Targeted Deletion of an Entire Chromosome Using CRISPR/Cas9

Adikusuma, Fatwa; Williams, Nicole; Grützner, Frank; Hughes, James; Thomas, Paul Q.

doi:10.1016/j.ymthe.2017.05.021

Cited by 80 publications

(67 citation statements)

References 9 publications

(15 reference statements)

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“…Recently, various genomic modifications with CRISPR/Cas9 technology have been reported, including genomic translocation, inversion, chromosome elimination, single point mutation and knock‐in . Combining these genome‐editing technologies with the RC platform will further facilitate diverse cancer modeling and functional assays in vivo, which should deepen our understanding of the mechanisms that govern various biological phenomena in mammals in vivo.…”

Section: Discussionmentioning

confidence: 99%

Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling

Sogabe

Yamada

et al. 2019

Cancer Science

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The emergence of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has dramatically advanced how we manipulate the genome. Regarding in vivo experiments, Cas9‐transgenic animals could provide efficient and complex genome editing. However, this potential has not been fully realized partly due to a lack of convenient platforms and limited examples of successful disease modeling. Here, we devised two doxycycline (Dox)‐inducible Cas9 platforms that efficiently enable conditional genome editing at multiple loci in vitro and in vivo. In these platforms, we took advantage of a site‐specific multi‐segment cloning strategy for rapid and easy integration of multiple single guide (sg)RNAs. We found that a platform containing rtTA at the Rosa26 locus and TRE‐Cas9 together with multiple sgRNAs at the Col1a1 locus showed higher efficiency of inducible insertions and deletions (indels) with minimal leaky editing. Using this platform, we succeeded to model Wilms’ tumor and the progression of intestinal adenomas with multiple mutations including an activating mutation with a large genomic deletion. Collectively, the established platform should make complicated disease modeling in the mouse easily attainable, extending the range of in vivo experiments in various biological fields including cancer research.

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Section: Discussionmentioning

confidence: 99%

Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling

Sogabe

Yamada

et al. 2019

Cancer Science

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“…Our study suggests that a DSB induced by even a single gRNA can result in the allele-specific removal of a chromosome in human embryos. Previous studies in mouse embryos demonstrated elimination of sex chromosomes by targeting Cas9 to centromeric repeats of the Y chromosome, or to multiple locations on the same chromosome (Adikusuma et al, 2017;Zuo et al, 2017). The finding that a single Cas9 induced cut can result in such outcome in human embryos suggests species-specific differences in repair or checkpoint control.…”

Section: Discussionmentioning

confidence: 99%

Reading frame restoration at the EYS locus, and allele-specific chromosome removal after Cas9 cleavage in human embryos

Zuccaro

Mitchell

et al. 2020

Preprint

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The correction of disease-causing mutations in human embryos could reduce the burden of inherited genetic disorders in the fetus and newborn, and improve the efficiency of fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here we evaluate the repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frame-shift mutation causing blindness.We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. However, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, upon entry into mitosis, loss of one or both chromosomal arms.Thus, Cas9 allows for the modification of chromosomal content in human embryos in a targeted manner, which may be useful for the prevention of trisomies.

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“…However, recent studies using genetic engineering have revealed the possibility of performing aneuploidy therapy in cultured cells. Here, we summarize some of the main studies aimed at eliminating an entire chromosome using the Cre/loxP system [79][80][81][82][83][84][85], the TKneo transgene for positive and negative antibiotic selection [86], CRISPR/Cas9 system-mediated multiple cleavage [87,88], and zinc finger nuclease (ZFN)-mediated knock-in of the XIST gene to silence one copy of chromosome 21 [89] (cited in Table 2).…”

Section: Gene Targeting-mediated Chromosome Eliminationmentioning

confidence: 99%

“…To eliminate extra chromosomes, multiple guide RNAs of the CRISPR/Cas9 system for introducing DNA double-strand breaks (DSBs) into the target site are designed for an entire region of the target chromosome [95][96][97][98][99]. Adikusuma et al [88] used this strategy to delete the mouse Y chromosome in vitro and in vivo ( Figure 1D). Guide RNAs corresponding to repetitive sequences (from 40 to 140 tandem repeats) of the centromere and long arm of chromosome Y were used in their study.…”

Section: Crispr/cas9 System To Introduce Multiple Dna Cleavages For Tmentioning

confidence: 99%

Applications of Genome Editing Technology in Research on Chromosome Aneuploidy Disorders

Akutsu

Fujita

Tomioka

et al. 2020

Cells

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Chromosomal segregation errors in germ cells and early embryonic development underlie aneuploidies, which are numerical chromosomal abnormalities causing fetal absorption, developmental anomalies, and carcinogenesis. It has been considered that human aneuploidy disorders cannot be resolved by radical treatment. However, recent studies have demonstrated that aneuploidies can be rescued to a normal diploid state using genetic engineering in cultured cells. Here, we summarize a series of studies mainly applying genome editing to eliminate an extra copy of human chromosome 21, the cause of the most common constitutional aneuploidy disorder Down syndrome. We also present findings on induced pluripotent stem cell reprogramming, which has been shown to be one of the most promising technologies for converting aneuploidies into normal diploidy without the risk of genetic alterations such as genome editing-mediated off-target effects.

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Targeted Deletion of an Entire Chromosome Using CRISPR/Cas9

Cited by 80 publications

References 9 publications

Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling

Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling

Reading frame restoration at the EYS locus, and allele-specific chromosome removal after Cas9 cleavage in human embryos

Applications of Genome Editing Technology in Research on Chromosome Aneuploidy Disorders

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