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16 pages, 1337 KiB

Open AccessArticle

Genome Editing of the SNAI1 Gene in Rhabdomyosarcoma: A Novel Model for Studies of Its Role

by Aleksandra Ulman, Klaudia Skrzypek, Paweł Konieczny, Claudio Mussolino, Toni Cathomen and Marcin Majka

Cells 2020, 9(5), 1095; https://doi.org/10.3390/cells9051095 - 28 Apr 2020

Cited by 6 | Viewed by 2876

Abstract

Genome editing (GE) tools and RNA interference technology enable the modulation of gene expression in cancer research. While GE mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 or transcription activator-like effector nucleases (TALEN) activity can be used to induce gene knockouts, shRNA [...] Read more.

Genome editing (GE) tools and RNA interference technology enable the modulation of gene expression in cancer research. While GE mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 or transcription activator-like effector nucleases (TALEN) activity can be used to induce gene knockouts, shRNA interacts with the targeted transcript, resulting in gene knockdown. Here, we compare three different methods for SNAI1 knockout or knockdown in rhabdomyosarcoma (RMS) cells. RMS is the most common sarcoma in children and its development has been previously associated with SNAI1 transcription factor activity. To investigate the role of SNAI1 in RMS development, we compared CRISPR/Cas9, TALEN, and shRNA tools to identify the most efficient tool for the modulation of SNAI1 expression with biological effects. Subsequently, the genome sequence, transcript levels, and protein expression of SNAI1 were evaluated. The modulation of SNAI1 using three different approaches affected the morphology of the cells and modulated the expression of myogenic factors and HDAC1. Our study revealed a similar effectiveness of the tested methods. Nevertheless, the low efficiency of the GE tools was a limiting factor in obtaining biallelic gene knockouts. To conclude, we established and characterized three different models of SNAI1 knockout and knockdown that might be used in further studies investigating the role of SNAI1 in RMS. Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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13 pages, 2835 KiB

Open AccessArticle

Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation

by Takuro Horii, Ryosuke Kobayashi, Mika Kimura, Sumiyo Morita and Izuho Hatada

Cells 2020, 9(5), 1088; https://doi.org/10.3390/cells9051088 - 28 Apr 2020

Cited by 4 | Viewed by 3562

Abstract

The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential [...] Read more.

The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage frequently induced blastomere fusion. These fused embryos cannot develop to term because they are tetraploidized. In this study, we examined the following three conditions to inhibit blastomere fusion by EP at the 2-cell stage: (1) hypertonic treatment, (2) Calcium (Ca²⁺)-free treatment, and (3) actin polymerization inhibition. Hypertonic treatment of 2-cell stage embryos prevented blastomere fusion and facilitated blastocyst development; however, KI efficiency was decreased. Ca²⁺-free treatment and actin polymerization inhibition by cytochalasin B (CB) reduced fusion rate, and did not have negative effects on development and KI efficiency. These results suggest that Ca²⁺-free and CB treatment at the 2-cell stage is effective to generate floxed mice in combination with a sequential EP method. Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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20 pages, 3152 KiB

Open AccessArticle

Effect of CRISPR/Cas9-Mediated PD-1-Disrupted Primary Human Third-Generation CAR-T Cells Targeting EGFRvIII on In Vitro Human Glioblastoma Cell Growth

by Tsutomu Nakazawa, Atsushi Natsume, Fumihiko Nishimura, Takayuki Morimoto, Ryosuke Matsuda, Mitsutoshi Nakamura, Shuichi Yamada, Ichiro Nakagawa, Yasushi Motoyama, Young-Soo Park, Takahiro Tsujimura, Toshihiko Wakabayashi and Hiroyuki Nakase

Cells 2020, 9(4), 998; https://doi.org/10.3390/cells9040998 - 16 Apr 2020

Cited by 64 | Viewed by 6471

Abstract

Glioblastoma (GBM), which is the most common malignant brain tumor, is resistant to standard treatments. Immunotherapy might be a promising alternative for the treatment of this cancer. Chimeric antigen receptor (CAR) is an artificially modified fusion protein that can be engineered to direct [...] Read more.

