CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

Xu, Shen; Kim, Jin Chul; Tang, Qinglian; Qu, Chao; Liu, Jingfeng; Xu, Yang; Fu, Xuemei

doi:10.1007/s13238-020-00699-6

Cited by 34 publications

(27 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…CRISPR/Cas9 has also also employed in a p53 genetic sensor system which specifically and efficiently killed p53-deficient cancer cells ( 120 ). However, the high risk of genome instabiliy induced by CRISPR/Cas9 should be rigorously considered ( 121 , 122 ). Small interference RNAs could specifically eliminate mutant p53 mRNA without affecting the wild-type one, However, the specificity and in vivo efficacy of such RNAi remains to be elucidated.…”

Section: Mutant P53 Facilitates Cancer Progressionmentioning

confidence: 99%

Mutant p53 in Cancer Progression and Targeted Therapies

et al. 2020

Self Cite

View full text Add to dashboard Cite

TP53 is the most frequently mutated tumor suppressor gene in human cancer. The majority of mutations of p53 are missense mutations, leading to the expression of the full length p53 mutant proteins. Mutant p53 (Mutp53) proteins not only lose wild-type p53dependent tumor suppressive functions, but also frequently acquire oncogenic gain-offunctions (GOF) that promote tumorigenesis. In this review, we summarize the recent advances in our understanding of the oncogenic GOF of mutp53 and the potential therapies targeting mutp53 in human cancers. In particular, we discuss the promising drugs that are currently under clinical trials as well as the emerging therapeutic strategies, including CRISPR/Cas9 based genome edition of mutant TP53 allele, small peptide mediated restoration of wild-type p53 function, and immunotherapies that directly eliminate mutp53 expressing tumor cells.

show abstract

Section: Mutant P53 Facilitates Cancer Progressionmentioning

confidence: 99%

Mutant p53 in Cancer Progression and Targeted Therapies

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…After Cas9 cleavage, the histone H2AX within nucleosomes located in the regions flanking the DSB ends is phosphorylated by the ATM kinase, generating γH2AX ( Iacovoni et al., 2010 ; Lee et al., 2014a ). Interestingly, a recent study showed that Cas9 is a genome mutator and induces γH2AX accumulation ( Xu et al., 2020 ). In addition, long-distance chromatin interactions are increased within the γH2AX chromatin domains ( Aymard et al., 2017 ).…”

Section: Toward Precise and Predictable Genome Editingmentioning

confidence: 99%

Toward precise CRISPR DNA fragment editing and predictable 3D genome engineering

Shou

2020

Journal of Molecular Cell Biology

View full text Add to dashboard Cite

Ever since gene targeting or specific modification of genome sequences in mice was achieved in the early 1980s, the reverse genetic approach of precise editing of any genomic locus has greatly accelerated biomedical research and biotechnology development. In particular, the recent development of the CRISPR/Cas9 system has greatly expedited genetic dissection of three-dimensional (3D) genomes. CRISPR gene editing results from targeted genome cleavage by ectopic bacterial Cas9 nuclease followed by presumed random ligations via the host double-strand break (DSB) repair machineries. Recent studies revealed, however, that the CRISPR genome editing system is precise and predictable because of cohesive Cas9 cleavage of targeting DNA. Here, we synthesize the current understanding of CRISPR DNA fragment editing mechanisms and recent progress in predictable outcomes from precise genetic engineering of 3D genomes. Specifically, we first briefly describe historical genetic studies leading to CRISPR and 3D genome engineering. We then summarize different types of chromosomal rearrangements by DNA fragment editing. Finally, we review significant progress from precise one-dimensional (1D) gene editing toward predictable 3D genome engineering and synthetic biology. The exciting and rapid advances in this emerging field provide new opportunities and challenges to understand or digest 3D genomes.

show abstract

“…Random integration of the selection marker may disrupt the function of non-target genes. Recently, CRISPR/Cas9 system sometimes causes genome instability 18 , off-targets from the genome manipulation 19 , reading frame restoration 20 , and gene conversion 21 . Although our method could delete a long genomic region from all alleles, which prevents reading frame restoration, loss-of-heterozygosity, and gene conversion, our method also had the possibility of off-target gRNA effects.…”

Section: Discussionmentioning

confidence: 99%

A simple method using CRISPR-Cas9 to knock-out genes in murine cancerous cell lines

Ishibashi

Saga

Hisatomi

et al. 2020

Sci Rep

View full text Add to dashboard Cite

CRISPR-Cas9 system can be used to generate knock-out cancer cell lines. An insertion or deletion induced by a single guide RNA (gRNA) is often used to generate knock-out cells, however, some cells express the target gene by skipping the disrupted exon, or by using a splicing variant, thus losing the target exon. To overcome this unexpected expression of the target gene, almost the entire gene can be swapped with a selection marker. However, it is time-consuming to create a targeting vector which contains 5′ and 3′ homology arms flanked by a selection marker. Here, we developed a simple and easy method called SUCCESS (Single-strand oligodeoxynucleotides, Universal Cassette, and CRISPR/Cas9 produce Easy Simple knock-out System), to knock-out a target gene without constructing a targeting vector. Our method removed the targeted large genomic region by using two pX330 plasmids encoding Cas9 and gRNA, two 80mer single strand oligodeoxynucleotides (ssODN), and a blunt-ended universal selection maker sequence in B16F10 murine cancer cell and ID8 murine ovarian cancer cell. SUCCESS generated knock-out clones in two murine cancer cell lines by homozygous deletion of the target genomic region, and without constructing targeting vectors. Thus, our method can be widely applied to generate homozygous knock-out cell lines, as well as knock-in cell lines.

show abstract

CAS9 is a genome mutator by directly disrupting DNA-PK dependent DNA repair pathway

Cited by 34 publications

References 34 publications

Mutant p53 in Cancer Progression and Targeted Therapies

Mutant p53 in Cancer Progression and Targeted Therapies

Toward precise CRISPR DNA fragment editing and predictable 3D genome engineering

A simple method using CRISPR-Cas9 to knock-out genes in murine cancerous cell lines

Contact Info

Product

Resources

About