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

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 (30 June 2022) | Viewed by 53729

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
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 enormous 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.

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

Manuscript Submission Information

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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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Keywords

  • 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.)

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Published Papers (16 papers)

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Research

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14 pages, 1662 KiB  
Article
Thermodynamic Swings: How Ideal Complex of Cas9–RNA/DNA Forms
by Polina V. Zhdanova, Alexander A. Lomzov, Daria V. Prokhorova, Grigory A. Stepanov, Alexander A. Chernonosov and Vladimir V. Koval
Int. J. Mol. Sci. 2022, 23(16), 8891; https://doi.org/10.3390/ijms23168891 - 10 Aug 2022
Cited by 1 | Viewed by 1528
Abstract
Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular [...] Read more.
Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR–Cas9 tool. The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing systems. The attainment of high-specificity and -efficiency Cas9 during targeted DNA cleavage is the main problem that limits the practical application of the CRISPR–Cas9 system. In this study, we analyzed the thermodynamics of interaction of a complex’s components of Cas9–RNA/DNA through experimental and computer simulation methods. We found that there is a small energetic preference during Cas9–RNA/DNA formation from the Cas9–RNA and DNA/DNA duplex. The small difference in binding energy is relevant for biological interactions and could be part of the sequence-specific recognition of double-stranded DNA by the CRISPR–Cas9 system. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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16 pages, 2041 KiB  
Article
Aberrant Splicing of INS Impairs Beta-Cell Differentiation and Proliferation by ER Stress in the Isogenic iPSC Model of Neonatal Diabetes
by Alexandra V. Panova, Natalia V. Klementieva, Anna V. Sycheva, Elena V. Korobko, Anastasia O. Sosnovtseva, Tatiana S. Krasnova, Maria R. Karpova, Petr M. Rubtsov, Yulia V. Tikhonovich, Anatoly N. Tiulpakov and Sergey L. Kiselev
Int. J. Mol. Sci. 2022, 23(15), 8824; https://doi.org/10.3390/ijms23158824 - 08 Aug 2022
Cited by 6 | Viewed by 2200
Abstract
One of the causes of diabetes in infants is the defect of the insulin gene (INS). Gene mutations can lead to proinsulin misfolding, an increased endoplasmic reticulum (ER) stress and possible beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain [...] Read more.
One of the causes of diabetes in infants is the defect of the insulin gene (INS). Gene mutations can lead to proinsulin misfolding, an increased endoplasmic reticulum (ER) stress and possible beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. We generated induced pluripotent stem cells (iPSCs) from a patient diagnosed with neonatal diabetes mellitus carrying the INS mutation in the 2nd intron (c.188-31G>A) and engineered isogenic CRISPR/Cas9 mutation-corrected cell lines. Differentiation into beta-like cells demonstrated that mutation led to the emergence of an ectopic splice site within the INS and appearance of the abnormal RNA transcript. Isogenic iPSC lines differentiated into beta-like cells showed a clear difference in formation of organoids at pancreatic progenitor stage of differentiation. Moreover, MIN6 insulinoma cell line expressing mutated cDNA demonstrated significant decrease in proliferation capacity and activation of ER stress and unfolded protein response (UPR)-associated genes. These findings shed light on the mechanism underlying the pathogenesis of monogenic diabetes. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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13 pages, 790 KiB  
Article
CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA
by Natalya V. Permyakova, Tatyana V. Marenkova, Pavel A. Belavin, Alla A. Zagorskaya, Yuriy V. Sidorchuk and Elena V. Deineko
