Homology arms of targeting vectors for gene insertions and CRISPR/Cas9 technology: size does not matter; quality control of targeted clones does

AbstractClustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles.

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Small Activating RNAs: Towards the Development of New Therapeutic Agents and Clinical Treatments

Cells ◽

10.3390/cells10030591 ◽

2021 ◽

Vol 10 (3) ◽

pp. 591

Author(s):

Hossein Ghanbarian ◽

Shahin Aghamiri ◽

Mohamad Eftekhary ◽

Nicole Wagner ◽

Kay-Dietrich Wagner

Keyword(s):

Gene Expression ◽

Dna Sequences ◽

Cultured Cells ◽

Exogenous Dna ◽

Evolutionarily Conserved ◽

Rna Activation ◽

Endogenous Genes ◽

Cellular Machinery ◽

Eukaryotic Organisms ◽

Activate Gene

Small double-strand RNA (dsRNA) molecules can activate endogenous genes via an RNA-based promoter targeting mechanism. RNA activation (RNAa) is an evolutionarily conserved mechanism present in diverse eukaryotic organisms ranging from nematodes to humans. Small activating RNAs (saRNAs) involved in RNAa have been successfully used to activate gene expression in cultured cells, and thereby this emergent technique might allow us to develop various biotechnological applications, without the need to synthesize hazardous construct systems harboring exogenous DNA sequences. Accordingly, this thematic issue aims to provide insights into how RNAa cellular machinery can be harnessed to activate gene expression leading to a more effective clinical treatment of various diseases.

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Fast and efficient generation of knock-in human organoids using homology-independent CRISPR/Cas9 precision genome editing

10.1101/2020.01.15.907766 ◽

2020 ◽

Author(s):

Benedetta Artegiani ◽

Delilah Hendriks ◽

Joep Beumer ◽

Rutger Kok ◽

Xuan Zheng ◽

...

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Mitotic Spindle ◽

Cell Types ◽

Intestinal Cell ◽

Precise Integration ◽

Exogenous Dna ◽

Knock Out ◽

Homology Directed Repair ◽

Efficient Generation

AbstractCRISPR/Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences in human organoids awaits robust knock-in approaches. Here, we describe CRISPR/Cas9-mediated Homology-independent Organoid Transgenesis (CRISPR-HOT), which allows efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences at desired loci, without the necessity to inactivate TP53 in untransformed cells, previously used to increase HDR-mediated knock-in. CRISPR-HOT was employed to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter labelling the mitotic spindle by tagged tubulin and the cell membrane by tagged E-cadherin uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed TP53 involvement in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.

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Transgenic mice

Genome ◽

10.1139/g89-165 ◽

1989 ◽

Vol 31 (2) ◽

pp. 938-949 ◽

Cited By ~ 31

Author(s):

C. Babinet ◽

D. Morello ◽

J. P. Renard

Keyword(s):

Homologous Recombination ◽

Transgenic Mice ◽

Dna Sequences ◽

Insertional Mutagenesis ◽

Germ Line ◽

Embryonic Stem ◽

Physiological Role ◽

Cis Acting ◽

Endogenous Genes

Stable integration into the mouse genome of exogenous genetic information has become, over the past few years, a very potent approach for different aspects of biology. It is a common feature that the integrated exogenous gene (the transgene) is expressed properly both spatially and temporally. Constructing different lines of transgenic mice carrying various versions of a gene, therefore, permits cis acting DNA sequences involved in the specificity of expression to be defined, in the context of the developing animal. This in turn opens the way to a variety of experiments in which a given gene product is targeted to one or another cell type, thus offering some insight into the physiological role of this product. Such a strategy has been used, for example, to address the questions of the role of oncogenes in malignant transformation. The insertion of foreign DNA per se may disrupt the function of endogenous genes, thus creating an insertional mutation. The corresponding affected genes may subsequently be cloned, using the transgene as a tag. Finally, the ability to perform homologous recombination, recently demonstrated with embryonic stem cells that can colonize the germ line of a foreign embryo, should constitute in the near future a unique way to analyse in detail the functioning of the mammalian genome.Key words: transgenic mice, oncogenes, insertional mutagenesis, cis-acting sequences, homologous recombination.

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Reliable CRISPR/Cas9 genome engineering in Caenorhabiditis elegans using a single efficient sgRNA and an easily selectable phenotype

10.1101/105718 ◽

2017 ◽

Author(s):

Sonia El Mouridi ◽

Claire Lecroisey ◽

Philippe Tardy ◽

Marine Mercier ◽

Alice Leclercq-Blondel ◽

...

Keyword(s):

Genome Engineering ◽

Fluorescent Protein ◽

Point Mutations ◽

Fold Increase ◽

Strand Break ◽

Phenotypic Analysis ◽

Fluorescent Reporter ◽

Knock Out ◽

C Elegans ◽

Fluorescent Tags

ABSTRACTCRISPR/Cas9 genome engineering strategies allow the directed modification of the C. elegans genome to introduce point mutations, generate knock-out mutants and insert coding sequences for epitope or fluorescent tags. Three practical aspects however complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an 8-fold increase in fluorescence in a direct comparison.

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Cas12a mediates efficient and precise endogenous gene tagging via MITI: microhomology-dependent targeted integrations

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03396-8 ◽

2019 ◽

Vol 77 (19) ◽

pp. 3875-3884 ◽

Cited By ~ 6

Author(s):

Pan Li ◽

Lijun Zhang ◽

Zhifang Li ◽

Chunlong Xu ◽

Xuguang Du ◽

...

