Rescuing Lethal Phenotypes Induced by Disruption of Genes in Mice: a Review of Novel Strategies

Approximately 35 % of the mouse genes are indispensable for life, thus, global knock-out (KO) of those genes may result in embryonic or early postnatal lethality due to developmental abnormalities. Several KO mouse lines are valuable human disease models, but viable homozygous mutant mice are frequently required to mirror most symptoms of a human disease. The site-specific gene editing systems, the transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs) and the clustered regularly interspaced short palindrome repeat-associated Cas9 nuclease (CRISPR/Cas9) made the generation of KO mice more efficient than before, but the homozygous lethality is still an undesired side-effect in case of many genes. The literature search was conducted using PubMed and Web of Science databases until June 30th, 2020. The following terms were combined to find relevant studies: “lethality”, “mice”, “knock-out”, “deficient”, “embryonic”, “perinatal”, “rescue”. Additional manual search was also performed to find the related human diseases in the Online Mendelian Inheritance in Man (OMIM) database and to check the citations of the selected studies for rescuing methods. In this review, the possible solutions for rescuing human disease-relevant homozygous KO mice lethal phenotypes were summarized.

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Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽

10.3390/biology10060530 ◽

2021 ◽

Vol 10 (6) ◽

pp. 530

Author(s):

Marlo K. Thompson ◽

Robert W. Sobol ◽

Aishwarya Prakash

Keyword(s):

Dna Repair ◽

Mammalian Cells ◽

Genome Stability ◽

Gene Editing ◽

Adaptive Immune System ◽

Zinc Finger Nucleases ◽

Specific Gene ◽

Target Sequence ◽

Transcription Activator ◽

Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

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The Role Of tet2 DNA Methylcytosine Dioxygenase In Zebrafish Early Hematopoiesis

Blood ◽

10.1182/blood.v122.21.1204.1204 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1204-1204

Author(s):

Huan-Chau Lin ◽

Ken-Hong Lim ◽

Yi-Hao Chiang ◽

Wei-Ting Wang ◽

Ching-Sung Lin ◽

...

Keyword(s):

Conflicts Of Interest ◽

Mendelian Inheritance ◽

Stem Cell Markers ◽

Loss Of Function ◽

Transcription Activator ◽

Hematopoietic Stem ◽

Mature Adult ◽

Knock Out ◽

Knock Down

Abstract Loss-of-function mutations in Ten-Eleven-Translocation 2 (TET2) gene have been identified in various human myeloid and lymphoid malignancies. Recently, the TET gene family (TET1, TET2, and TET3) was found to function as DNA methylcytosine dioxygenase that is able to oxidize 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). In Tet2-deficient mouse models, Tet2 has been shown to play an important role in regulating self-renewal and differentiation of hematopoietic stem cells. These Tet2-deficient mice would gradually develop a chronic myeloid neoplasm resembling human chronic myelomonocytic leukemia suggesting that TET2 may function as a tumor suppressor. In the present study, we investigated the role of tet2 in zebrafish early hematopoiesis. During zebrafish early development, the expression of tet1, tet2, and tet3 by qRT-PCR can be detected mainly after the segmentation stage (26-somite), with fluctuated expression levels thereafter. Whole-mount in situ hybridization revealed that tet2 expression was strong over aorta-gonad-mesonephros region at 48 hours post-fertilization (hpf). Morpholino oligonucleotide (MO) knock-down of tet2 increased the expression of tet1, tet3, dnmt3aa, gata-1, alpha-Hb and fli1a (48 hpf) as well as rag2 and lck (4 days post-fertilization), and the expression of spi1b and mpo decreased (48 hpf). The expression of primitive hematopoietic stem cell markers scl and lmo2, as well as dnmt3ab, beta-Hb, l-plastin, and rag1 were unaffected. The levels of 5-mC and 5-hmC measured by ELISA were also decreased after MO knock-down of tet2. The number of gata-1 expressing red blood cells increased after tet2 MO knock-down as evaluated by flow-cytometry indicating that tet2 deficiency increased erythropoiesis. These preliminary results suggest that tet2 might play a role in the epigenetic regulation of zebrafish early hematopoiesis including erythropoiesis. Recently, transcription activator-like effector nuclease (TALEN) has been shown to generate targeted genomic editing in zebrafish. To validate our observation, we therefore utilized customized TALENs pair to generate tet2 knock-out zebrafish animal model. We designed a pair of TALENs targeting first exon of tet2 and our tet2 TALENs were able to generate insertion and/or deletion in targeted region of tet2 exon 1 in 25% to 44% zebrafish embryos. We obtained a total of fifteen different tet2 mutation genotypes F1 fish, and seven of them were predicted to cause early termination of transcription. The in-cross of these F1 genotypes matched the Mendelian inheritance. The tet2-/- knock-out F2 zebrafish is not embryonic lethal and can grow to sexually mature adult fish. The detailed analysis of tet2-/- knock-out zebrafish early hematopoiesis will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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CRISPR/Cas9 gene-editing strategies in cardiovascular cells

