Engineering Point Mutant and Epitope‐Tagged Alleles in Mice Using Cas9 RNA‐Guided Nuclease

Gertsenstein, Marina; Nutter, Lauryl M. J.

doi:10.1002/cpmo.40

Cited by 24 publications

(21 citation statements)

References 78 publications

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“…Blue and orange bars represent deletions and insertions, respectively.), in agreement with reports that analyzed deletion or knock-in alleles in mice [35,36,37]. Droplet digital PCR or qPCR can be used as an alternative to WGS or standard PCR for copy counting of donor template DNA in founder mouse offspring, as template oligonucleotides can be randomly inserted into the genome [36, 38,39,40]. A report using several cell lines, including mouse ES cells, showed that Cas9 activity at on-target sites resulted in large deletions up to several kilobases long or complex lesions with segments from another chromosome in over 10% of the recovered alleles [11].…”

Section: How Can We Screen Out Unexpected “On-target Effects”?supporting

confidence: 75%

Off- and on-target effects of genome editing in mouse embryos

Ayabe

Nakashima

Yoshiki

2019

Journal of Reproduction and Development

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based genome editing technology has enabled manipulation of the embryonic genome. Unbiased whole genome sequencing comparing parents to progeny has revealed that the rate of Cas9-induced mutagenesis in mouse embryos is indistinguishable from the background rate of de novo mutation. However, establishing the best practice to confirm on-target alleles of interest remains a challenge. We believe that improvement in editing strategies and screening methods for founder mice will contribute to the generation of quality-controlled animals, thereby ensuring reproducibility of results in animal studies and advancing the 3Rs (replacement, reduction, and refinement). effect (J. Reprod. Dev. 65: 1-5, 2019) 2. Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 2013; 31: 839-843. [Medline] [CrossRef] 3. Willi M, Smith HE, Wang C, Liu C, Hennighausen L. Mutation frequency is not increased in CRISPR-Cas9edited mice. Nat Methods 2018; 15: 756-758. [Medline] [CrossRef] 4.

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Section: How Can We Screen Out Unexpected “On-target Effects”?supporting

confidence: 75%

Off- and on-target effects of genome editing in mouse embryos

Ayabe

Nakashima

Yoshiki

2019

Journal of Reproduction and Development

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“…All single guide (sgRNAs) were synthesized as described by Gertsenstein and Nutter (2018). Injection mixes for pronuclear injections included 10 ng/µl of each sgRNA and 20 ng/µl of Cas9 mRNA (Life Technologies; A23978) (Gertsenstein and Nutter, 2018).…”

Section: Synthesis Of Sgrnas and Cas9 Mrnamentioning

confidence: 99%

A novel mouse model of Duchenne muscular dystrophy carrying a multi-exonic Dmd deletion exhibits progressive muscular dystrophy and early-onset cardiomyopathy

Wong

Ahmed

Yang

et al. 2020

Disease Models &Amp; Mechanisms

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Duchenne muscular dystrophy (DMD) is a life-threatening neuromuscular disease caused by the lack of dystrophin, resulting in progressive muscle wasting and locomotor dysfunctions. By adulthood, almost all patients also develop cardiomyopathy, which is the primary cause of death in DMD. Although there has been extensive effort in creating animal models to study treatment strategies for DMD, most fail to recapitulate the complete skeletal and cardiac disease manifestations that are presented in affected patients. Here, we generated a mouse model mirroring a patient deletion mutation of exons 52-54 (Dmd Δ52-54). The Dmd Δ52-54 mutation led to the absence of dystrophin, resulting in progressive muscle deterioration with weakened muscle strength. Moreover, Dmd Δ52-54 mice present with early-onset hypertrophic cardiomyopathy, which is absent in current pre-clinical dystrophin-deficient mouse models. Therefore, Dmd Δ52-54 presents itself as an excellent pre-clinical model to evaluate the impact on skeletal and cardiac muscles for both mutation-dependent and -independent approaches.

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“…Post-birth, founders can be screened by long-range genotyping PCR using one primer within the insert and one primer outside the homology arm, similar to the procedure we have previously described (Gu, Posfai, & Rossant, 2018). Positive founders should be further validated by Sanger sequencing of the knock-in allele and copy number analysis (Current Protocols article: Gertsenstein & Nutter, 2018). , pH 7.4, 0.25…”

Section: Of 15mentioning

confidence: 99%

Efficient Generation of Large‐Fragment Knock‐In Mouse Models Using 2‐Cell (2C)‐Homologous Recombination (HR)‐CRISPR

Pósfai

Gertsenstein

et al. 2020

CP Mouse Biology

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Generating large‐fragment knock‐ins, such as reporters, conditional alleles, or humanized alleles, directly in mouse embryos is still a challenging feat. We have developed 2C‐HR‐CRISPR, a technology that allows highly efficient (10‐50%) and rapid (generating founders in 2 months) targeting of large DNA fragments. Key to this strategy is the delivery of CRISPR reagents into 2‐cell‐stage mouse embryos, taking advantage of the high homologous recombination activity during the long G2 cell cycle phase at this stage. Furthermore, by exploiting a Cas9–monomeric streptavidin (Cas‐mSA) and biotinylated PCR template (BioPCR) system to localize the repair template to specific double strand breaks, the efficiency can be further improved to up to 95%. Here we provide a procedure to generate large‐fragment knock‐in mouse models using 2C‐HR‐CRISPR. We first describe the principles for designing single guide RNAs and repair templates but refer to published manuscripts and protocols for molecular cloning methods or commercial sources for these reagents. We then describe two unique aspects of 2C‐HR‐CRISPR that are critical for success: (1) production of the CRISPR reagents for 2C‐HR‐CRISPR, particularly for applying the Cas9‐mSA/BioPCR method, and (2) microinjection of mouse embryos at the 2‐cell stage. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Single guide RNA and repair template design Basic Protocol 2: Preparing reagents for 2C‐HR‐CRISPR Basic Protocol 3: Microinjecting 2‐cell‐stage mouse embryos

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Engineering Point Mutant and Epitope‐Tagged Alleles in Mice Using Cas9 RNA‐Guided Nuclease

Cited by 24 publications

References 78 publications

Off- and on-target effects of genome editing in mouse embryos

Off- and on-target effects of genome editing in mouse embryos

A novel mouse model of Duchenne muscular dystrophy carrying a multi-exonic Dmd deletion exhibits progressive muscular dystrophy and early-onset cardiomyopathy

Efficient Generation of Large‐Fragment Knock‐In Mouse Models Using 2‐Cell (2C)‐Homologous Recombination (HR)‐CRISPR

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