CRISPR-Cas is an adaptive defense system against invading phages and plasmids that is prevalent in bacteria and archaea. The CRISPR-Cas immunity is based on spacer sequences recognizing specific target DNA, eventually leading to cleavage of the target DNA. Although the mechanism of action of CRISPR interference was largely deciphered, the adaptation stage, by which foreign DNA is recognized and new spacers are added to the CRISPR array, is still poorly understood. Here, we establish a CRISPR adaptation assay in combination with next generation sequencing that enables a large-scale, genome-wide evaluation of in vivo spacer acquisition into the Escherichia coli CRISPR array. This approach recorded over 20 million new spacer acquisition events in multiple conditions. Computational analysis of the spacer origin reveals a strong bias (up to 1000 fold) toward plasmid versus the bacterial chromosome, suggesting a preferential recognition mechanism for plasmid DNA. Analysis of the spacers derived from the E. coli chromosome revealed prominent hotspots for protospacers found within the chromosome terminus (ter) sites, where high number of replication forks are known to be localized. In addition, the leading strand of replication has higher tendency to yield spacers than the lagging strand. Finally, a strong protospacer hotspot was discovered just upstream of the CRISPR locus itself. These results link spacer acquisition to the process of DNA replication, thus shedding more light on the process of CRISPR adaptation.
