Knockout of the IFITM3 Gene Increases the Sensitivity of WI-38 VA13 Cells to the Influenza A Virus

by Natalya Eshchenko, Mariia Sergeeva, Evgenii Zhuravlev, Kira Kudria, Elena Goncharova, Andrey Komissarov and Grigory Stepanov

Int. J. Mol. Sci. 2024, 25(1), 625; https://doi.org/10.3390/ijms25010625 - 3 Jan 2024

Viewed by 2331

Abstract

One of the ways to regulate the sensitivity of human cells to the influenza virus is to knock out genes of the innate immune response. Promising targets for the knockout are genes of the interferon-inducible transmembrane protein (IFITM) family, in particular the IFITM3 [...] Read more.

One of the ways to regulate the sensitivity of human cells to the influenza virus is to knock out genes of the innate immune response. Promising targets for the knockout are genes of the interferon-inducible transmembrane protein (IFITM) family, in particular the IFITM3 gene, whose product limits the entry of a virus into the cell by blocking the fusion of the viral and endosomal membranes. In this study, by means of genome-editing system CRISPR/Cas9, monoclonal cell lines with an IFITM3 knockout were obtained based on WI-38 VA13 cells (human origin). It was found that such cell lines are more sensitive to infection by influenza A viruses of various subtypes. Nevertheless, this feature is not accompanied by an increased titer of newly formed viral particles in a culture medium. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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21 pages, 7212 KiB

Open AccessArticle

Intron Editing Reveals SNORD-Dependent Maturation of the Small Nucleolar RNA Host Gene GAS5 in Human Cells

by Anastasiya Matveeva, Dmitry Vinogradov, Evgenii Zhuravlev, Dmitriy Semenov, Valentin Vlassov and Grigory Stepanov

Int. J. Mol. Sci. 2023, 24(24), 17621; https://doi.org/10.3390/ijms242417621 - 18 Dec 2023

Cited by 3 | Viewed by 1923

Abstract

The GAS5 gene encodes a long non-coding RNA (lncRNA) and intron-located small nucleolar RNAs (snoRNAs). Its structure, splice variants, and diverse functions in mammalian cells have been thoroughly investigated. However, there are still no data on a successful knockout of GAS5 in human [...] Read more.

The GAS5 gene encodes a long non-coding RNA (lncRNA) and intron-located small nucleolar RNAs (snoRNAs). Its structure, splice variants, and diverse functions in mammalian cells have been thoroughly investigated. However, there are still no data on a successful knockout of GAS5 in human cells, with most of the loss-of-function experiments utilizing standard techniques to produce knockdowns. By using CRISPR/Cas9 to introduce double-strand breaks in the terminal intronic box C/D snoRNA genes (SNORDs), we created monoclonal cell lines carrying continuous deletions in one of the GAS5 alleles. The levels of GAS5-encoded box C/D snoRNAs and lncRNA GAS5 were assessed, and the formation of the novel splice variants was analyzed. To comprehensively evaluate the influence of specific SNORD mutations, human cell lines with individual mutations in SNORD74 and SNORD81 were obtained. Specific mutations in SNORD74 led to the downregulation of all GAS5-encoded SNORDs and GAS5 lncRNA. Further analysis revealed that SNORD74 contains a specific regulatory element modulating the maturation of the GAS5 precursor transcript. The results demonstrate that the maturation of GAS5 occurs through the m6A-associated pathway in a SNORD-dependent manner, which is a quite intriguing epitranscriptomic mechanism. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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20 pages, 3159 KiB

Open AccessArticle

Expression and Functional Analysis of the Compact Thermophilic Anoxybacillus flavithermus Cas9 Nuclease

by Anastasiya Matveeva, Alexander Ryabchenko, Viktoria Petrova, Daria Prokhorova, Evgenii Zhuravlev, Alexander Zakabunin, Artem Tikunov and Grigory Stepanov

Int. J. Mol. Sci. 2023, 24(23), 17121; https://doi.org/10.3390/ijms242317121 - 4 Dec 2023

Viewed by 1681

Abstract

Research on Cas9 nucleases from different organisms holds great promise for advancing genome engineering and gene therapy tools, as it could provide novel structural insights into CRISPR editing mechanisms, expanding its application area in biology and medicine. The subclass of thermophilic Cas9 nucleases [...] Read more.

