Simple Protocol for Generating and Genotyping Genome‐Edited Mice With CRISPR‐Cas9 Reagents

Fernández, Almudena; Morín, Matías; Muñoz-Santos, Diego; Josa, Santiago; Montero, Andrea C.; Rubio‐Fernández, Marcos; Cantero, Marta; Fernández, J.; Hierro, María Jesús del; Castrillo, Marta; Moreno-Pelayo, Miguel A.; Montoliu, Lluı́s

doi:10.1002/cpmo.69

Cited by 22 publications

(17 citation statements)

References 20 publications

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“…Protocols using sgRNA in connection with Cas9 for a restriction enzyme‐like genotyping assay similar to the one described here have also been reported (Kim et al, 2014; Liang et al., 2018; Zhou, Peng, Zhang, & Li, 2018), but with a time‐consuming protocol limited to short PCR fragments which renders it difficult to test several sgRNAs with the same amplified target. Next generation sequencing of amplicons derived from CRISPR/Cas9‐edited populations can be used to screen for desired mutations (Fernández et al, 2020), but this requires costly library preparation and sequence data analysis and cannot be routinely used in every lab. Our design, with its proven robustness in different buffers, allows for a versatile choice of template fragments, is suitable for testing multiple sgRNA sites at the same time, and can be combined with other enzymatic reactions.…”

Section: Discussionmentioning

confidence: 99%

Versatile in vitro assay to recognize Cas9‐induced mutations

2020

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Mutagenesis is a powerful method to alter an organism's phenotype or to study the function of gene products by disrupting their genomic template. Classical mutagenesis is based on the random distribution of genetic lesions induced by radiation or genotoxic chemicals. While these procedures are valuable approaches for unbiased forward genetic screens or for generating genetic diversity for plant breeding without requiring particular reagents or procedures, they come at the price of creating multiple, unpredictable, and untargeted mutations that need to be screened, separated from unwanted lesions, mapped, and characterized in laborious, time-and cost-intensive procedures. Recently, the precise editing of genomic information within living organisms at single, pre-defined sites has tremendously enriched the mutagenesis toolbox (Bortesi et al., 2016; Wang, La Russa, & Qi, 2016). While gene replacement remains difficult in plants, working efficiently in only a limited number of experimental systems, targeted disruption of

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Section: Discussionmentioning

confidence: 99%

Versatile in vitro assay to recognize Cas9‐induced mutations

2020

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“…Finally, thanks to CRISPR genome‐editing methods, it is now possible to reproduce patient‐specific OCA1 mutations in mouse models, in order to study their role in the establishment of the albinism phenotype (Fernández et al., 2020).…”

Section: Oculocutaneous Albinism Typementioning

confidence: 99%

Genetics of non‐syndromic and syndromic oculocutaneous albinism in human and mouse

Fernández

Hayashi

Garrido

et al. 2021

Pigment Cell Melanoma Res

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Oculocutaneous albinism (OCA) is the most frequent presentation of albinism, a heterogeneous rare genetic condition generally associated with variable alterations in pigmentation and with a profound visual impairment. There are non‐syndromic and syndromic types of OCA, depending on whether the gene product affected impairs essentially the function of melanosomes or, in addition, that of other lysosome‐related organelles (LROs), respectively. Syndromic OCA can be more severe and associated with additional systemic consequences, beyond pigmentation and vision alterations. In addition to OCA, albinism can also be presented without obvious skin and hair pigmentation alterations, in ocular albinism (OA), and a related genetic condition known as foveal hypoplasia, optic nerve decussation defects, and anterior segment dysgenesis (FHONDA). In this review, we will focus only in the genetics of skin pigmentation in OCA, both in human and mouse, updating our current knowledge on this subject.