Int. J. Mol. Sci. 2022, 23(15), 8636; https://doi.org/10.3390/ijms23158636 - 03 Aug 2022
Cited by 3 | Viewed by 1597
Abstract
Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in [...] Read more.
Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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13 pages, 2350 KiB  
Article
CRISPR/Cas9–Mediated Genome Editing for Pseudomonas fulva, a Novel Pseudomonas Species with Clinical, Animal, and Plant–Associated Isolates
by Nan Zhang, Jintao He, Abrar Muhammad and Yongqi Shao
Int. J. Mol. Sci. 2022, 23(10), 5443; https://doi.org/10.3390/ijms23105443 - 13 May 2022
Cited by 3 | Viewed by 2271
Abstract
As one of the most widespread groups of Gram–negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant–associated isolates, [...] Read more.
As one of the most widespread groups of Gram–negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant–associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time–consuming and laborious endeavor. Here, by using the CRISPR–Cas system, a versatile gene–editing tool with a two–plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic–related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked–in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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15 pages, 6450 KiB  
Article
Prime Editor 3 Mediated Beta-Thalassemia Mutations of the HBB Gene in Human Erythroid Progenitor Cells
by Haokun Zhang, Qinlinglan Zhou, Hongyan Chen and Daru Lu
Int. J. Mol. Sci. 2022, 23(9), 5002; https://doi.org/10.3390/ijms23095002 - 30 Apr 2022
Cited by 6 | Viewed by 2654
Abstract
Recently developed Prime Editor 3 (PE3) has been implemented to induce genome editing in various cell types but has not been proven in human hematopoietic stem and progenitor cells. Using PE3, we successfully installed the beta-thalassemia (beta-thal) mutations in the HBB gene in [...] Read more.
Recently developed Prime Editor 3 (PE3) has been implemented to induce genome editing in various cell types but has not been proven in human hematopoietic stem and progenitor cells. Using PE3, we successfully installed the beta-thalassemia (beta-thal) mutations in the HBB gene in the erythroid progenitor cell line HUDEP-2. We inserted the mCherry reporter gene cassette into editing plasmids, each including the prime editing guide RNA (pegRNA) and nick sgRNA. The plasmids were electroporated into HUDEP-2 cells, and the PE3 modified cells were identified by mCherry expression and collected using fluorescence-activated cell sorting (FACS). Sanger sequencing of the positive cells confirmed that PE3 induced precise beta-thal mutations with editing ratios from 4.55 to 100%. Furthermore, an off-target analysis showed no unintentional edits occurred in the cells. The editing ratios and parameters of pegRNA and nick sgRNA were also analyzed and summarized and will contribute to enhanced PE3 design in future studies. The characterization of the HUDEP-2 beta-thal cells showed typical thalassemia phenotypes, involving ineffective erythropoiesis, abnormal erythroid differentiation, high apoptosis rate, defective alpha-globin colocalization, cell viability deterioration, and ROS resisting deficiency. These HUDEP-2 beta-thal cells could provide ideal models for future beta-thal gene therapy studies. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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23 pages, 5209 KiB  
Article
Employing CRISPR-Cas9 to Generate CD133 Synthetic Lethal Melanoma Stem Cells
by Cynthia M. Simbulan-Rosenthal, Yogameenakshi Haribabu, Sahar Vakili, Li-Wei Kuo, Havens Clark, Ryan Dougherty, Ryyan Alobaidi, Bonnie Carney, Peter Sykora and Dean S. Rosenthal
Int. J. Mol. Sci. 2022, 23(4), 2333; https://doi.org/10.3390/ijms23042333 - 20 Feb 2022
Cited by 5 | Viewed by 2603
Abstract
Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and is characterized by the elevated expression of stem cell markers, including CD133. The siRNA knockdown of CD133 enhances apoptosis induced by the MEK inhibitor [...] Read more.