Research on Cas9 nucleases from different organisms holds great promise for advancing genome engineering and gene therapy tools, as it could provide novel structural insights into CRISPR editing mechanisms, expanding its application area in biology and medicine. The subclass of thermophilic Cas9 nucleases is actively expanding due to the advances in genome sequencing allowing for the meticulous examination of various microorganisms’ genomes in search of the novel CRISPR systems. The most prominent thermophilic Cas9 effectors known to date are GeoCas9, ThermoCas9, IgnaviCas9, AceCas9, and others. These nucleases are characterized by a varying temperature range of the activity and stringent PAM preferences; thus, further diversification of the naturally occurring thermophilic Cas9 subclass presents an intriguing task. This study focuses on generating a construct to express a compact Cas9 nuclease (AnoCas9) from the thermophilic microorganism Anoxybacillus flavithermus displaying the nuclease activity in the 37–60 °C range and the PAM preference of 5′-NNNNCDAA-3′ in vitro. Here, we highlight the close relation of AnoCas9 to the GeoCas9 family of compact thermophilic Cas9 effectors. AnoCas9, beyond broadening the repertoire of Cas9 nucleases, suggests application in areas requiring the presence of thermostable CRISPR/Cas systems in vitro, such as sequencing libraries’ enrichment, allele-specific isothermal PCR, and others. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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11 pages, 1420 KiB

Open AccessArticle

Influence of N1-Methylpseudouridine in Guide RNAs on CRISPR/Cas9 Activity

by Daria Prokhorova, Anastasiya Matveeva, Alexander Zakabunin, Alexander Ryabchenko and Grigory Stepanov

Int. J. Mol. Sci. 2023, 24(23), 17116; https://doi.org/10.3390/ijms242317116 - 4 Dec 2023

Cited by 3 | Viewed by 2745

Abstract

At present, there are many strategies to improve the activity of CRISPR/Cas9. A well-known and effective approach is guide RNA modification. Many chemical guide RNA modifications have been studied, whereas naturally occurring RNA modifications are largely unexplored. N1-methylpseudouridine (m1Ψ) is an RNA base [...] Read more.

At present, there are many strategies to improve the activity of CRISPR/Cas9. A well-known and effective approach is guide RNA modification. Many chemical guide RNA modifications have been studied, whereas naturally occurring RNA modifications are largely unexplored. N1-methylpseudouridine (m1Ψ) is an RNA base modification widely used in mRNA therapy, and it holds great promise for application in genome editing systems. The present study focuses on investigating the effect of N1-methylpseudouridine on the functioning of CRISPR/Cas9. In vitro cleavage assays helped determine the level of m1Ψ guide RNA modification that is sufficient to cleave the target substrate. By analyzing FAM-labeled dsDNA substrate cleavage, we calculated the kinetic parameters and the specificity scores of modified guide RNAs. Neon transfection and digital PCR enabled us to assess the activity of modified guide RNAs in mammalian cells. Our study shows that the presence of m1Ψ in guide RNAs can help preserve on-target genome editing while significantly reducing the off-target effects of CRISPR/Cas9 in vitro. We also demonstrate that Cas9 complexes with guide RNAs containing m1Ψ allow for genome editing in human cells. Thus, the incorporation of m1Ψ into guide RNAs supports CRISPR/Cas9 activity both in vitro and in cells. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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22 pages, 4845 KiB

Open AccessArticle

ASCL1 Is Involved in the Pathogenesis of Schizophrenia by Regulation of Genes Related to Cell Proliferation, Neuronal Signature Formation, and Neuroplasticity

by Dmitrii A. Abashkin, Dmitry S. Karpov, Artemii O. Kurishev, Ekaterina V. Marilovtseva and Vera E. Golimbet

Int. J. Mol. Sci. 2023, 24(21), 15746; https://doi.org/10.3390/ijms242115746 - 30 Oct 2023

Cited by 3 | Viewed by 2442

Abstract

Schizophrenia (SZ) is a common psychiatric neurodevelopmental disorder with a complex genetic architecture. Genome-wide association studies indicate the involvement of several transcription factors, including ASCL1, in the pathogenesis of SZ. We aimed to identify ASCL1-dependent cellular and molecular mechanisms associated with SZ. We [...] Read more.

