Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells*

Canver, Matthew C.; Bauer, Daniel E.; Dass, Abhishek; Yien, Yvette Y.; Chung, Joon; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H.; Orkin, Stuart H.

doi:10.1074/jbc.m114.564625

Cited by 307 publications

(335 citation statements)

References 35 publications

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“…In cases when single clones were isolated, they were retrieved at a surprisingly high frequency (;0.5%) , given the fact that TALENs are generally believed to be less efficient than the CRISPR/Cas9 system. When finalizing this manuscript, we noticed several recent reports showing large deletions (Canver et al 2014) or chromosomal rearrangements (Choi and Meyerson 2014) as a consequence of paired CRISPR/Cas9 cleavage. Efficiencies reported for monoallelic, megabase-scale deletions were in the range of 1% (Canver et al 2014) and are thus comparable to our findings.…”

Section: Discussionmentioning

confidence: 99%

Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line

Essletzbichler¹,

Konopka

Santoro³

et al. 2014

Genome Res.

244

207

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Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.

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Section: Discussionmentioning

confidence: 99%

Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line

Essletzbichler¹,

Konopka

Santoro³

et al. 2014

Genome Res.

244

207

View full text Add to dashboard Cite

show abstract

“…Using zinc‐finger nucleases and transcription activator‐like effector nucleases (TALENS), inversions and duplications extending to several 100 kb in mammalian cell lines and up to 1 Mb in zebrafish have been generated (Gupta et al ., 2013; Xiao et al ., 2013). More recently, CRISPR/Cas9 technology has been used to create large structural rearrangements in several mammalian cell lines (Canver et al ., 2014; Choi and Meyerson, 2014). This success has now been extended to ES cells in a process termed CRISVar to generate large deletions, duplications, and inversions of up to 1.6 Mb which were then used to establish one of these alleles in the germ line of mice (Kraft et al ., 2015).…”

Section: Introductionmentioning

confidence: 99%

Chromosome engineering in zygotes with CRISPR/Cas9

Boroviak

Doe

Banerjee

et al. 2016

Genesis

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SUMMARYDeletions, duplications, and inversions of large genomic regions covering several genes are an important class of disease causing variants in humans. Modeling these structural variants in mice requires multistep processes in ES cells, which has limited their availability. Mutant mice containing small insertions, deletions, and single nucleotide polymorphisms can be reliably generated using CRISPR/Cas9 directly in mouse zygotes. Large structural variants can be generated using CRISPR/Cas9 in ES cells, but it has not been possible to generate these directly in zygotes. We now demonstrate the direct generation of deletions, duplications and inversions of up to one million base pairs by zygote injection. genesis 54:78–85, 2016. © 2016 The Authors. genesis Published by Wiley Periodicals, Inc.

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“…18,19 Briefly, sgRNA-specifying oligos were chosen using publicly available online tools (supplemental Table 1 and supplemental Figure 1; see the Blood Web site). 20 Oligos were phosphorylated, annealed, and cloned into pSpCas9(BB) (pX330; Addgene plasmid ID: 42230) using a Golden Gate Assembly strategy.…”

Section: Generation Of Ehmt2 Knockout Clonesmentioning

confidence: 99%