Malignant melanoma is a lethal skin cancer containing melanoma-initiating cells (MIC) implicated in tumorigenesis, invasion, and drug resistance, and is characterized by the elevated expression of stem cell markers, including CD133. The siRNA knockdown of CD133 enhances apoptosis induced by the MEK inhibitor trametinib in melanoma cells. This study investigates the underlying mechanisms of CD133’s anti-apoptotic activity in patient-derived BAKP and POT cells, harboring difficult-to-treat NRASQ61K and NRASQ61R drivers, after CRISPR-Cas9 CD133 knockout or Dox-inducible expression of CD133. MACS-sorted CD133(+) BAKP cells were conditionally reprogrammed to derive BAKR cells with sustained CD133 expression and MIC features. Compared to BAKP, CD133(+) BAKR exhibit increased cell survival and reduced apoptosis in response to trametinib or the chemotherapeutic dacarbazine (DTIC). CRISPR-Cas9-mediated CD133 knockout in BAKR cells (BAKR-KO) re-sensitized cells to trametinib. CD133 knockout in BAKP and POT cells increased trametinib-induced apoptosis by reducing anti-apoptotic BCL-xL, p-AKT, and p-BAD and increasing pro-apoptotic BAX. Conversely, Dox-induced CD133 expression diminished apoptosis in both trametinib-treated cell lines, coincident with elevated p-AKT, p-BAD, BCL-2, and BCL-xL and decreased activation of BAX and caspases-3 and -9. AKT1/2 siRNA knockdown or inhibition of BCL-2 family members with navitoclax (ABT-263) in BAKP-KO cells further enhanced caspase-mediated apoptotic PARP cleavage. CD133 may therefore activate a survival pathway where (1) increased AKT phosphorylation and activation induces (2) BAD phosphorylation and inactivation, (3) decreases BAX activation, and (4) reduces caspases-3 and -9 activity and caspase-mediated PARP cleavage, leading to apoptosis suppression and drug resistance in melanoma. Targeting nodes of the CD133, AKT, or BCL-2 survival pathways with trametinib highlights the potential for combination therapies for NRAS-mutant melanoma stem cells for the development of more effective treatments for patients with high-risk melanoma. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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15 pages, 5521 KiB  
Article
Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen-Deuterium Exchange Mass Spectrometry
by Polina V. Zhdanova, Alexander A. Chernonosov, Daria V. Prokhorova, Grigory A. Stepanov, Lyubov Yu. Kanazhevskaya and Vladimir V. Koval
Int. J. Mol. Sci. 2022, 23(3), 1129; https://doi.org/10.3390/ijms23031129 - 20 Jan 2022
Cited by 2 | Viewed by 2279
Abstract
The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR–Cas9 system. A deep understanding of [...] Read more.
The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR–Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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24 pages, 23223 KiB  
Article
CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation
by Panayiota L. Papasavva, Petros Patsali, Constantinos C. Loucari, Ryo Kurita, Yukio Nakamura, Marina Kleanthous and Carsten W. Lederer
Int. J. Mol. Sci. 2022, 23(3), 1082; https://doi.org/10.3390/ijms23031082 - 19 Jan 2022
Cited by 5 | Viewed by 2841
Abstract
Molecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate γ-globin [...] Read more.
Molecular therapies and functional studies greatly benefit from spatial and temporal precision of genetic intervention. We therefore conceived and explored tag-activated microRNA (miRNA)-mediated endogene deactivation (TAMED) as a research tool and potential lineage-specific therapy. For proof of principle, we aimed to deactivate γ-globin repressor BCL11A in erythroid cells by tagging the 3′ untranslated region (UTR) of BCL11A with miRNA recognition sites (MRSs) for the abundant erythromiR miR-451a. To this end, we employed nucleofection of CRISPR/Cas9 ribonucleoprotein (RNP) particles alongside double- or single-stranded oligodeoxynucleotides for, respectively, non-homologous-end-joining (NHEJ)- or homology-directed-repair (HDR)-mediated MRS insertion. NHEJ-based tagging was imprecise and inefficient (≤6%) and uniformly produced knock-in- and indel-containing MRS tags, whereas HDR-based tagging was more efficient (≤18%), but toxic for longer donors encoding concatenated and thus potentially more efficient MRS tags. Isolation of clones for robust HEK293T cells tagged with a homozygous quadruple MRS resulted in 25% spontaneous reduction in BCL11A and up to 36% reduction after transfection with an miR-451a mimic. Isolation of clones for human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells tagged with single or double MRS allowed detection of albeit weak γ-globin induction. Our study demonstrates suitability of TAMED for physiologically relevant modulation of gene expression and its unsuitability for therapeutic application in its current form. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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15 pages, 4648 KiB  