Schizophrenia (SZ) is a common psychiatric neurodevelopmental disorder with a complex genetic architecture. Genome-wide association studies indicate the involvement of several transcription factors, including ASCL1, in the pathogenesis of SZ. We aimed to identify ASCL1-dependent cellular and molecular mechanisms associated with SZ. We used Capture-C, CRISPR/Cas9 systems and RNA-seq analysis to confirm the involvement of ASCL1 in SZ-associated pathogenesis, establish a mutant SH-SY5Y line with a functional ASCL1 knockout (ASCL1-del) and elucidate differentially expressed genes that may underlie ASCL1-dependent pathogenic mechanisms. Capture-C confirmed the spatial interaction of the ASCL1 promoter with SZ-associated loci. Transcriptome analysis showed that ASCL1 regulation may be through a negative feedback mechanism. ASCL1 dysfunction affects the expression of genes associated with the pathogenesis of SZ, as well as bipolar and depressive disorders. Genes differentially expressed in ASCL1-del are involved in cell mitosis, neuronal projection, neuropeptide signaling, and the formation of intercellular contacts, including the synapse. After retinoic acid (RA)-induced differentiation, ASCL1 activity is restricted to a small subset of genes involved in neuroplasticity. These data suggest that ASCL1 dysfunction promotes SZ development predominantly before the onset of neuronal differentiation by slowing cell proliferation and impeding the formation of neuronal signatures. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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16 pages, 5888 KiB

Open AccessArticle

Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems

by Dmitry S. Karpov, Natalia A. Demidova, Kirill A. Kulagin, Anastasija I. Shuvalova, Maxim A. Kovalev, Ruslan A. Simonov, Vadim L. Karpov, Anastasiya V. Snezhkina, Anna V. Kudryavtseva, Regina R. Klimova and Alla A. Kushch

Int. J. Mol. Sci. 2022, 23(23), 14847; https://doi.org/10.3390/ijms232314847 - 27 Nov 2022

Cited by 6 | Viewed by 5438

Abstract

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms [...] Read more.

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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Review

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20 pages, 1653 KiB

Open AccessReview

Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches

by Andrés Felipe Leal, Angelica María Herreno-Pachón, Eliana Benincore-Flórez, Amali Karunathilaka and Shunji Tomatsu

Int. J. Mol. Sci. 2024, 25(5), 2456; https://doi.org/10.3390/ijms25052456 - 20 Feb 2024

Cited by 8 | Viewed by 10885

Abstract

Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with [...] Read more.

Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient’s death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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71 pages, 34184 KiB

Open AccessReview

Editing Metabolism, Sex, and Microbiome: How Can We Help Poplar Resist Pathogens?

by Maxim A. Kovalev, Natalya S. Gladysh, Alina S. Bogdanova, Nadezhda L. Bolsheva, Mikhail I. Popchenko and Anna V. Kudryavtseva

Int. J. Mol. Sci. 2024, 25(2), 1308; https://doi.org/10.3390/ijms25021308 - 21 Jan 2024

Cited by 1 | Viewed by 3064

Abstract

Poplar (Populus) is a genus of woody plants of great economic value. Due to the growing economic importance of poplar, there is a need to ensure its stable growth by increasing its resistance to pathogens. Genetic engineering can create organisms with [...] Read more.

Poplar (Populus) is a genus of woody plants of great economic value. Due to the growing economic importance of poplar, there is a need to ensure its stable growth by increasing its resistance to pathogens. Genetic engineering can create organisms with improved traits faster than traditional methods, and with the development of CRISPR/Cas-based genome editing systems, scientists have a new highly effective tool for creating valuable genotypes. In this review, we summarize the latest research data on poplar diseases, the biology of their pathogens and how these plants resist pathogens. In the final section, we propose to plant male or mixed poplar populations; consider the genes of the MLO group, transcription factors of the WRKY and MYB families and defensive proteins BbChit1, LJAMP2, MsrA2 and PtDef as the most promising targets for genetic engineering; and also pay attention to the possibility of microbiome engineering. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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21 pages, 3825 KiB

