CRISPR-C: circularization of genes and chromosome by CRISPR in human cells

Nucleic Acids Res. 2018 Dec 14;46(22):e131. doi: 10.1093/nar/gky767.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Biosensing Techniques
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Cell Line
  • Chromosomes, Human, Pair 18 / chemistry
  • Chromosomes, Human, Pair 18 / metabolism
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA End-Joining Repair
  • DNA, Circular / genetics*
  • DNA, Circular / metabolism
  • Fibroblasts
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism
  • Gene Editing / methods*
  • Genes, Reporter
  • Genetic Loci
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Genome, Human
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • RNA, Guide / genetics
  • RNA, Guide / metabolism

Substances

  • DNA, Circular
  • Fluorescent Dyes
  • Luminescent Proteins
  • RNA, Guide
  • enhanced green fluorescent protein
  • fluorescent protein 583
  • Green Fluorescent Proteins
  • CRISPR-Associated Protein 9