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Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0

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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 (10 April 2023) | Viewed by 27620

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Dr. Timofey S. Rozhdestvensky

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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
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Dr. Alexander Kondrashov

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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

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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

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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 (12 papers)

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Research

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21 pages, 5167 KiB

Open AccessArticle

CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B

by Qiyu Tang, Zhiqing Hu, Junya Zhao, Tao Zhou, Shuqing Tang, Peiyun Wang, Rou Xiao, Yan Chen, Lingqian Wu, Miaojin Zhou and Desheng Liang

Int. J. Mol. Sci. 2023, 24(10), 9013; https://doi.org/10.3390/ijms24109013 - 19 May 2023

Cited by 2 | Viewed by 1712

Abstract

Hemophilia B (HB) is an X-linked recessive disease caused by F9 gene mutation and functional coagulation factor IX (FIX) deficiency. Patients suffer from chronic arthritis and death threats owing to excessive bleeding. Compared with traditional treatments, gene therapy for HB has obvious advantages, especially when the hyperactive FIX mutant (FIX-Padua) is used. However, the mechanism by which FIX-Padua works remains ambiguous due to a lack of research models. Here, in situ introduction of F9-Padua mutation was performed in human induced pluripotent stem cells (hiPSCs) via CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs). The hyperactivity of FIX-Padua was confirmed to be 364% of the normal level in edited hiPSCs-derived hepatocytes, providing a reliable model for exploring the mechanism of the hyperactivity of FIX-Padua. Moreover, the F9 cDNA containing F9-Padua was integrated before the F9 initiation codon by CRISPR/Cas9 in iPSCs from an HB patient (HB-hiPSCs). Integrated HB-hiPSCs after off-target screening were differentiated into hepatocytes. The FIX activity in the supernatant of integrated hepatocytes showed a 4.2-fold increase and reached 63.64% of the normal level, suggesting a universal treatment for HB patients with various mutations in F9 exons. Overall, our study provides new approaches for the exploration and development of cell-based gene therapy for HB. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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13 pages, 3069 KiB

Open AccessArticle

A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing

by Jochen M. Wettengel, Lea Hansen-Palmus, Sofiya Yusova, Lauren Rust, Sreya Biswas, Julien Carson, Junghyun Ryu, Benjamin N. Bimber, Jon D. Hennebold and Benjamin J. Burwitz

Int. J. Mol. Sci. 2023, 24(9), 8271; https://doi.org/10.3390/ijms24098271 - 05 May 2023

Cited by 1 | Viewed by 1536

Abstract

CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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14 pages, 2119 KiB

Open AccessArticle

A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts

by Azamat V. Karginov, Marina G. Tarutina, Anastasia R. Lapteva, Maria D. Pakhomova, Artur A. Galliamov, Sergey Y. Filkin, Alexey N. Fedorov and Michael O. Agaphonov

Int. J. Mol. Sci. 2023, 24(9), 8173; https://doi.org/10.3390/ijms24098173 - 03 May 2023

Cited by 3 | Viewed by 2090

Abstract

Methylotrophic yeasts such as Ogataea polymorpha and Komagataella phaffii (sin. Hansenula polymorpha and Pichia pastoris, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and LEU2 auxotrophic selectable markers. The functionality of this setup has been demonstrated in O. polymorpha, O. parapolymorpha, O. haglerorum and Komagataella phaffii. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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16 pages, 6149 KiB

Open AccessArticle

CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes

by Valentina Muto, Federica Benigni, Valentina Magliocca, Rossella Borghi, Elisabetta Flex, Valentina Pallottini, Alessandro Rosa, Claudia Compagnucci and Marco Tartaglia

Int. J. Mol. Sci. 2023, 24(9), 7988; https://doi.org/10.3390/ijms24097988 - 28 Apr 2023

Cited by 1 | Viewed by 1656

Abstract

Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its “stemness”. The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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10 pages, 1152 KiB

