Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli

Abstract

The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.

Figure 1.

Detection of spacer acquisition in BL21-AI and IYB5101. (A) Schematics of CRISPR I arrays and leader sequences from E. coli BL21-AI and IYB5101 (based on NCBI reference sequences of NC_012947.1 positions 1002800–1003800 and NC_000913.2 positions 2875600–2876800, respectively). Repeats are marked as gray diamonds for IYB5101 consensus and black diamonds for BL21-AI consensus. Last repeat in IYB5101 is marked as half a diamond to indicate that only half a repeat is present. Spacers are marked as numbered rectangles. The leader and repeat sequences of each strain are specified. Asterisk below or above a base indicates its conservation between the two strains. Gray highlighting indicates the (-10)-TATA box (18). (B) Schematics and results of the spacer-acquisition assay. Cultures of E. coli BL21-AI or IYB5101 harboring the indicated plasmids were grown in the presence of inducers (0.2% l-arabinose and 0.1 mM IPTG) for the indicated cycles. Each cycle represents dilution of 1:300 of a previously 10–16 h induced culture grown for additional 10–16 h in medium containing inducers. PCR products using the primers indicated as flagged arrows, from samples taken from cultures at the indicated cycle, were electrophoresed on an agarose gel and imaged. Parental and expanded bands are indicated. Gels are representative of two experiments yielding similar results.

Figure 2.

Position and orientation of protospacers on plasmid DNA and analysis of protospacer adjacent motifs (PAMs). (A) Protospacers matching the sequenced spacers are marked on the pCas1+2 plasmid map according to their position and orientation (blue arrows pointing clockwise and red arrows pointing counterclockwise). Protospacers are numbered according to the clone from which the spacers were sequenced, as listed in Table S3. Spacer position in a sequenced array is separated from the clone number by a period, where applicable. (B) WebLogo (22) was used to analyze PAMs from sequenced spacers that were 32 bp long. The first nt of the protospacer is at position 0. Protospacer region is highlighted in gray. Weblogo of the protospacer as well as 10 nt upstream and downstream of the protospacer are shown. Relative letter size indicates the base frequency in that position.

Figure 3.

Minimal repeat sequence essential for spacer acquisition. Schematics of the constructs in CRISPR array I of genetically engineered E. coli BL21-AI. Repeats are marked as diamonds, spacers as rectangles. Kanamycin-resistance cassette used to construct the different strains is marked as gray chevron. Parental: non-manipulated E. coli BL21-AI; 2-rep: one spacer between two repeats, other repeats removed. 1-rep: only one repeat, no spacer, other repeats removed. 0-rep: complete array replaced with a kanamycin-resistance cassette. Parallel lines on the kan cassette indicate that only a partial gene is depicted. Gel shows PCR products amplified from the indicated cultures of E. coli BL21-AI harboring plasmid pCas1+2 and grown in the presence or absence of inducers (0.2% l-arabinose and 0.1 mM IPTG) for 10–16 h for three cycles. Gel is representative of two experiments yielding similar results.

Figure 4.

Regions in the leader sequence essential for spacer acquisition. (A) Schematics of the constructs in CRISPR array I of genetically engineered E. coli BL21-AI. Repeats are marked as diamonds, and spacers as rectangles. Parental: non-manipulated array; LN: N – number of nt upstream of first repeat left intact. Parallel lines on the kan cassette indicate that only a partial gene is depicted. Gel shows PCR products amplified from the indicated cultures of E. coli BL21-AI harboring plasmid pCas1+2 and grown in the presence or absence of inducers (0.2% l-arabinose and 0.1 mM IPTG) for 10–16 h for three cycles. (B) Schematics of constructs as above. Scram40: 40 nt upstream of first repeat scrambled, other nucleotides not manipulated; Scram20: 20 nt upstream of first repeat scrambled, other nucleotides not manipulated. Gel shows PCR products amplified from the indicated cultures of E. coli BL21-AI harboring plasmid pCas1+2 and grown in the presence or absence of inducers (0.2% l-arabinose and 0.1 mM IPTG) for 10–16 h for three cycles. Gels are representative of two experiments yielding similar results.

Figure 5.

Determination of the DNA template for newly inserted repeats. Schematic of the parental and expanded arrays in CRISPR I of E. coli BL21-AI. Repeats are marked as black or gray diamonds, depending on the nucleotide ‘label’, parental spacer as a blank rectangle whereas newly inserted spacer as gray rectangle marked ‘new’. R*-S-R: first repeat from BL21-AI consensus, second from K-12 consensus; R-S-R*: first repeat from K-12 consensus, second repeat from BL21-AI consensus. Indicated cultures of E. coli BL21-AI harboring plasmid pCas1+2 and grown in the presence or absence of inducers (0.2% l-arabinose and 0.1 mM IPTG) for 10–16 h for three cycles were streaked to isolate single colonies. Five isolated colonies from each culture showing insertion of a spacer in a PCR were DNA-sequenced. Five out of five colonies of each strain harbored labeled repeats, as indicated.