CRISPR-C: circularization of genes and chromosome by CRISPR in human cells.
Nucleic Acids Res
; 46(22): e131, 2018 12 14.
Article
in En
| MEDLINE
| ID: mdl-30551175
ABSTRACT
Extrachromosomal circular DNA (eccDNA) and ring chromosomes are genetic alterations found in humans with genetic disorders. However, there is a lack of genetic engineering tools to recapitulate and study the biogenesis of eccDNAs. Here, we created a dual-fluorescence biosensor cassette, which upon the delivery of pairs of CRISPR/Cas9 guide RNAs, CRISPR-C, allows us to study the biogenesis of a specific fluorophore expressing eccDNA in human cells. We show that CRISPR-C can generate functional eccDNA, using the novel eccDNA biosensor system. We further reveal that CRISPR-C also can generate eccDNAs from intergenic and genic loci in human embryonic kidney 293T cells and human mammary fibroblasts. EccDNAs mainly forms by end-joining mediated DNA-repair and we show that CRISPR-C is able to generate endogenous eccDNAs in sizes from a few hundred base pairs and ranging up to 207 kb. Even a 47.4 megabase-sized ring chromosome 18 can be created by CRISPR-C. Our study creates a new territory for CRISPR gene editing and highlights CRISPR-C as a useful tool for studying the cellular impact, persistence and function of eccDNAs.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA, Circular
/
Clustered Regularly Interspaced Short Palindromic Repeats
/
CRISPR-Cas Systems
/
Gene Editing
/
CRISPR-Associated Protein 9
Limits:
Humans
Language:
En
Journal:
Nucleic Acids Res
Year:
2018
Document type:
Article
Affiliation country:
Denmark