Glioblastoma (GBM), which is the most common malignant brain tumor, is resistant to standard treatments. Immunotherapy might be a promising alternative for the treatment of this cancer. Chimeric antigen receptor (CAR) is an artificially modified fusion protein that can be engineered to direct the specificity and function of T cells against tumor antigens. However, the antitumor effects of EGFRvIII-targeting CAR-T (EvCAR-T) cells in GBM are limited. The inhibitory effect is induced by the interaction between programmed cell death protein 1 (PD-1) on activated EvCAR-T cells and its ligands on GBM cells. In the present study, PD-1-disrupted EvCAR-T cells were established using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The sgRNA/Cas9 expression vectors designed precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. The PD-1-disrupted EvCAR-T cells had an in vitro growth inhibitory effect on EGFRvIII-expressing GBM cells without altering the T-cell phenotype and the expression of other checkpoint receptors. In the future, the in vivo antitumor effect of this vector should be evaluated in order to determine if it could be applied clinically for improving the efficacy of EvCAR-T cell-based adoptive immunotherapy for GBM. Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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16 pages, 1996 KiB

Open AccessArticle

Modification of i-GONAD Suitable for Production of Genome-Edited C57BL/6 Inbred Mouse Strain

by Yukari Kobayashi, Takuya Aoshima, Ryota Ito, Ryota Shinmura, Masato Ohtsuka, Eri Akasaka, Masahiro Sato and Shuji Takabayashi

Cells 2020, 9(4), 957; https://doi.org/10.3390/cells9040957 - 13 Apr 2020

Cited by 10 | Viewed by 4397

Abstract

Improved genome editing via oviductal nucleic acid delivery (i-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. i-GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins [...] Read more.

Improved genome editing via oviductal nucleic acid delivery (i-GONAD) is a novel method for producing genome-edited mice in the absence of ex vivo handling of zygotes. i-GONAD involves the intraoviductal injection of clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins via the oviductal wall of pregnant females at 0.7 days post-coitum, followed by in vivo electroporation (EP). Unlike outbred Institute of Cancer Research (ICR) and hybrid mouse strains, genome editing of the most widely used C57BL/6J (B6) strain with i-GONAD has been considered difficult but, recently, setting a constant current of 100 mA upon EP enabled successful i-GONAD in this strain. Unfortunately, the most widely used electroporators employ a constant voltage, and thus we explored conditions allowing the generation of a 100 mA current using two electroporators: NEPA21 (Nepa Gene Co., Ltd.) and GEB15 (BEX Co., Ltd.). When the current and resistance were set to 40 V and 350–400 Ω, respectively, the current was fixed to 100 mA. Another problem in using B6 mice for i-GONAD is the difficulty in obtaining pregnant B6 females consistently because estrous females often fail to be found. A single intraperitoneal injection of low-dose pregnant mare’s serum gonadotrophin (PMSG) led to synchronization of the estrous cycle of these mice. Consequently, approximately 51% of B6 females had plugs upon mating with males 2 days after PMSG administration, which contrasts with the case (≈26%) when B6 females were subjected to natural mating. i-GONAD performed on PMSG-treated pregnant B6 females under conditions of average resistance of 367 Ω and average voltage of 116 mA resulted in the production of pregnant females at a rate of 56% (5/9 mice), from which 23 fetuses were successfully delivered. Nine (39%) of these fetuses exhibited successful genome editing at the target locus. Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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19 pages, 3052 KiB

Open AccessArticle

Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice

by Masahiro Sato, Rico Miyagasako, Shuji Takabayashi, Masato Ohtsuka, Izuho Hatada and Takuro Horii

Cells 2020, 9(3), 546; https://doi.org/10.3390/cells9030546 - 26 Feb 2020

Cited by 13 | Viewed by 4855

Abstract

Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a [...] Read more.

Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4–1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4–1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called “sequential i-GONAD (si-GONAD).” Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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15 pages, 3173 KiB

Open AccessArticle

uPAR Knockout Results in a Deep Glycolytic and OXPHOS Reprogramming in Melanoma and Colon Carcinoma Cell Lines

by Alessio Biagioni, Anna Laurenzana, Anastasia Chillà, Mario Del Rosso, Elena Andreucci, Martina Poteti, Daniele Bani, Daniele Guasti, Gabriella Fibbi and Francesca Margheri

Cells 2020, 9(2), 308; https://doi.org/10.3390/cells9020308 - 28 Jan 2020

Cited by 14 | Viewed by 3713

Abstract

Urokinase Plasminogen Activator (uPA) Receptor (uPAR) is a well-known GPI-anchored three-domain membrane protein with pro-tumor roles largely shown in all the malignant tumors where it is over-expressed. Here we have exploited the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene knock out approach [...] Read more.

