Molecular recordings by directed CRISPR spacer acquisition

Science. 2016 Jul 29;353(6298):aaf1175. doi: 10.1126/science.aaf1175. Epub 2016 Jun 9.

Abstract

The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system of Escherichia coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution so as to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device.

MeSH terms

  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / metabolism
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Directed Molecular Evolution
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Genome, Bacterial*
  • Synthetic Biology / methods*

Substances

  • CRISPR-Associated Proteins
  • Escherichia coli Proteins
  • Cas2 protein, E coli
  • Endodeoxyribonucleases
  • Endonucleases
  • YgbT protein, E coli