Cardiovascular Research ◽

10.1093/cvr/cvz250 ◽

2019 ◽

Vol 116 (5) ◽

pp. 894-907 ◽

Cited By ~ 1

Author(s):

Eva Vermersch ◽

Charlène Jouve ◽

Jean-Sébastien Hulot

Keyword(s):

Cardiovascular Diseases ◽

Genome Editing ◽

Zinc Finger Nucleases ◽

Public Health Issue ◽

Transcription Activator ◽

Knock Out ◽

Cardiovascular Cells ◽

Induced Pluripotent

Abstract Cardiovascular diseases are among the main causes of morbidity and mortality in Western countries and considered as a leading public health issue. Therefore, there is a strong need for new disease models to support the development of novel therapeutics approaches. The successive improvement of genome editing tools with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has enabled the generation of genetically modified cells and organisms with much greater efficiency and precision than before. The simplicity of CRISPR/Cas9 technology made it especially suited for different studies, both in vitro and in vivo, and has been used in multiple studies evaluating gene functions, disease modelling, transcriptional regulation, and testing of novel therapeutic approaches. Notably, with the parallel development of human induced pluripotent stem cells (hiPSCs), the generation of knock-out and knock-in human cell lines significantly increased our understanding of mutation impacts and physiopathological mechanisms within the cardiovascular domain. Here, we review the recent development of CRISPR–Cas9 genome editing, the alternative tools, the available strategies to conduct genome editing in cardiovascular cells with a focus on its use for correcting mutations in vitro and in vivo both in germ and somatic cells. We will also highlight that, despite its potential, CRISPR/Cas9 technology comes with important technical and ethical limitations. The development of CRISPR/Cas9 genome editing for cardiovascular diseases indeed requires to develop a specific strategy in order to optimize the design of the genome editing tools, the manipulation of DNA repair mechanisms, the packaging and delivery of the tools to the studied organism, and the assessment of their efficiency and safety.

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CRISPER/CAS: A potential tool for genomes editing

Biomedical Letters ◽

10.47262//bl/7.2.20210711 ◽

2021 ◽

Vol 7 (2) ◽

pp. 122-129

Keyword(s):

Genome Editing ◽

Basic Mechanism ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

End Joining ◽

Knock Out ◽

Homology Directed Repair ◽

Wide Range ◽

Non Homologous End Joining ◽

Cas A

The ability to engineer genomes presents a significant opportunity for applied biology research. In 2050, the population of this world is expected to reach 9.6 billion residents; rising food with better quality is the most promising approach to food security. Compared to earlier methodologies including Zinc Finger Nucleases (ZFNs) plus Transcription Activator-Like Effector Nucleases (TALENs), which were expensive as well as time-consuming, innovation in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and related CRISPR (Cas) protein classifications allowed selective editing of genes for the enhancement of food. The basic mechanism of CRISPR Cas9 process and its applications on genome editing has been summarized in this manuscript. The method relies on Sequence-Specific Nucleases (SSNs) to create Double Stranded Breaks (DSB) of DNA at the locus of genome defined by user, mended by using one of two DNA mending ways: Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR). Cas9, an RNA-guided endonuclease, was used to produce stable knock-in and knock-out mutants. The focus of this effort is to explore the CRISPR Cas9 genome editing to manage gene expression and improve future editing success. This adaptable technique can be consumed for a wide range of applications of genome editing requiring high precision. Advances in this technology have sparked renewed interest in the possibilities for editing genome in plants.

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243 EFFICIENT EDITION OF THE BOVINE PRNP PRION GENE IN SOMATIC CELLS AND IVF EMBRYOS USING THE CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 SYSTEM

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab243 ◽

2016 ◽

Vol 28 (2) ◽

pp. 253

Author(s):

R. J. Bevacqua ◽

R. Fernandez-Martín ◽

V. Savy ◽

N. G. Canel ◽

M. I. Gismondi ◽

...