Article
A Novel Isogenic Human Cell-Based System for MEN1 Syndrome Generated by CRISPR/Cas9 Genome Editing
by Natalia Klementieva, Daria Goliusova, Julia Krupinova, Vladislav Yanvarev, Alexandra Panova, Natalia Mokrysheva and Sergey L. Kiselev
Int. J. Mol. Sci. 2021, 22(21), 12054; https://doi.org/10.3390/ijms222112054 - 08 Nov 2021
Cited by 2 | Viewed by 2537
Abstract
Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome that manifests differently among various patients. Despite the mutations in the MEN1 gene that commonly predispose tumor development, there are no obvious phenotype–genotype correlations. The existing animal and in vitro models do [...] Read more.
Multiple endocrine neoplasia type 1 (MEN1) is a rare tumor syndrome that manifests differently among various patients. Despite the mutations in the MEN1 gene that commonly predispose tumor development, there are no obvious phenotype–genotype correlations. The existing animal and in vitro models do not allow for studies of the molecular genetics of the disease in a human-specific context. We aimed to create a new human cell-based model, which would consider the variability in genetic or environmental factors that cause the complexity of MEN1 syndrome. Here, we generated patient-specific induced pluripotent stem cell lines carrying the mutation c.1252G>T, D418Y in the MEN1 gene. To reduce the genetically determined variability of the existing cellular models, we created an isogenic cell system by modifying the target allele through CRISPR/Cas9 editing with great specificity and efficiency. The high potential of these cell lines to differentiate into the endodermal lineage in defined conditions ensures the next steps in the development of more specialized cells that are commonly affected in MEN1 patients, such as parathyroid or pancreatic islet cells. We anticipate that this isogenic system will be broadly useful to comprehensively study MEN1 gene function across different contexts, including in vitro modeling of MEN1 syndrome. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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10 pages, 1866 KiB  
Communication
Comparative Analysis of Genome Editors Efficiency on a Model of Mice Zygotes Microinjection
by Olga A. Averina, Oleg A. Permyakov, Olga O. Grigorieva, Alexey S. Starshin, Alexander M. Mazur, Egor B. Prokhortchouk, Olga A. Dontsova and Petr V. Sergiev
Int. J. Mol. Sci. 2021, 22(19), 10221; https://doi.org/10.3390/ijms221910221 - 23 Sep 2021
Viewed by 2186
Abstract
Genome editing is an indispensable tool for functional genomics. The caveat of the genome-editing pipeline is a prevalence of error-prone non-homologous end joining over homologous recombination, while only the latter is suitable to introduce particularly desired genetic variants. To overcome this problem, a [...] Read more.
Genome editing is an indispensable tool for functional genomics. The caveat of the genome-editing pipeline is a prevalence of error-prone non-homologous end joining over homologous recombination, while only the latter is suitable to introduce particularly desired genetic variants. To overcome this problem, a toolbox of genome engineering was appended by a variety of improved instruments. In this work, we compared the efficiency of a number of recently suggested improved systems for genome editing applied to the same genome regions on a murine zygote model via microinjection. As a result, we observed that homologous recombination utilizing an ssDNA template following sgRNA directed Cas9 cleavage is still the method of choice for the creation of animals with precise genome alterations. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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Review