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“…Expensive to implement unless large numbers of samples analyzed [26,58] Trends in Genetics sequence rearrangements. Given the variability of the outcome and the length of the modified segments, a fuller examination requires more elaborate and expensive assays, such as targeted locus amplification (TLA) [56] or, in the case of material with a complex genetic make-up, high-throughput short-read [57] or long-read [26,58] sequencing. For the latter two, targeted sequencing can rely on the isolation of the loci of interest by simple PCR [26,57,58], but this limits the size of the interval that can be interrogated.…”

Section: Capturing the Variability Of Genome Editing Outcome On Targetmentioning

confidence: 99%

“…Given the variability of the outcome and the length of the modified segments, a fuller examination requires more elaborate and expensive assays, such as targeted locus amplification (TLA) [56] or, in the case of material with a complex genetic make-up, high-throughput short-read [57] or long-read [26,58] sequencing. For the latter two, targeted sequencing can rely on the isolation of the loci of interest by simple PCR [26,57,58], but this limits the size of the interval that can be interrogated. Other approaches for larger template isolation are emerging to lift this constraint (e.g., biotin-labeled probes [59] and Cas9-aided capture [60,61]) (see Table 1).…”

Section: Capturing the Variability Of Genome Editing Outcome On Targetmentioning

confidence: 99%

Anticipating and Identifying Collateral Damage in Genome Editing

Burgio

Teboul

2020

Trends in Genetics

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Genome editing has powerful applications in research, healthcare, and agriculture. However, the range of possible molecular events resulting from genome editing has been underestimated and the technology remains unpredictable on, and away from, the target locus. This has considerable impact in providing a safe approach for therapeutic genome editing, agriculture, and other applications. This opinion article discusses how to anticipate and detect those editing events by a combination of assays to capture all possible genomic changes. It also discusses strategies for preventing unwanted effects, critical to appraise the benefit or risk associated with the use of the technology. Anticipating and verifying the result of genome editing are essential for the success for all applications. Genome Editing: A Transformative TechnologyThe application of genome editing is transforming agriculture, biomedical research, and healthcare. The many proposed purposes include the generation of more productive or robust crops and farm animals, animal hosts for the production of tissues for graft purposes and therapies that use ex vivo or somatic tissue engineering [1-3]. The promise of applicability is turning into reality, as illustrated by the first nonrandomized Phase I clinical trial i in which the use of clustered regularly interspaced short palindromic repeats (CRISPR)-engineered T cells was recently found to be safe [4]. To date, N20 Phase I/II human clinical trials are underway for a broad range of diseases, including cancers, β-thalassemia, sickle cell disease, and Duchenne muscular dystrophy (summarized and discussed in [1,5]).Genome editing is generally based on either zinc finger nucleases [6], transcription activator-like effector nucleases [7], or the CRISPR/CRISPR-associated (Cas) system (see Glossary) [8]. These molecules act by inducing a double-stranded cut in a specific DNA sequence, which results in a genetic alteration as the gap is being repaired. In the clinic, the initial applications aim for deletions of genomic DNA intervals and do not yet involve precision at the nucleotide level; thus, these can be executed through the sole delivery of a genome-editing nuclease. However, for more precise editing, such as the generation of point mutations or more intricate changes, or even accurate deletion of a genomic segment, single-or double-stranded DNA templates are also delivered, together with the nucleases, to direct the repair to result in a given sequence by homology directed repair (HDR) [9][10][11] or nonhomologous end-joining [12]. Base editors [13] and prime editors [14] are alternative strategies for more precise editing. Overall, the range of genome editing tools is ever increasing and their transformative potential across a range of fields of application is immense. Genome Editing: A Disruptive but Still Erratic TechnologyThe safety of genome-editing technologies is just as critical as their efficiency for their successful application in health or agriculture. Common to all fields of application are the ri...

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Simple Protocol for Generating and Genotyping Genome‐Edited Mice With CRISPR‐Cas9 Reagents

Cited by 22 publications

References 20 publications

Versatile in vitro assay to recognize Cas9‐induced mutations

Versatile in vitro assay to recognize Cas9‐induced mutations

Genetics of non‐syndromic and syndromic oculocutaneous albinism in human and mouse

Anticipating and Identifying Collateral Damage in Genome Editing

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