Keyword(s):

Gene Editing ◽

Prnp Gene ◽

Zinc Finger Nucleases ◽

Spongiform Encephalopathy ◽

Specific Gene ◽

Transcription Activator ◽

Domestic Species ◽

Fetal Fibroblasts ◽

High Level

The rapid introduction of engineered nucleases technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR), provides new opportunities for editing genes in a targeted and rather simple fashion. Few reports are available regarding CRISPR efficiency in domestic species. Here, the CRISPR/Cas9 system was employed to develop knockout and knock-in alleles of the bovine PRNP gene, responsible for bovine spongiform encephalopathy (mad cow disease), both in bovine fetal fibroblasts and in IVF embryos. Five sgRNAs were designed to target a 875-bp region within prnp exon 3; all 5 were co-delivered with hCas9 and a homologous recombination vector carrying gfp (pHRegfp). For cells, 3 transfection conditions were compared: 2 μg of hCas9 + 1 μg of sgRNAs mix ± 2 μg pHREGFP (1X) versus 4 μg of hCas9 + 2 μg of sgRNAs mix ± 4 μg of pHREGFP (2X). For IVF zygotes, cytoplasmic injection was conducted with 2 RNA concentrations: (a) 50 ng μL–1 hCas9 RNA + 25 ng μL–1 sgRNAs mix (RNA1X), ±50 ng μL–1 pHREGFP, and (b) 100 ng μL–1 hCas9 + 50 ng μL–1 sgRNAs mix (RNA2X), ±100 ng μL–1 pHREGFP, which were compared with plasmid injections with 100 ng μL–1 pCMVCas9 + 50 ng μL–1 pU6sgRNAs mix (DNA2X), ±100 ng μL–1 pHREGFP. The pHREGFP was always injected as plasmid, under the same conditions as hCas9. DNA from cells was subjected to PCR, Surveyor assay, and sequence analysis. Embryo analysis was conducted on whole-genome-amplified DNA from blastocysts, followed by PCR assays and sequencing. In cells, 2X transfection resulted in indels and amplification of PCR products of lower MW than the wild-type, indicative of the deletion of a part of the targeted PRNP region. However, it was not possible to detect an effect for 1X transfection. For the group transfected with pHREGFP, insertion of a partial EGFP sequence was detected (383 bp). Regarding embryo injection, higher blastocyst rates were obtained in all groups injected with RNA (Table 1). In 48% (21/43) of the sequenced blastocysts specific gene editing was detected (Table 1). Modifications varied among single base pair shift (3/43; 7%), high level of mismatches all over the targeted sequence and vicinity (12/43; 27.9%), full deletion of the 875-bp region (1/43; 2.3%), and partial insertion of 100–498 bp pHREGFP fragments between the HR arms (5/24; 20.8%). Most of these modifications occurred in a mosaic fashion (76%). Results demonstrate that CRISPR/Cas can be efficiently applied for site-specific edition of domestic species genomes. Table 1.In vitro development and gene editing efficiency of embryos injected with plasmids or RNA coding for CRISPR/Cas9 system targeting PRNP

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INDEL detection, the ‘Achilles heel’ of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels

Nucleic Acids Research ◽

10.1093/nar/gkaa975 ◽

2020 ◽

Vol 48 (21) ◽

pp. 11958-11981

Author(s):

Eric Paul Bennett ◽

Bent Larsen Petersen ◽

Ida Elisabeth Johansen ◽

Yiyuan Niu ◽

Zhang Yang ◽

...

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Strand Break ◽

Zinc Finger Nucleases ◽

Great Promise ◽

Transcription Activator ◽

End Joining ◽

Knock Out ◽

Indel Detection ◽

In Cells

Abstract Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

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From Descriptive to Functional Genomics of Leukemias Focusing on Genome Engineering Techniques

International Journal of Molecular Sciences ◽

10.3390/ijms221810065 ◽

2021 ◽

Vol 22 (18) ◽

pp. 10065

Author(s):

Beata Balla ◽

Florin Tripon ◽

Claudia Banescu

Keyword(s):