Jump to: Research

21 pages, 1659 KiB  
Review
CRISPR/Cas9—A Promising Therapeutic Tool to Cure Blindness: Current Scenario and Future Prospects
by Irshad Ahmad
Int. J. Mol. Sci. 2022, 23(19), 11482; https://doi.org/10.3390/ijms231911482 - 29 Sep 2022
Cited by 7 | Viewed by 4443
Abstract
CRISPR-based targeted genome editing is bringing revolutionary changes in the research arena of biological sciences. CRISPR/Cas9 has been explored as an efficient therapeutic tool for the treatment of genetic diseases. It has been widely used in ophthalmology research by using mouse models to [...] Read more.
CRISPR-based targeted genome editing is bringing revolutionary changes in the research arena of biological sciences. CRISPR/Cas9 has been explored as an efficient therapeutic tool for the treatment of genetic diseases. It has been widely used in ophthalmology research by using mouse models to correct pathogenic mutations in the eye stem cells. In recent studies, CRISPR/Cas9 has been used to correct a large number of mutations related to inherited retinal disorders. In vivo therapeutic advantages for retinal diseases have been successfully achieved in some rodents. Current advances in the CRISPR-based gene-editing domain, such as modified Cas variants and delivery approaches have optimized its application to treat blindness. In this review, recent progress and challenges of the CRISPR-Cas system have been discussed to cure blindness and its prospects. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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13 pages, 954 KiB  
Review
Improving Homology-Directed Repair in Genome Editing Experiments by Influencing the Cell Cycle
by Svetlana A. Smirnikhina, Milyausha I. Zaynitdinova, Vasilina A. Sergeeva and Alexander V. Lavrov
Int. J. Mol. Sci. 2022, 23(11), 5992; https://doi.org/10.3390/ijms23115992 - 26 May 2022
Cited by 10 | Viewed by 3453
Abstract
Genome editing is currently widely used in biomedical research; however, the use of this method in the clinic is still limited because of its low efficiency and possible side effects. Moreover, the correction of mutations that cause diseases in humans seems to be [...] Read more.
Genome editing is currently widely used in biomedical research; however, the use of this method in the clinic is still limited because of its low efficiency and possible side effects. Moreover, the correction of mutations that cause diseases in humans seems to be extremely important and promising. Numerous attempts to improve the efficiency of homology-directed repair-mediated correction of mutations in mammalian cells have focused on influencing the cell cycle. Homology-directed repair is known to occur only in the late S and G2 phases of the cell cycle, so researchers are looking for safe ways to enrich the cell culture with cells in these phases of the cell cycle. This review surveys the main approaches to influencing the cell cycle in genome editing experiments (predominantly using Cas9), for example, the use of cell cycle synchronizers, mitogens, substances that affect cyclin-dependent kinases, hypothermia, inhibition of p53, etc. Despite the fact that all these approaches have a reversible effect on the cell cycle, it is necessary to use them with caution, since cells during the arrest of the cell cycle can accumulate mutations, which can potentially lead to their malignant transformation. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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11 pages, 2641 KiB  
Review
A New Generation of Lineage Tracing Dynamically Records Cell Fate Choices
by Mingze Yao, Tinglin Ren, Yuanqing Pan, Xiaoqing Xue, Rong Li, Lei Zhang, Yuhang Li and Ke Huang
Int. J. Mol. Sci. 2022, 23(9), 5021; https://doi.org/10.3390/ijms23095021 - 30 Apr 2022
Cited by 7 | Viewed by 4207
Abstract
Reconstructing the development of lineage relationships and cell fate mapping has been a fundamental problem in biology. Using advanced molecular biology and single-cell RNA sequencing, we have profiled transcriptomes at the single-cell level and mapped cell fates during development. Recently, CRISPR/Cas9 barcode editing [...] Read more.
Reconstructing the development of lineage relationships and cell fate mapping has been a fundamental problem in biology. Using advanced molecular biology and single-cell RNA sequencing, we have profiled transcriptomes at the single-cell level and mapped cell fates during development. Recently, CRISPR/Cas9 barcode editing for large-scale lineage tracing has been used to reconstruct the pseudotime trajectory of cells and improve lineage tracing accuracy. This review presents the progress of the latest CbLT (CRISPR-based Lineage Tracing) and discusses the current limitations and potential technical pitfalls in their application and other emerging concepts. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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17 pages, 9294 KiB  