Open AccessArticle

Bridging Gaps in HDR Improvement: The Role of MAD2L2, SCAI, and SCR7

by Arina A. Anuchina, Milyausha I. Zaynitdinova, Anna G. Demchenko, Nadezhda A. Evtushenko, Alexander V. Lavrov and Svetlana A. Smirnikhina

Int. J. Mol. Sci. 2023, 24(7), 6704; https://doi.org/10.3390/ijms24076704 - 04 Apr 2023

Cited by 3 | Viewed by 1632

Abstract

This study aimed to enhance homology-directed repair (HDR) efficiency in CRISPR/Cas-mediated genome editing by targeting three key factors regulating the balance between HDR and non-homologous end joining (NHEJ): MAD2L2, SCAI, and Ligase IV. In order to achieve this, a cellular model using mutated eGFP was designed to monitor HDR events. Results showed that MAD2L2 knockdown and SCR7 treatment significantly improved HDR efficiency during Cas9-mediated HDR repair of the mutated eGFP gene in the HEK293T cell line. Fusion protein Cas9-SCAI did not improve HDR. This study is the first to demonstrate that MAD2L2 knockdown during CRISPR-mediated gene editing in HEK293T cells can increase precise correction by up to 10.2 times. The study also confirmed a moderate but consistent effect of SCR7, an inhibitor of Ligase IV, which increased HDR by 1.7 times. These findings provide valuable insights into improving HDR-based genome editing efficiency. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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15 pages, 4866 KiB

Open AccessArticle

Effects of Different Gene Editing Modes of CRISPR/Cas9 on Soybean Fatty Acid Anabolic Metabolism Based on GmFAD2 Family

by Junming Zhou, Zeyuan Li, Yue Li, Qiuzhu Zhao, Xinchao Luan, Lixue Wang, Yixuan Liu, Huijing Liu, Jun Zhang and Dan Yao

Int. J. Mol. Sci. 2023, 24(5), 4769; https://doi.org/10.3390/ijms24054769 - 01 Mar 2023

Cited by 2 | Viewed by 1650

Abstract

Δ¹²-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean FAD2 gene family—GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C—and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T₁ generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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17 pages, 3831 KiB

Open AccessArticle

CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines

by Trygve A. H. Strømsnes, Sebastian E. Schmidke, Mitra Azad, Øyvind Singstad, Idun M. Grønsberg, Roy A. Dalmo and Arinze S. Okoli

Int. J. Mol. Sci. 2022, 23(24), 16218; https://doi.org/10.3390/ijms232416218 - 19 Dec 2022

Cited by 2 | Viewed by 2624

Abstract

Finfish production has seen over three-fold increase in the past 30 years (1990–2020), and Atlantic salmon (A. salmon; salmo salar) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish species globally. This growth in production is, however, threatened by a number of problems which can be solved using the CRISPR/Cas technology. In vitro applications of CRISPR/Cas using cell lines can complement its in vivo applications, but salmonids-derived cell lines are difficult to gene edit because they grow slowly, are difficult to transfect and isolate single clones of gene-edited cells. While clonal isolation of the gene-edited Chinook salmon cell line (CHSE-214) has successfully been performed, there is no report of successful clonal isolation of the gene-edited A. salmon ASK-1 and SHK-1cell lines. In the current study, two gene loci—cr2 and mmp9 of A. salmon—were efficiently edited using the ribonucleoprotein (RNP) and plasmid CRISPR/Cas9 strategies. Edited cells were enriched using flow cytometer-activated cell sorting (FACS), followed by clonal isolation and expansion of edited cells. The study both confirms the recent report of the highly efficient editing of these widely used model cell lines, as well as extends the frontline in the single-cell cloning of gene-edited salmonids cells. The report also highlights the pitfalls and future directions in the application of CRISPR/Cas9 in these cells. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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13 pages, 888 KiB

