Specificity of Prespacer Choice during CRISPR Adaptation

CRISPR arrays comprising unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. Spacers are acquired into arrays as fragments of foreign DNA (prespacers) [1]. Different fragments are acquired into CRISPR arrays with widely different efficiencies but factors responsible for acquisition efficiency are not fully known.

In this work, we studied the effects of prespacer sequence alterations on the acquisition efficiency by the type I-E CRISPR-Cas system using library-based approaches as well as by introducing specific mutations in prespacer sequences. Both naïve and primed adaptation was investigated.

Our work revealed structures and motives that were depleted from spacers acquired with a high frequency. Most prominently, the presence of internal consensus PAM trinucleotides in prespacers had a strong negative effect on spacer acquisition efficiency during primed but not naïve adaptation [2]. Similar trends of avoidance of cognate PAMs were observed when spacers acquired by different CRISPR-Cas systems (type I-B, III-B, I-F, II-A) were analyzed.

[1] Savitskaya E, Semenova E, Dedkov V, Metlitskaya A, Severinov K. High-throughput analysis of type IE CRISPR-Cas spacer acquisition in E. coli. RNA biology. 2013; 10(5): 716-725.

[2] Musharova O, Vyhovskyi D, Medvedeva S, Guzina J, Zhitnyuk Y, Djordjevic M, Savitskaya E. Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation. mBio. 2018; 9(6), e02169-18.