Urokinase Plasminogen Activator (uPA) Receptor (uPAR) is a well-known GPI-anchored three-domain membrane protein with pro-tumor roles largely shown in all the malignant tumors where it is over-expressed. Here we have exploited the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene knock out approach to investigate its role in the oxidative metabolism in human melanoma and colon cancer as the consequences of its irreversible loss. Knocking out PLAUR, a uPAR-encoding gene, in A375p, A375M6 and HCT116, which are two human melanoma and a colon carcinoma, respectively, we have observed an increased number of mitochondria in the two melanoma cell lines, while we evidenced an immature biogenesis of mitochondria in the colon carcinoma culture. Such biological diversity is, however, reflected in a significant enhancement of the mitochondrial spare respiratory capacity, fueled by an increased expression of GLS2, and in a decreased glycolysis paired with an increased secretion of lactate by all uPAR KO cells. We speculated that this discrepancy might be explained by an impaired ratio between LDHA and LDHB. Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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24 pages, 5528 KiB

Open AccessArticle

Knockout of the HvCKX1 or HvCKX3 Gene in Barley (Hordeum vulgare L.) by RNA-Guided Cas9 Nuclease Affects the Regulation of Cytokinin Metabolism and Root Morphology

by Sebastian Gasparis, Mateusz Przyborowski, Maciej Kała and Anna Nadolska-Orczyk

Cells 2019, 8(8), 782; https://doi.org/10.3390/cells8080782 - 26 Jul 2019

Cited by 51 | Viewed by 5582

Abstract

Barley is among four of the most important cereal crops with respect to global production. Increasing barley yields to desired levels can be achieved by the genetic manipulation of cytokinin content. Cytokinins are plant hormones that regulate many developmental processes and have a [...] Read more.

Barley is among four of the most important cereal crops with respect to global production. Increasing barley yields to desired levels can be achieved by the genetic manipulation of cytokinin content. Cytokinins are plant hormones that regulate many developmental processes and have a strong influence on grain yield. Cytokinin homeostasis is regulated by members of several multigene families. CKX genes encode the cytokinin oxidase/dehydrogenase enzyme, which catalyzes the irreversible degradation of cytokinin. Several recent studies have demonstrated that the RNAi-based silencing of CKX genes leads to increased grain yields in some crop species. To assess the possibility of increasing the grain yield of barley by knocking out CKX genes, we used an RNA-guided Cas9 system to generate ckx1 and ckx3 mutant lines with knockout mutations in the HvCKX1 and HvCKX3 genes, respectively. Homozygous, transgene-free mutant lines were subsequently selected and analyzed. A significant decrease in CKX enzyme activity was observed in the spikes of the ckx1 lines, while in the ckx3 lines, the activity remained at a similar level to that in the control plants. Despite these differences, no changes in grain yield were observed in either mutant line. In turn, differences in CKX activity in the roots between the ckx1 and ckx3 mutants were reflected via root morphology. The decreased CKX activity in the ckx1 lines corresponded to greater root length, increased surface area, and greater numbers of root hairs, while the increased CKX activity in the ckx3 mutants gave the opposite results. RNA-seq analysis of the spike and root transcriptomes revealed an altered regulation of genes controlling cytokinin metabolism and signaling, as well as other genes that are important during seed development, such as those that encode nutrient transporters. The observed changes suggest that the knockout of a single CKX gene in barley may be not sufficient for disrupting cytokinin homeostasis or increasing grain yields. Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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Review

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20 pages, 4523 KiB

Open AccessReview

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

by Masahiro Sato, Shuji Takabayashi, Eri Akasaka and Shingo Nakamura

Cells 2020, 9(4), 799; https://doi.org/10.3390/cells9040799 - 26 Mar 2020

Cited by 24 | Viewed by 5238

Abstract

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of [...] Read more.

The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of efficient and simple methods to produce genome-edited (GE) animals would accelerate research in this field. The CRISPR/Cas9 system was initially employed in early embryos, utilizing classical gene delivery methods such as microinjection or electroporation, which required ex vivo handling of zygotes before transfer to recipients. Recently, novel in vivo methods such as genome editing via oviductal nucleic acid delivery (GONAD), improved GONAD (i-GONAD), or transplacental gene delivery for acquiring genome-edited fetuses (TPGD-GEF), which facilitate easy embryo manipulation, have been established. Studies utilizing these techniques employed pregnant female mice for direct introduction of the genome-editing components into the oviduct or were dependent on delivery via tail-vein injection. In mice, embryogenesis occurs within the oviducts and the uterus, which often hampers the genetic manipulation of embryos, especially those at early postimplantation stages (days 6 to 8), owing to a thick surrounding layer of tissue called decidua. In this review, we have surveyed the recent achievements in the production of GE mice and have outlined the advantages and disadvantages of the process. We have also referred to the past achievements in gene delivery to early postimplantation stage embryos and germ cells such as primordial germ cells and spermatogonial stem cells, which will benefit relevant research. Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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15 pages, 1804 KiB

Open AccessReview

Applications of Genome Editing Technology in Research on Chromosome Aneuploidy Disorders

by Silvia Natsuko Akutsu, Kazumasa Fujita, Keita Tomioka, Tatsuo Miyamoto and Shinya Matsuura

Cells 2020, 9(1), 239; https://doi.org/10.3390/cells9010239 - 17 Jan 2020

Cited by 6 | Viewed by 5504

Abstract

Chromosomal segregation errors in germ cells and early embryonic development underlie aneuploidies, which are numerical chromosomal abnormalities causing fetal absorption, developmental anomalies, and carcinogenesis. It has been considered that human aneuploidy disorders cannot be resolved by radical treatment. However, recent studies have demonstrated [...] Read more.

