Rapidly evolving homing CRISPR barcodes

Nat Methods. 2017 Feb;14(2):195-200. doi: 10.1038/nmeth.4108. Epub 2016 Dec 5.

Abstract

We present an approach for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9-hgRNA complex to the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show that they can be used to record lineage history and that the barcode RNA can be amplified in situ, a prerequisite for in situ sequencing. This integrated approach will have wide-ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Genetic Vectors
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Lentivirus / genetics
  • RNA, Guide, CRISPR-Cas Systems

Substances

  • Bacterial Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • Green Fluorescent Proteins
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases