CRISPR-Cas9 enables conditional mutagenesis of challenging loci

Schick, Joel; Seisenberger, Claudia; Beig, Joachim; Bürger, Antje; Iyer, Vivek; Maier, Viola; Perera, Sajith; Rosen, Barry; Skarnes, William C.; Wurst, Wolfgang

doi:10.1038/srep32326

Cited by 11 publications

(9 citation statements)

References 27 publications

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“…For example, it is fairly efficient to produce knockins into the genomic ROSA26 locus in mice, while other loci are targeted less efficiently, and thus refractory to knockins. This accessibility to CRISPR/Cas9 complexes mirrors observations in mouse ES cell gene targeting technology, in which it was reported that some genes are not as efficiently targeted as others [105].…”

Section: Locus-specific Genetic Engineering Vectors In Mouse and Rat supporting

confidence: 65%

Principles of Genetic Engineering

Lanigan

Kopera

Saunders

2020

Genes

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Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

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Section: Locus-specific Genetic Engineering Vectors In Mouse and Rat supporting

confidence: 65%

Principles of Genetic Engineering

Lanigan

Kopera

Saunders

2020

Genes

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“…more than two orders of magnitude higher than reported in previous studies (Brinster et al 1989). The CRISPR/Cas9 treatment of ES cells dramatically increased homologous recombination with DNA donors (Gennequin et al 2013; Schick et al 2016, and author’s unpublished observations). Thus, the probability that correct integration of the synthetic DNA donor molecule occurred after CRISPR/Cas9-induced chromosome break is very high and the most likely explanation for the transgene knockin in Prokr2 sequence.…”

Section: Discussionmentioning

confidence: 95%

Sexually dimorphic distribution of Prokr2 neurons revealed by the Prokr2-Cre mouse model

et al. 2017

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Prokineticin receptor 2 (PROKR2) is predominantly expressed in the mammalian central nervous system. Loss-of-function mutations of PROKR2 in humans are associated with Kallmann syndrome due to the disruption of gonadotropin releasing hormone neuronal migration and deficient olfactory bulb morphogenesis. PROKR2 has been also implicated in the neuroendocrine control of GnRH neurons post-migration and other physiological systems. However, the brain circuitry and mechanisms associated with these actions have been difficult to investigate mainly due to the widespread distribution of Prokr2-expressing cells, and the lack of animal models and molecular tools. Here, we describe the generation, validation and characterization of a new mouse model that expresses Cre recombinase driven by the Prokr2 promoter, using CRISPR-Cas9 technology. Cre expression was visualized using reporter genes, tdTomato and GFP, in males and females. Expression of Cre-induced reporter genes was found in brain sites previously described to express Prokr2, e.g., the paraventricular and the suprachiasmatic nuclei, and the area postrema. The Prokr2-Cre mouse model was further validated by colocalization of Cre-induced GFP and Prokr2 mRNA. No disruption of Prokr2 expression, GnRH neuronal migration or fertility was observed. Comparative analysis of Prokr2-Cre expression in male and female brains revealed a sexually dimorphic distribution confirmed by in situ hybridization. In females, higher Cre activity was found in the medial preoptic area, ventromedial nucleus of the hypothalamus, arcuate nucleus, medial amygdala and lateral parabrachial nucleus. In males, Cre was higher in the amygdalo-hippocampal area. The sexually dimorphic pattern of Prokr2 expression indicates differential roles in reproductive function and, potentially, in other physiological systems.

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“…The purpose of gene editing in a hiPSCs workflow may be the insertion of a specific sequence from a HDR donor template, or the deletion of a genomic sequence. Templates for HDR-mediated knockin may use donor vectors that include drug selection or fluorescent marker genes enabling the efficient isolation of targeted colonies harboring stable vector integrations [33,34]. In some cases, especially for gene therapeutic purposes, the marker gene needs to be removed.…”

Section: Gene Editing Approachmentioning

confidence: 99%

Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9

Yumlu

Stumm

Bashir

et al. 2017

Methods

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Human induced pluripotent stem cells (hiPSCs) represent an ideal in vitro platform to study human genetics and biology. The recent advent of programmable nucleases makes also the human genome amenable to experimental genetics through either the correction of mutations in patient-derived iPSC lines or the de novo introduction of mutations into otherwise healthy iPSCs. The production of specific and sometimes complex genotypes in multiple cell lines requires efficient and streamlined gene editing technologies. In this article we provide protocols for gene editing in hiPSCs. We presently achieve high rates of gene editing at up to three loci using a modified iCRISPR system. This system includes a doxycycline inducible Cas9 and sgRNA/reporter plasmids for the enrichment of transfected cells by fluorescence-activated cell sorting (FACS). Here we cover the selection of target sites, vector construction, transfection, and isolation and genotyping of modified hiPSC clones.

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CRISPR-Cas9 enables conditional mutagenesis of challenging loci

Cited by 11 publications

References 27 publications

Principles of Genetic Engineering

Principles of Genetic Engineering

Sexually dimorphic distribution of Prokr2 neurons revealed by the Prokr2-Cre mouse model

Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9

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