Open AccessArticle

Structure- and Content-Dependent Efficiency of Cas9-Assisted DNA Cleavage in Genome-Editing Systems

by Svetlana V. Baranova, Polina V. Zhdanova, Alexander A. Lomzov, Vladimir V. Koval and Alexander A. Chernonosov

Int. J. Mol. Sci. 2022, 23(22), 13889; https://doi.org/10.3390/ijms232213889 - 11 Nov 2022

Viewed by 1145

Abstract

Genome-editing systems, being some of the key tools of molecular biologists, represent a reasonable hope for progress in the field of personalized medicine. A major problem with such systems is their nonideal accuracy and insufficient selectivity. The selectivity of CRISPR-Cas9 systems can be improved in several ways. One efficient way is the proper selection of the consensus sequence of the DNA to be cleaved. In the present work, we attempted to evaluate the effect of formed non-Watson–Crick pairs in a DNA duplex on the efficiency of DNA cleavage in terms of the influence of the structure of the formed partially complementary pairs. We also studied the effect of the location of such pairs in DNA relative to the PAM (protospacer-adjacent motif) on the cleavage efficiency. We believe that the stabilization of the Cas9-sgRNA complex with a DNA substrate containing noncomplementary pairs is due to loop reorganization in the RuvC domain of the enzyme. In addition, PAM-proximal mismatches in the DNA substrate lower enzyme efficiency because the “seed” region is involved in binding and cleavage, whereas PAM-distal mismatches have no significant impact on target DNA cleavage. Our data suggest that in the case of short duplexes with mismatches, the stages of recognition and binding of dsDNA substrates by the enzyme determine the reaction rate and time rather than the thermodynamic parameters affected by the “unwinding” of DNA. The results will provide a theoretical basis for predicting the efficiency and accuracy of CRISPR-Cas9 systems at cleaving target DNA. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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Review

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33 pages, 4734 KiB

Open AccessReview

Recent Advances in Genome-Editing Technology with CRISPR/Cas9 Variants and Stimuli-Responsive Targeting Approaches within Tumor Cells: A Future Perspective of Cancer Management

by Khaled S. Allemailem, Saleh A. Almatroodi, Ahmad Almatroudi, Faris Alrumaihi, Waleed Al Abdulmonem, Wafa Abdullah I. Al-Megrin, Adel Nasser Aljamaan, Arshad Husain Rahmani and Amjad Ali Khan

Int. J. Mol. Sci. 2023, 24(8), 7052; https://doi.org/10.3390/ijms24087052 - 11 Apr 2023

Cited by 6 | Viewed by 2948

Abstract

The innovative advances in transforming clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) into different variants have taken the art of genome-editing specificity to new heights. Allosteric modulation of Cas9-targeting specificity by sgRNA sequence alterations and protospacer adjacent motif (PAM) modifications have been a good lesson to learn about specificity and activity scores in different Cas9 variants. Some of the high-fidelity Cas9 variants have been ranked as Sniper-Cas9, eSpCas9 (1.1), SpCas9-HF1, HypaCas9, xCas9, and evoCas9. However, the selection of an ideal Cas9 variant for a given target sequence remains a challenging task. A safe and efficient delivery system for the CRISPR/Cas9 complex at tumor target sites faces considerable challenges, and nanotechnology-based stimuli-responsive delivery approaches have significantly contributed to cancer management. Recent innovations in nanoformulation design, such as pH, glutathione (GSH), photo, thermal, and magnetic responsive systems, have modernized the art of CRISPR/Cas9 delivery approaches. These nanoformulations possess enhanced cellular internalization, endosomal membrane disruption/bypass, and controlled release. In this review, we aim to elaborate on different CRISPR/Cas9 variants and advances in stimuli-responsive nanoformulations for the specific delivery of this endonuclease system. Furthermore, the critical constraints of this endonuclease system on clinical translations towards the management of cancer and prospects are described. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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21 pages, 2778 KiB