Open AccessReview

CRISPR/Cas9 as a New Antiviral Strategy for Treating Hepatitis Viral Infections

by Ulyana I. Bartosh, Anton S. Dome, Natalya V. Zhukova, Polina E. Karitskaya and Grigory A. Stepanov

Int. J. Mol. Sci. 2024, 25(1), 334; https://doi.org/10.3390/ijms25010334 - 26 Dec 2023

Cited by 7 | Viewed by 3468

Abstract

Hepatitis is an inflammatory liver disease primarily caused by hepatitis A (HAV), B (HBV), C (HCV), D (HDV), and E (HEV) viruses. The chronic forms of hepatitis resulting from HBV and HCV infections can progress to cirrhosis or hepatocellular carcinoma (HCC), while acute [...] Read more.

Hepatitis is an inflammatory liver disease primarily caused by hepatitis A (HAV), B (HBV), C (HCV), D (HDV), and E (HEV) viruses. The chronic forms of hepatitis resulting from HBV and HCV infections can progress to cirrhosis or hepatocellular carcinoma (HCC), while acute hepatitis can lead to acute liver failure, sometimes resulting in fatality. Viral hepatitis was responsible for over 1 million reported deaths annually. The treatment of hepatitis caused by viral infections currently involves the use of interferon-α (IFN-α), nucleoside inhibitors, and reverse transcriptase inhibitors (for HBV). However, these methods do not always lead to a complete cure for viral infections, and chronic forms of the disease pose significant treatment challenges. These facts underscore the urgent need to explore novel drug developments for the treatment of viral hepatitis. The discovery of the CRISPR/Cas9 system and the subsequent development of various modifications of this system have represented a groundbreaking advance in the quest for innovative strategies in the treatment of viral infections. This technology enables the targeted disruption of specific regions of the genome of infectious agents or the direct manipulation of cellular factors involved in viral replication by introducing a double-strand DNA break, which is targeted by guide RNA (spacer). This review provides a comprehensive summary of our current knowledge regarding the application of the CRISPR/Cas system in the regulation of viral infections caused by HAV, HBV, and HCV. It also highlights new strategies for drug development aimed at addressing both acute and chronic forms of viral hepatitis. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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33 pages, 3266 KiB

Open AccessReview

Challenges of CRISPR/Cas-Based Cell Therapy for Type 1 Diabetes: How Not to Engineer a “Trojan Horse”

by Dmitry S. Karpov, Anastasiia O. Sosnovtseva, Svetlana V. Pylina, Asya N. Bastrich, Darya A. Petrova, Maxim A. Kovalev, Anastasija I. Shuvalova, Anna K. Eremkina and Natalia G. Mokrysheva

Int. J. Mol. Sci. 2023, 24(24), 17320; https://doi.org/10.3390/ijms242417320 - 10 Dec 2023

Cited by 11 | Viewed by 5571

Abstract

Type 1 diabetes mellitus (T1D) is an autoimmune disease caused by the destruction of insulin-producing β-cells in the pancreas by cytotoxic T-cells. To date, there are no drugs that can prevent the development of T1D. Insulin replacement therapy is the standard care for [...] Read more.

Type 1 diabetes mellitus (T1D) is an autoimmune disease caused by the destruction of insulin-producing β-cells in the pancreas by cytotoxic T-cells. To date, there are no drugs that can prevent the development of T1D. Insulin replacement therapy is the standard care for patients with T1D. This treatment is life-saving, but is expensive, can lead to acute and long-term complications, and results in reduced overall life expectancy. This has stimulated the research and development of alternative treatments for T1D. In this review, we consider potential therapies for T1D using cellular regenerative medicine approaches with a focus on CRISPR/Cas-engineered cellular products. However, CRISPR/Cas as a genome editing tool has several drawbacks that should be considered for safe and efficient cell engineering. In addition, cellular engineering approaches themselves pose a hidden threat. The purpose of this review is to critically discuss novel strategies for the treatment of T1D using genome editing technology. A well-designed approach to β-cell derivation using CRISPR/Cas-based genome editing technology will significantly reduce the risk of incorrectly engineered cell products that could behave as a “Trojan horse”. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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14 pages, 855 KiB

