Priming in a permissive type I-C CRISPR-Cas system reveals distinct dynamics of spacer acquisition and loss

Abstract

16CRISPR-Cas is a bacterial and archaeal adaptive immune system that uses short, invader-derived 17 sequences termed spacers to target invasive nucleic acids. Upon recognition of previously encountered 18 invaders, the system can stimulate secondary spacer acquisitions, a process known as primed adaptation. 19Previous studies of primed adaptation have been complicated by intrinsically high interference efficiency 20 of most systems against bona fide targets. As such, most primed adaptation to date has been studied 21 within the context of imperfect sequence complementarity between spacers and targets. Here, we take 22 advantage of a native type I-C CRISPR-Cas system in Legionella pneumophila that displays robust 23 primed adaptation even within the context of a perfectly matched target. Using next-generation 24 sequencing to survey acquired spacers, we observe strand bias and positional preference that are 25 consistent with a 3′ to 5′ translocation of the adaptation machinery. We show that spacer acquisition 26 happens in a wide range of frequencies across the plasmid, including a remarkable hotspot that 27 predominates irrespective of the priming strand. We systematically characterize protospacer sequence 28 constraints in both adaptation and interference and reveal extensive flexibilities regarding the protospacer 29 adjacent motif in both processes. Lastly, in a strain with a genetically truncated CRISPR array, we 30 observe greatly increased interference efficiency coupled with a dramatic shift away from spacer 31 acquisition towards spacer loss. Based on these observations, we propose that the Legionella type I-C 32 system represents a powerful model to study primed adaptation and the interplay between CRISPR 33 interference and adaptation.

CRISPR-Cas in Streptococcus pyogenes

“…Nevertheless, the information arising from recent studies of type I-C in other bacteria may provide insights into the adaptation mechanism of streptococcal type I-C. Type I-C CRISPR-Cas from the pathogenic bacterium Legionella pneumophila exhibits robust primed adaptation with the selection of new spacers preferentially from the 5′ region relative to the priming site of the non-complementary strand [42,43].…”

Type I-F CRISPR-Cas distribution and array dynamics in Legionella pneumophila

“…previously (Rao et al 2017). Briefly, the raw paired-end reads were merged using FLASH 251 (Magoc and Salzberg 2011), and any unpaired reads were subsequently quality trimmed using 252…”

“…A common mechanism of escape is the accumulation of random mutations within the 50 foreign element which prevent complementary base pairing with crRNAs during interference 51 Semenova et al 2011;Datsenko et al 2012). CRISPR-Cas systems can 52 overcome this escape by acquiring new spacers; in fact, imperfect CRISPR targeting often leads 53 to a highly efficient "primed" acquisition response, providing an intrinsic mechanism to protect 54 against mutational escape (Swarts et type I-C system (Rao et al 2017). While much of our work to date has focused on the I-C 69 systems of L. pneumophila, the type I-F systems of this pathogen are highly protective, 70 remarkably diverse with respect to spacer content, and are frequently found on plasmids -71 suggesting that they may be circulated via horizontal gene transfer (Rao et al 2016).…”

“…To characterize the patterns of targeted 134 spacer acquisition in the chromosomal and pLPL CRISPR-Cas systems, we amplified the leader-135 proximal region of each CRISPR array from wild-type populations that had been transformed 136 with the plasmids targeted by their relatively permissive mid-array spacers (chromosomal: spacer 137 23; pLPL: spacer 50). We Illumina sequenced these PCR products and used an established 138 bioinformatics pipeline (Rao et al 2017) to identify newly acquired spacer sequences within 139 each read (Table 1). We then mapped the target of each new spacer to the priming plasmid 140 Since the chromosomal and pLPL CRISPR-Cas systems function in a very similar 158 manner during targeted acquisition and share a high degree of homology within their Cas genes 159 and repeat sequences, we speculated that priming of one system might lead to spacer acquisition 160 in the other.…”

Fidelity of Prespacer Capture and Processing is Governed by the PAM Mediated Interaction of Cas1-2 Adaptation complex in Escherichia coli

“…This observation perhaps explains the reason for fractional uptake of spacers that are devoid of PAM in E. coli (Wt in Figure 4E). Uptake of prespacers with erroneous PAM preference is not uncommon among various type I systems (Datsenko et al, 2012;Rao et al, 2017;Savitskaya et al, 2013;Shmakov et al, 2014). Such imprecision in spacer acquisition was shown to heighten the immune response by switching to primed adaptation (Datsenko et al, 2012;Jackson et al, 2019;Musharova et al, 2019;Musharova et al, 2018).…”