Open AccessReview

Building Blocks of Artificial CRISPR-Based Systems beyond Nucleases

by Andrey A. Kuzmin and Alexey N. Tomilin

Int. J. Mol. Sci. 2023, 24(1), 397; https://doi.org/10.3390/ijms24010397 - 26 Dec 2022

Cited by 2 | Viewed by 2135

Abstract

Tools developed in the fields of genome engineering, precise gene regulation, and synthetic gene networks have an increasing number of applications. When shared with the scientific community, these tools can be used to further unlock the potential of precision medicine and tissue engineering. A large number of different genetic elements, as well as modifications, have been used to create many different systems and to validate some technical concepts. New studies have tended to optimize or improve existing elements or approaches to create complex synthetic systems, especially those based on the relatively new CRISPR technology. In order to maximize the output of newly developed approaches and to move from proof-of-principle experiments to applications in regenerative medicine, it is important to navigate efficiently through the vast number of genetic elements to choose those most suitable for specific needs. In this review, we have collected information regarding the main genetic elements and their modifications, which can be useful in different synthetic systems with an emphasis of those based on CRISPR technology. We have indicated the most suitable elements and approaches to choose or combine in planning experiments, while providing their deeper understanding, and have also stated some pitfalls that should be avoided. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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12 pages, 2738 KiB

Open AccessReview

A Decade of CRISPR-Cas Gnome Editing in C. elegans

by Hyun-Min Kim, Yebin Hong and Jiani Chen

Int. J. Mol. Sci. 2022, 23(24), 15863; https://doi.org/10.3390/ijms232415863 - 14 Dec 2022

Cited by 1 | Viewed by 3603

Abstract

CRISPR-Cas allows us to introduce desired genome editing, including mutations, epitopes, and deletions, with unprecedented efficiency. The development of CRISPR-Cas has progressed to such an extent that it is now applicable in various fields, with the help of model organisms. C. elegans is one of the pioneering animals in which numerous CRISPR-Cas strategies have been rapidly established over the past decade. Ironically, the emergence of numerous methods makes the choice of the correct method difficult. Choosing an appropriate selection or screening approach is the first step in planning a genome modification. This report summarizes the key features and applications of CRISPR-Cas methods using C. elegans, illustrating key strategies. Our overview of significant advances in CRISPR-Cas will help readers understand the current advances in genome editing and navigate various methods of CRISPR-Cas genome editing. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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16 pages, 2501 KiB

Open AccessReview

Selecting for CRISPR-Edited Knock-In Cells

by Nina Reuven and Yosef Shaul

Int. J. Mol. Sci. 2022, 23(19), 11919; https://doi.org/10.3390/ijms231911919 - 07 Oct 2022

Cited by 5 | Viewed by 3232

Abstract

CRISPR technology affords a simple and robust way to edit the genomes of cells, providing powerful tools for basic research and medicine. While using Cas9 to target a genomic site is very efficient, making a specific mutation at that site is much less so, as it depends on the endogenous DNA repair machinery. Various strategies have been developed to increase the efficiency of knock-in mutagenesis, but often the desired cells remain a small percentage of the total population. To improve efficiency, strategies to select edited cells have been developed. In some applications, a selectable foreign gene is linked directly to the gene of interest (GOI). Alternatively, co-editing, where the GOI is edited along with a selectable gene, enriches the desired cells since the cells that successfully edited the selectable gene are likely to have also edited the GOI. To minimize perturbations of the host genome, “scarless” selection strategies have been developed, where the modified cells are mutated solely in the GOI. In this review, we will discuss strategies employed to improve specific genome editing in mammalian cells, focusing on ways to select successfully edited cells. Full article

(This article belongs to the Special Issue Advances, Pitfalls and Future Perspectives for CRISPR/Cas9 Mediated Genome Editing 2.0)

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