Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition

Lina Amlinger,Magnus Lundgren,Mirthe Hoekzema,E Gerhart H Wagner,Sanna Koskiniemi

doi:10.1038/s41598-017-10876-z

Abstract

CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

Highlights

clustered regularly interspaced short palindromic repeats (CRISPR)-Cas are prokaryotic adaptive immune systems that defend against e.g. bacteriophages[1]
In the most common approach, spacer integration is detected by PCR-amplification of the CRISPR arrays followed by gel electrophoresis, where the longer products generated from expanded arrays can be distinguished from the short products of unexpanded arrays[9, 10, 13, 20, 21]
Translational stop codons (TAA) in the leader sequence are in frame with the ATG

Summary

Introduction

CRISPR-Cas are prokaryotic adaptive immune systems that defend against e.g. bacteriophages[1] They consist of a clustered regularly interspaced short palindromic repeats (CRISPR) array and CRISPR-associated (cas) genes that encode proteins required for immunity. Integration of a new 32 bp spacer results in duplication of the 29 bp leader-proximal repeat and the array is expanded by 61 bp[9]. Other assays are based on functionality, where new spacers mediate plasmid curing[10, 13] or survival of phage infection[10, 20, 21, 24] Such methods do not detect non-functional or self-targeting adaptation events, as they may result in lack of phage protection or cytotoxicity[25]

Methods

Results

Conclusion