Open AccessReview

CRISPR-Cas Technology for Bioengineering Conventional and Non-Conventional Yeasts: Progress and New Challenges

by Yuanyuan Xia, Yujie Li, Wei Shen, Haiquan Yang and Xianzhong Chen

Int. J. Mol. Sci. 2023, 24(20), 15310; https://doi.org/10.3390/ijms242015310 - 18 Oct 2023

Cited by 4 | Viewed by 3840

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) system has undergone substantial and transformative progress. Simultaneously, a spectrum of derivative technologies has emerged, spanning both conventional and non-conventional yeast strains. Non-conventional yeasts, distinguished by their robust metabolic pathways, formidable [...] Read more.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) system has undergone substantial and transformative progress. Simultaneously, a spectrum of derivative technologies has emerged, spanning both conventional and non-conventional yeast strains. Non-conventional yeasts, distinguished by their robust metabolic pathways, formidable resilience against diverse stressors, and distinctive regulatory mechanisms, have emerged as a highly promising alternative for diverse industrial applications. This comprehensive review serves to encapsulate the prevailing gene editing methodologies and their associated applications within the traditional industrial microorganism, Saccharomyces cerevisiae. Additionally, it delineates the current panorama of non-conventional yeast strains, accentuating their latent potential in the realm of industrial and biotechnological utilization. Within this discourse, we also contemplate the potential value these tools offer alongside the attendant challenges they pose. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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15 pages, 887 KiB

Open AccessReview

Evolution of CRISPR/Cas Systems for Precise Genome Editing

by Magdalena Hryhorowicz, Daniel Lipiński and Joanna Zeyland

Int. J. Mol. Sci. 2023, 24(18), 14233; https://doi.org/10.3390/ijms241814233 - 18 Sep 2023

Cited by 23 | Viewed by 7628

Abstract

The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges [...] Read more.

The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges remain, such as the risk of immunological reactions or off-target effects. To overcome these problems, many new methods and CRISPR/Cas-based tools have been developed. In this review, we describe the current classification of CRISPR systems and new precise genome-editing technologies, summarize the latest applications of this technique in several fields of research, and, finally, discuss CRISPR/Cas system limitations, ethical issues, and challenges. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

17 pages, 1429 KiB

Open AccessReview

Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice

by Elena McBeath, Keigi Fujiwara and Marie-Claude Hofmann

Int. J. Mol. Sci. 2023, 24(12), 10258; https://doi.org/10.3390/ijms241210258 - 17 Jun 2023

Viewed by 2774

Abstract

Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues, several labs have started successfully using a [...] Read more.

Up until recently, methods for generating floxed mice either conventionally or by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 (CRISPR-associated protein 9) editing have been technically challenging, expensive and error-prone, or time-consuming. To circumvent these issues, several labs have started successfully using a small artificial intron to conditionally knockout (KO) a gene of interest in mice. However, many other labs are having difficulty getting the technique to work. The key problem appears to be either a failure in achieving correct splicing after the introduction of the artificial intron into the gene or, just as crucial, insufficient functional KO of the gene’s protein after Cre-induced removal of the intron’s branchpoint. Presented here is a guide on how to choose an appropriate exon and where to place the recombinase-regulated artificial intron (rAI) in that exon to prevent disrupting normal gene splicing while maximizing mRNA degradation after recombinase treatment. The reasoning behind each step in the guide is also discussed. Following these recommendations should increase the success rate of this easy, new, and alternative technique for producing tissue-specific KO mice. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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20 pages, 954 KiB

Open AccessReview

Precision Editing as a Therapeutic Approach for β-Hemoglobinopathies

by Kiriaki Paschoudi, Evangelia Yannaki and Nikoletta Psatha

Int. J. Mol. Sci. 2023, 24(11), 9527; https://doi.org/10.3390/ijms24119527 - 31 May 2023

Cited by 9 | Viewed by 4459

Abstract

Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the β-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to [...] Read more.

Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the β-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic β- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with β-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for β-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies. Full article

(This article belongs to the Special Issue CRISPR-Cas Systems and Genome Editing)

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