CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice

Oji, Asami; Noda, Taichi; Fujihara, Yoshitaka; Miyata, Haruhiko; Kim, Yeon Joo; Muto, Masanaga; Nozawa, Kaori; Matsumura, Takafumi; Isotani, Ayako; Ikawa, Masahito

doi:10.1038/srep31666

Cited by 87 publications

(103 citation statements)

References 37 publications

(53 reference statements)

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“…There has been an intensive effort made by the community during the past 3 years to use CRISPR-based strategies for developing floxed models through zygote injections [11, 12, 17], and also through ES cell targeting strategies [20, 24, 25]. The primary objective of this work was to develop a CRISPR targeting strategy suitable for both high- and low-throughput generation of floxed animal models.…”

Section: Discussionmentioning

confidence: 99%

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

et al. 2017

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BackgroundConditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient.ResultsHere we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring.Conclusions Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1220-4) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

et al. 2017

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“…The spermatozoa of patients with DNAJB13 mutations were similar to the findings in chimeric mice (Oji et al ., ) but not the tailless and short‐tailed phenotype. Guan J suggested that the silencing of RSP16 in Chlamydomonas reinhardtii did not massively alter the structure of the RSs or the axoneme but delayed the initiation and propagation of flagellar bending (Toure et al ., ), which could explain why patients with mutations have sperm flagellar motility and display structure disordered and twitching in sperm flagellar and low sperm motility.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, Oji et al . generated chimeric mice with biallelic mutant embryonic stem cells (ESCs) for the Dnajb13 gene; they found suppression of the phenotype of hydrocephalus and sperm malformation (Oji et al ., ). These findings suggest that this protein may participate in the motility of mature mouse spermatozoa.…”

Section: Introductionmentioning

confidence: 97%

Missense mutation in DNAJB13 gene correlated with male fertility in asthenozoospermia

Zhu

Jia

et al. 2019

Andrology

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Background The most common type of male infertility is asthenospermia. We cloned DnaJ heat shock protein family member B13 (Dnajb13/DNAJB13), a type II HSP40 family member that is highly expressed in the testis. DNAJB13 plays a crucial role in sperm flagellar function. Objectives The aim of this study was to investigate whether a correlation exists between DNAJB13 and low sperm motility in infertile men. Materials and methods In the present study, we performed a mutation screening of the DNAJB13 gene in 92 idiopathic asthenozoospermia patients and 200 men with normal fertility. Additionally, we used immunoelectron microscopy, co‐immunoprecipitation, mass spectrometric detection, indirect immunofluorescence assay, transmission electron microscopy studies, isobaric tags for relative and absolute quantitation, and multiple reaction monitoring studies to analyze changes in DNAJB13 protein. Results A novel c.106T>C mutation of DNAJB13 was present in nearly 10% (9/92) of idiopathic asthenozoospermia patients and was absent in 200 fertile men. A computer‐assisted sperm analyzer and transmission electron microscopy analysis using samples from 9 patients with DNAJB13 mutations demonstrated that most spermatozoa were immotile due to sperm tail defects. Multiple reaction monitoring results indicated that DNAJB13 protein levels were reduced after gene mutation. We achieved a pregnancy rate of 100% in 8 patients with DNAJB13 mutations using ICSI. Discussion and conclusion The DNAJB13 heterozygous variant may affect fertility. ICSI can help these patients with low fertility to father children.

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“…Embryonic stem (ES) cells (EGR‐101) (Fujihara, Kaseda, Inoue, Ikawa, & Okabe, ) were transfected with linearized 3xFLAG‐dCas9‐IG/pSKII‐ROSA, as described previously (Oji et al., ). ES cells retaining the transgene in the Rosa26 locus were injected into blastocysts (ICR × ICR) to generate chimeric mice.…”

Section: Methodsmentioning

confidence: 99%

Transgenic mouse lines expressing the 3xFLAG‐dCas9 protein for enChIP analysis

et al. 2018

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We developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technology to isolate specific genomic regions while retaining their molecular interactions. In enChIP, the locus of interest is tagged with an engineered DNA-binding molecule, such as a modified form of the clustered regularly interspaced short palindromic repeats (CRISPR) system containing a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). The locus is then affinity-purified to enable identification of associated molecules. In this study, we generated transgenic mice expressing 3xFLAG-tagged Streptococcus pyogenes dCas9 (3xFLAG-dCas9) and retrovirally transduced gRNA into primary CD4 T cells from these mice for enChIP. Using this approach, we achieved high yields of enChIP at the targeted genomic region. Our novel transgenic mouse lines provide a valuable tool for enChIP analysis in primary mouse cells.

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CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice

Cited by 87 publications

References 37 publications

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

Missense mutation in DNAJB13 gene correlated with male fertility in asthenozoospermia

Transgenic mouse lines expressing the 3xFLAG‐dCas9 protein for enChIP analysis

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