Chromosomal segregation errors in germ cells and early embryonic development underlie aneuploidies, which are numerical chromosomal abnormalities causing fetal absorption, developmental anomalies, and carcinogenesis. It has been considered that human aneuploidy disorders cannot be resolved by radical treatment. However, recent studies have demonstrated that aneuploidies can be rescued to a normal diploid state using genetic engineering in cultured cells. Here, we summarize a series of studies mainly applying genome editing to eliminate an extra copy of human chromosome 21, the cause of the most common constitutional aneuploidy disorder Down syndrome. We also present findings on induced pluripotent stem cell reprogramming, which has been shown to be one of the most promising technologies for converting aneuploidies into normal diploidy without the risk of genetic alterations such as genome editing-mediated off-target effects. Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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20 pages, 1118 KiB

Open AccessReview

Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses

by Oliver Patrick March, Thomas Kocher and Ulrich Koller

Cells 2020, 9(1), 112; https://doi.org/10.3390/cells9010112 - 02 Jan 2020

Cited by 29 | Viewed by 5797

Abstract

The skin provides direct protection to the human body from assault by the harsh external environment. The crucial function of this organ is significantly disrupted in genodermatoses patients. Genodermatoses comprise a heterogeneous group of largely monogenetic skin disorders, typically involving mutations in genes [...] Read more.

The skin provides direct protection to the human body from assault by the harsh external environment. The crucial function of this organ is significantly disrupted in genodermatoses patients. Genodermatoses comprise a heterogeneous group of largely monogenetic skin disorders, typically involving mutations in genes encoding structural proteins. Therapeutic options for this debilitating group of diseases, including epidermolysis bullosa, primarily consist of wound management. Genome editing approaches co-opt double-strand break repair pathways to introduce desired sequence alterations at specific loci. Rapid advances in genome editing technologies have the potential to propel novel genetic therapies into the clinic. However, the associated phenotypes of many mutations may be treated via several genome editing strategies. Therefore, for potential clinical applications, implementation of efficient approaches based upon mutation, gene and disease context is necessary. Here, we describe current genome editing approaches for the treatment of genodermatoses, along with a discussion of the optimal strategy for each genetic context, in order to achieve enhanced genome editing approaches. Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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39 pages, 3178 KiB

Open AccessReview

Genome Editing in Plants: Exploration of Technological Advancements and Challenges

by Sanskriti Vats, Surbhi Kumawat, Virender Kumar, Gunvant B. Patil, Trupti Joshi, Humira Sonah, Tilak Raj Sharma and Rupesh Deshmukh

Cells 2019, 8(11), 1386; https://doi.org/10.3390/cells8111386 - 04 Nov 2019

Cited by 97 | Viewed by 15702

Abstract

Genome-editing, a recent technological advancement in the field of life sciences, is one of the great examples of techniques used to explore the understanding of the biological phenomenon. Besides having different site-directed nucleases for genome editing over a decade ago, the CRISPR/Cas (clustered [...] Read more.

Genome-editing, a recent technological advancement in the field of life sciences, is one of the great examples of techniques used to explore the understanding of the biological phenomenon. Besides having different site-directed nucleases for genome editing over a decade ago, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) based genome editing approach has become a choice of technique due to its simplicity, ease of access, cost, and flexibility. In the present review, several CRISPR/Cas based approaches have been discussed, considering recent advances and challenges to implicate those in the crop improvement programs. Successful examples where CRISPR/Cas approach has been used to improve the biotic and abiotic stress tolerance, and traits related to yield and plant architecture have been discussed. The review highlights the challenges to implement the genome editing in polyploid crop plants like wheat, canola, and sugarcane. Challenges for plants difficult to transform and germline-specific gene expression have been discussed. We have also discussed the notable progress with multi-target editing approaches based on polycistronic tRNA processing, Csy4 endoribonuclease, intron processing, and Drosha ribonuclease. Potential to edit multiple targets simultaneously makes it possible to take up more challenging tasks required to engineer desired crop plants. Similarly, advances like precision gene editing, promoter bashing, and methylome-editing will also be discussed. The present review also provides a catalog of available computational tools and servers facilitating designing of guide-RNA targets, construct designs, and data analysis. The information provided here will be useful for the efficient exploration of technological advances in genome editing field for the crop improvement programs. Full article

(This article belongs to the Special Issue Genome Editing Systems, Methods, Techniques and Their Application)

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