Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes

Mizuno-Iijima, Saori; Ayabe, Shin-ichi; Kato, Kayoko; Matoba, Shogo; Ikeda, Yoshihisa; Dinh, Tra Thi Huong; Le, Hoai Thu; Suzuki, Hayate; Nakashima, Ken‐ichi; Hasegawa, Yoshikazu; Hamada, Yuko; Tanimoto, Yoko; Daitoku, Yoko; Iki, Natsumi; Ishida, Miyuki; Ibrahim, Elzeftawy Abdelaziz Elsayed; Hamada, Michito; Murata, Katsuyuki; Miwa, Yoshiro; Okada-Iwabu, Miki; Iwabu, Masato; Yagami, Ken‐ichi; Ogura, Atsuo; Obata, Yuichi; Takahashi, Satoru; Mizuno, Seiya; Yoshiki, Atsushi; Sugiyama, Fumihiro

doi:10.1016/j.ymeth.2020.04.007

Cited by 31 publications

(34 citation statements)

References 31 publications

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“…The electroporated embryos that developed into the 2-cell stage were transferred to oviducts of pseudopregnant ICR female mice. The floxed mice were generated using the microinjection method [ 25 ]. Each gRNA target sequence ( S1 Table ) was inserted into the entry site of pX330-mC carrying both the gRNA and Cas9 expression units.…”

Section: Methodsmentioning

confidence: 99%

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

et al. 2022

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Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

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Section: Methodsmentioning

confidence: 99%

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

et al. 2022

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“…and Rosa26 [24] target sequences were designed using CRISPOR (http://crispor.tefor.net). sgRNA was transcribed in vitro using the MEGAshortscript T7 transcription kit (Thermo Fisher Scientific).…”

Section: In Vitro Transcription Of Mrna and Single Grna (Sgrna)mentioning

confidence: 99%

The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator

et al. 2022

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Technique for Animal Knockout system by Electroporation (TAKE) is a simple and efficient method to generate genetically modified (GM) mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems. To reinforce the versatility of electroporation used for gene editing in mice, the electric condition was optimized for vitrified-warmed mouse embryos, and applied to the fresh embryos from widely used inbred strains (C57BL/6NCr, BALB/cCrSlc, FVB/NJcl, and C3H/HeJJcl). The electric pulse settings (poring pulse: voltage, 150 V; pulse width, 1.0 ms; pulse interval, 50 ms; number of pulses, +4; transfer pulse: voltage, 20 V; pulse width, 50 ms; pulse interval, 50 ms; number of pulses, ±5) were optimal for vitrified-warmed mouse embryos, which could efficiently deliver the gRNA/Cas9 complex into the zygotes without zona pellucida thinning process and edit the target locus. These electric condition efficiently generated GM mice in widely used inbred mouse strains. In addition, electroporation using the electrode with a 5 mm gap could introduce more than 100 embryos within 5 min without specific pretreatment and sophisticated technical skills, such as microinjection, and exhibited a high developmental rate of embryos and genomeediting efficiency in the generated offspring, leading to the rapid and efficient generation of genome editing mice. The electric condition used in this study is highly versatile and can contribute to understanding human diseases and gene functions by generating GM mice more easily and efficiently.

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“…Where a DNA template is used, tipping the balance in favor of HDR against nonhomologous end-joining and other repair events is beneficial to ensuring quality. This may be achieved by codelivery of HDR effectors [79], by pharmacological intervention using small-molecule compounds [80] (although this may reduce cell viability), or by directing Cas protein expression to specific cell-cycle phases [81,82]. The choice of the repair template is also of primary importance to reduce or eliminate the prevalence of ectopic insertions.…”

Section: Preventing the Damagementioning

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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Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes

Cited by 31 publications

References 31 publications

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator

Anticipating and Identifying Collateral Damage in Genome Editing

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