Review
CRISPR Approaches for the Diagnosis of Human Diseases
by Pilar Puig-Serra, Maria Cruz Casado-Rosas, Marta Martinez-Lage, Beatriz Olalla-Sastre, Alejandro Alonso-Yanez, Raul Torres-Ruiz and Sandra Rodriguez-Perales
Int. J. Mol. Sci. 2022, 23(3), 1757; https://doi.org/10.3390/ijms23031757 - 03 Feb 2022
Cited by 10 | Viewed by 5241
Abstract
CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, [...] Read more.
CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, since they are based on four of the six major subtypes of CRISPR systems. In just 5 years, the CRISPR diagnostic field has rapidly expanded, growing from a set of specific molecular biology discoveries to multiple FDA-authorized COVID-19 tests and the establishment of several companies. CRISPR-based detection methods are coupled with pre-existing preamplification and readout technologies, achieving sensitivity and reproducibility comparable to the current gold standard nucleic acid detection methods. Moreover, they are very versatile, can be easily implemented to detect emerging pathogens and new clinically relevant mutations, and offer multiplexing capability. The advantages of the CRISPR-based diagnostic approaches are a short sample-to-answer time and no requirement of laboratory settings; they are also much more affordable than current nucleic acid detection procedures. In this review, we summarize the applications and development trends of the CRISPR/Cas13 system in the identification of particular pathogens and mutations and discuss the challenges and future prospects of CRISPR-based diagnostic platforms in biomedicine. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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23 pages, 1529 KiB  
Review
Challenges of CRISPR-Based Gene Editing in Primary T Cells
by Alaleh Rezalotfi, Lea Fritz, Reinhold Förster and Berislav Bošnjak
Int. J. Mol. Sci. 2022, 23(3), 1689; https://doi.org/10.3390/ijms23031689 - 01 Feb 2022
Cited by 13 | Viewed by 6393
Abstract
Adaptive T-cell immunotherapy holds great promise for the successful treatment of leukemia, as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells [...] Read more.
Adaptive T-cell immunotherapy holds great promise for the successful treatment of leukemia, as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T-cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system, allowing for the deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs that were important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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19 pages, 3561 KiB  
Review
Stimulus-Responsive Smart Nanoparticles-Based CRISPR-Cas Delivery for Therapeutic Genome Editing
by Muhammad Naeem, Mubasher Zahir Hoque, Muhammad Ovais, Chanbasha Basheer and Irshad Ahmad
Int. J. Mol. Sci. 2021, 22(20), 11300; https://doi.org/10.3390/ijms222011300 - 19 Oct 2021
Cited by 13 | Viewed by 4302
Abstract
The innovative research in genome editing domains such as CRISPR-Cas technology has enabled genetic engineers to manipulate the genomes of living organisms effectively in order to develop the next generation of therapeutic tools. This technique has started the new era of “genome surgery”. [...] Read more.
The innovative research in genome editing domains such as CRISPR-Cas technology has enabled genetic engineers to manipulate the genomes of living organisms effectively in order to develop the next generation of therapeutic tools. This technique has started the new era of “genome surgery”. Despite these advances, the barriers of CRISPR-Cas9 techniques in clinical applications include efficient delivery of CRISPR/Cas9 and risk of off-target effects. Various types of viral and non-viral vectors are designed to deliver the CRISPR/Cas9 machinery into the desired cell. These methods still suffer difficulties such as immune response, lack of specificity, and efficiency. The extracellular and intracellular environments of cells and tissues differ in pH, redox species, enzyme activity, and light sensitivity. Recently, smart nanoparticles have been synthesized for CRISPR/Cas9 delivery to cells based on endogenous (pH, enzyme, redox specie, ATP) and exogenous (magnetic, ultrasound, temperature, light) stimulus signals. These methodologies can leverage genome editing through biological signals found within disease cells with less off-target effects. Here, we review the recent advances in stimulus-based smart nanoparticles to deliver the CRISPR/Cas9 machinery into the desired cell. This review article will provide extensive information to cautiously utilize smart nanoparticles for basic biomedical applications and therapeutic genome editing. Full article
(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing)
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