Functional Genomics ◽

Dna Sequences ◽

Genome Engineering ◽

Leukemic Cell ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Knock Out ◽

Leukemic Cell Lines ◽

Immune Therapies ◽

A Cell

Genome engineering makes the precise manipulation of DNA sequences possible in a cell. Therefore, it is essential for understanding gene function. Meganucleases were the start of genome engineering, and it continued with the discovery of Zinc finger nucleases (ZFNs), followed by Transcription activator-like effector nucleases (TALENs). They can generate double-strand breaks at a desired target site in the genome, and therefore can be used to knock in mutations or knock out genes in the same way. Years later, genome engineering was transformed by the discovery of clustered regularly interspaced short palindromic repeats (CRISPR). Implementation of CRISPR systems involves recognition guided by RNA and the precise cleaving of DNA molecules. This property proves its utility in epigenetics and genome engineering. CRISPR has been and is being continuously successfully used to model mutations in leukemic cell lines and control gene expression. Furthermore, it is used to identify targets and discover drugs for immune therapies. The descriptive and functional genomics of leukemias is discussed in this study, with an emphasis on genome engineering methods. The CRISPR/Cas9 system’s challenges, viewpoints, limits, and solutions are also explored.

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Genetics in Ophthalmology

Nauchno-prakticheskii zhurnal «Medicinskaia genetika» ◽

10.25557/2073-7998.2020.08.44-45 ◽

2020 ◽

pp. 44-45

Author(s):

А.Ю. Рудник ◽

М.А. Федяков ◽

О.С. Глотов

Keyword(s):

Genetic Basis ◽

Genetic Diseases ◽

Mendelian Inheritance ◽

Diagnostic Screening ◽

Online Mendelian Inheritance ◽

Clinical Diagnostic ◽

Omim Database

На сегодняшний день в базе данных Online Mendelian Inheritance in Man (OMIM) описано более 6613 заболеваний и фенотипов, 4241 имеют доказанную генетическую основу, не менее 45% вкючают офтальмологические проявления. В статье приведен ряд клинический примеров пациентов с офтальмологическими симптомами различных генетических заболеваний (алкаптонурия, болезнь Штаргардта, синдром микроцефалии с или без хориоретинопатии; астроцитарная гамартома) с целью демонстрации эффективного клинико-диагностического скрининга генетической патологии у пациентов. So far, the Online Mendelian Inheritance in Man (OMIM) database describes more than 6613 diseases and phenotypes, 4241 have a proven genetic basis, 45% of which are combined with ophthalmological manifestations. The article provides a number of clinical examples of patients with ophthalmological manifestations of various genetic diseases (alcaptonuria, Stadgart ‘s disease, microcephaly syndrome with or without choriretinopathy; Astrocytic gamartoma) to demonstrate effective clinical-diagnostic screening of genetic pathology in patients.

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Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

Scientific Reports ◽

10.1038/s41598-021-89546-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yuu Asano ◽

Kensuke Yamashita ◽

Aoi Hasegawa ◽

Takanori Ogasawara ◽

Hoshie Iriki ◽

...

Keyword(s):

Genome Editing ◽

Dictyostelium Discoideum ◽

Streptococcus Pyogenes ◽

Nucleotide Substitution ◽

Point Mutations ◽

Targeted Mutagenesis ◽

Single Amino Acid ◽

Specific Gene ◽

Knock Out ◽

Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

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Mini-Review Regarding the Applicability of Genome Editing Techniques Developed for Studying Infertility

Diagnostics ◽

10.3390/diagnostics11020246 ◽

2021 ◽

Vol 11 (2) ◽

pp. 246

Author(s):

Bogdan Doroftei ◽

Ovidiu-Dumitru Ilie ◽

Maria Puiu ◽

Alin Ciobica ◽

Ciprian Ilea

Keyword(s):

Motor Activity ◽

Experimental Model ◽

Zinc Finger ◽

Biomedical Research ◽

Zinc Finger Nucleases ◽

Loss Of Function ◽

Transcription Activator ◽

Phenotypic Changes ◽

Wide Range ◽

Short Palindromic Repeat

Infertility is a highly debated topic today. It has been long hypothesized that infertility has an idiopathic cause, but recent studies demonstrated the existence of a genetic substrate. Fortunately, the methods of editing the human genome proven to be revolutionary. Following research conducted, we identified a total of 21 relevant studies; 14 were performed on mice, 5 on zebrafish and 2 on rats. We concluded that over forty-four genes in total are dispensable for fertility in both sexes without affecting host homeostasis. However, there are genes whose loss-of-function induces moderate to severe phenotypic changes in both sexes. There were situations in which the authors reported infertility, exhibited by the experimental model, or other pathologies such as cryptorchidism, cataracts, or reduced motor activity. Overall, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 are techniques that offer a wide range of possibilities for studying infertility, even to create mutant variants. It can be concluded that ZFNs, TALENs, and CRISPR/Cas9 are crucial tools in biomedical research.

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