Keyword(s):

Negative Selection ◽

Gene Editing ◽

Genome Engineering ◽

Integration Method ◽

Specific Gene ◽

End Joining ◽

Exogenous Dna ◽

Dna Integration ◽

Endogenous Gene ◽

Endogenous Genes

AbstractEfficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications.

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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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Retention of relict satellite DNA sequences in Anemone (Ranunculaceae)

Acta Botanica Croatica ◽

10.2478/v10184-012-0010-z ◽

2013 ◽

Vol 72 (1) ◽

pp. 1-133 ◽

Cited By ~ 2

Author(s):

Višnja Besendorfer ◽

Jelena Mlinarec

Keyword(s):

Dna Sequences ◽

Southern Hybridization ◽

Repeat Unit ◽

Similar Species ◽

Genomic Component ◽

Library Hypothesis ◽

Species Specific ◽

Eukaryotic Organisms ◽

Fish Signal

Abstract Satellite DNAis a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNAis an important element in genome organization and evolution in plants. Here we study the presence, physical distribution and abundance of the satellite DNAfamily AhTR1 in Anemone. Twenty-two Anemone accessions were analyzed by PCR to assess the presence of AhTR1, while fluorescence in situ hybridization and Southern hybridization were used to determine the abundance and genomic distribution of AhTR1. The AhTR1 repeat unit was PCR-amplified only in eight phylogenetically related European Anemone taxa of the Anemone section. FISH signal with AhTR1 probe was visible only in A. hortensis and A. pavonina, showing localization of AhTR1 in the regions of interstitial heterochromatin in both species. The absence of a FISH signal in the six other taxa as well as weak signal after Southern hybridization suggest that in these species AhTR1 family appears as relict sequences. Thus, the data presented here support the »library hypothesis« for AhTR1 satellite evolution in Anemone. Similar species-specific satellite DNAprofiles in A. hortensis and A. pavonina support the treatment of A. hortensis and A. pavonina as one species, i.e. A. hortensis s.l.

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Impact of CRISPR-Cas9-Based Genome Engineering in Farm Animals

Veterinary Sciences ◽

10.3390/vetsci8070122 ◽

2021 ◽

Vol 8 (7) ◽

pp. 122

Author(s):

Parul Singh ◽

Syed Azmal Ali

Keyword(s):

Disease Resistance ◽

Genome Engineering ◽

Animal Health ◽

Fundamental Principle ◽

Farm Animals ◽

Web Based ◽

Knock Out ◽

Guide Rna ◽

Female Birth ◽

Comprehensive Information

Humans are sorely over-dependent on livestock for their daily basic need of food in the form of meat, milk, and eggs. Therefore, genetic engineering and transgenesis provide the opportunity for more significant gains and production in a short span of time. One of the best strategies is the genetic alteration of livestock to enhance the efficiency of food production (e.g., meat and milk), animal health, and welfare (animal population and disease). Moreover, genome engineering in the bovine is majorly focused on subjects such as disease resistance (e.g., tuberculosis), eradicate allergens (e.g., beta-lactoglobulin knock-out), products generation (e.g., meat from male and milk from female), male or female birth specifically (animal sexing), the introduction of valuable traits (e.g., stress tolerance and disease resistance) and their wellbeing (e.g., hornlessness). This review addressed the impressive genome engineering method CRISPR, its fundamental principle for generating highly efficient target-specific guide RNA, and the accompanying web-based tools. However, we have covered the remarkable roadmap of the CRISPR method from its conception to its use in cattle. Additionally, we have updated the comprehensive information on CRISPR-based gene editing in cattle.

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Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs

Development ◽

10.1242/dev.99.1.15 ◽

1987 ◽

Vol 99 (1) ◽

pp. 15-23

Author(s):

L.D. Etkin ◽

B. Pearman

Keyword(s):

Xenopus Laevis ◽

Dna Sequences ◽

Copy Number ◽

Germ Line ◽

Gene Promoter ◽

Early Gene ◽

Mosaic Pattern ◽

Exogenous Dna ◽

Gene Coding ◽

Germ Line Transmission

We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis. DNA was injected into fertilized eggs within 1 h following fertilization. The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6. Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues. We detected the exogenous DNA sequences in 60% of injected frogs. Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers. The copy number varied from 2 to 30 copies/cell in various tissues of the same frog. Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene. The pattern of expression, however, varied between different animals and could not be correlated with copy number. We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

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Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells

Development ◽

10.1242/dev.121.9.2853 ◽

1995 ◽

Vol 121 (9) ◽

pp. 2853-2859 ◽

Cited By ~ 1

Author(s):

A. Weng ◽

T. Magnuson ◽

U. Storb

Keyword(s):

De Novo ◽

Es Cells ◽

Embryonic Stem ◽

Dependent Manner ◽

Mouse Development ◽

Partial Methylation ◽

Distal Chromosome ◽

Before And After ◽

Endogenous Genes ◽

De Novo Methylation

A murine transgene, HRD, is methylated only when carried in certain inbred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain-specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryos revealed that strain-specific methylation is initiated prior to embryonic day (E) 6.5. Strain-specific transgene methylation is all-or-none in pattern and occurs exclusively in the primitive ectoderm lineage. A strain-independent pattern of partial methylation occurs in the primitive endoderm and trophectoderm lineages. To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and examined for transgene methylation before and after differentiation. Though the transgene had already acquired some methylation in undifferentiated ES cells, differentiation induced further, de novo methylation in a strain-dependent manner. Analysis of methylation in ES cultures suggests that the transgene and endogenous genes (such as immunoglobulin genes) are synchronously methylated during early development. These results are interpreted in the context of a model in which Ssm1-like modifier genes produce alterations in chromatin structure during and/or shortly after implantation, thereby marking target loci for de novo methylation with the rest of the genome during gastrulation.

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