Molecular recordings by directed CRISPR spacer acquisition

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-Cas system of E. 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 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.

CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria

“…Protospacer electroporations were performed as previously described 8 . Briefly, after overnight outgrowth from a single colony, Cas1+2 were induced in a 3 ml dilution of the culture (containing 80ul of the overnight), and grown at 37C for 2 hours (L-arabinose 0.2% w/w, Sigma-Aldrich; isopropyl-beta-D-thiogalactopyranoside 1mM, Sigma-Aldrich).…”

“…Using the Cas1-Cas2 integrase, the CRISPR-Cas microbial immune system stores the nucleotide content of invading viruses to confer adaptive immunity 7 . Harnessed, this system has the potential to write arbitrary information into the genome 8 . Here, we use the CRISPR-Cas system to encode images and a short movie into the genomes of a population of living bacteria.…”

“…In prokaryotic viral defense, the CRISPR associated (Cas) proteins, Cas1 and Cas2, function as an integrase complex to acquire nucleotides from invading viruses and store them in the CRISPR array 7,9,10 . In previous work, we found that we could direct the system to acquire synthetic sequences into the CRISPR array if those sequences are supplied as oligonucleotides 8 . Using this approach we showed simple molecular recordings by supplying different oligo sequences over time.…”

“…1d). We included a protospacer adjacent motif (PAM) on each protospacer, which increases the efficiency of acquisition and determines orientation of spacer insertion 8,13–15 . After adding the PAM and pixet, we were left with 28 bases per protospacer to encode pixel values.…”

Five big mysteries about CRISPR’s origins

“…Some work has shown that cells containing CRISPRCas machinery could serve as a recording device of sorts, cataloguing DNA and RNA sequences that they have encountered 3 . This might allow researchers to track a cell's gene expression or exposure to environmental chemicals over time.…”