Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system

Hara, Satoshi; Kato, Tomoko; Goto, Yuji; Kubota, Shin; Tamano, Moe; Terao, Miho; Takada, Shuji

doi:10.1262/jrd.2016-058

Cited by 23 publications

(23 citation statements)

References 21 publications

(27 reference statements)

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“…Correcting certain genetic disorders, such as T cell lymphomas, will require the introduction of two or more mutations in the same homologous chromosome (linked mutations) 30 , 31 . This can be achieved using base editing 32 but is more challenging to accomplish using nucleases such as Cas9, which introduce deletions or translocations of sequences between multiple cleavage sites 3 , 7 , 33 , and provoke undesired cellular responses to the presence of double-stranded breaks 7 , 8 , 34 , 35 . The remarkable rarity of proximal off-target mutations and the predominance of anticipated on-target and bystander edits in embryos exposed to ABE and to preferred CBE variants (Fig.…”

Section: Discussionmentioning

confidence: 99%

Targeting fidelity of adenine and cytosine base editors in mouse embryos

et al. 2018

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Base editing directly converts a target base pair into a different base pair in the genome of living cells without introducing double-stranded DNA breaks. While cytosine base editors (CBE) and adenine base editors (ABE) are used to install and correct point mutations in a wide range of organisms, the extent and distribution of off-target edits in mammalian embryos have not been studied in detail. We analyze on-target and proximal off-target editing at 13 loci by a variety of CBEs and ABE in more than 430 alleles generated from mouse zygotic injections using newly generated and published sequencing data. ABE predominantly generates anticipated A•T-to-G•C edits. Among CBEs, SaBE3 and BE4, result in the highest frequencies of anticipated C•G-to-T•A products relative to editing byproducts. Together, these findings highlight the remarkable fidelity of ABE in mouse embryos and identify preferred CBE variants when fidelity in vivo is critical.

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Section: Discussionmentioning

confidence: 99%

Targeting fidelity of adenine and cytosine base editors in mouse embryos

et al. 2018

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“…Wang et al [30] reported a 95 kb deletion in mice. Recently, we demonstrated generation of mice carrying deletions of 0.5, 2, and 5 Mb and an inversion of 5 Mb [31,32]. To induce the 2 and the 5 Mb deletions, we microinjected Cas9/two sgRNAs and a donor plasmid carrying the breakpoint sequences so that the breakpoint sequence could be artificially defined.…”

Section: Reproduction Of Diseases In Mice Carrying Chromosome Rearranmentioning

confidence: 99%

Genome editing for the reproduction and remedy of human diseases in mice

Hara

Takada

2017

J Hum Genet

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With the recent progress in genome-editing technologies, such as the CRISPR/Cas9 system, genetically modified animals carrying nucleotide substitutions or large chromosomal rearrangements can be produced rapidly and at low cost. Such genome-editing techniques have been applied in the generation of animal models, especially mice, for reproducing human disease mutations, such as single-nucleotide polymorphisms (SNPs) or large chromosomal rearrangements identified by genome-wide screening analyses. While application methods are under development for various complex mutations involving genome editing for mimicking human disease-causing mutations in mice, functional studies of mouse models carrying replicated human mutations are gradually being published. In this review, we discuss the recent progress in application methods of the CRISPR/Cas9 system, focusing on the production of mouse models of diseases.

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“…In contrast, in the CRISPR-Cas9 system, the sgRNAs can share the same Cas9 nucleases, enabling the targeting of multiple genes in a single step by the injection of multiple sgRNAs with Cas9 nucleases into 1-cell embryos. Although these types of reports are currently limited (Table 1), [55][56][57][58][59][60][61] the simultaneous targeting of multiple genes by the CRISPR-Cas9 system is likely to become widely used for understanding complex genetic events.…”

Section: Multiplex Modificationmentioning

confidence: 99%

Applications of the CRISPR-Cas9 system in kidney research

Higashijima

Hirano

Nangaku

et al. 2017

Kidney International

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The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) is an RNA-guided DNA nuclease, and has been harnessed for the development of simple, efficient, and relatively inexpensive technologies to precisely manipulate the genomic information in virtually all cell types and organisms. The CRIPSR-Cas9 systems have already been effectively used to disrupt multiple genes simultaneously, create conditional alleles, and generate reporter proteins, even in vivo. The ability of Cas9 to target a specific genomic region has also been exploited for various applications, such as transcriptional regulation, epigenetic control, and chromosome labeling. Here we first describe the molecular mechanism of the RNA-guided DNA targeting by the CRISPR-Cas9 system and then outline the current applications of this system as a genome-editing tool in mice and other species, to better model and study human diseases. We also discuss the practical and potential uses of the CRISPR-Cas9 system in kidney research and highlight the further applications of this technology beyond genome editing. Undoubtedly, the CRISPR-Cas9 system holds enormous potential for revolutionizing and accelerating kidney research and therapeutic applications in the future.

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Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system

Cited by 23 publications

References 21 publications

Targeting fidelity of adenine and cytosine base editors in mouse embryos

Targeting fidelity of adenine and cytosine base editors in mouse embryos

Genome editing for the reproduction and remedy of human diseases in mice

Applications of the CRISPR-Cas9 system in kidney research

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