Molecular recordings by directed CRISPR spacer acquisition

Abstract

The ability to write a stable record of identified molecular events into a specific genomic locus would enable the examination of long cellular histories and have many applications, ranging from developmental biology to synthetic devices. We show that the type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system of Escherichia coli can mediate acquisition of defined pieces of synthetic DNA. We harnessed this feature to generate records of specific DNA sequences into a population of bacterial genomes. We then applied directed evolution so as to alter the recognition of a protospacer adjacent motif by the Cas1-Cas2 complex, which enabled recording in two modes simultaneously. We used this system to reveal aspects of spacer acquisition, fundamental to the CRISPR-Cas adaptation process. These results lay the foundations of a multimodal intracellular recording device.

Fig. 1

Acquisition of synthetic spacers. (A) Schematic of the minimal elements of the type I-E CRISPR acquisition system, used including Cas1, Cas2, and array with leader (L), repeat (R), and spacer (S) along with PCR detection of an expanded array following the overnight induction of Cas1-Cas2. (B) Origin of new spacers (plasmid or genome) mean ±SEM. (C) Genome- and plasmid-derived spacers following overnight induction are mapped back to the approximate location of their protospacer (marked in red). (D) Array expansion (top) and specific acquisition of synthetic oligo protospacer (bottom) following electroporation. Top schematic shows the experimental outline. Schematics under each gel show specific PCR strategy. (E) Sequence-specific acquisition in either the forward (top) or reverse (bottom) orientation following electroporation with various single- and double-stranded oligos. 5′PT indicates phosphorothioate modifications to the oligos at the 5′ ends. (F) Time course of expansion following electroporation, mean ±SEM. (G) Percent of arrays expanded by spacer source as a function of electroporated oligo concentration, mean ±SEM. (H) Position of new spacers relative to the leader, mean ±SEM. (I) Size of new spacers in base-pairs, mean ±SEM. All gels are representative of ≥ 3 biological replicates, * indicates p<0.05, additional statistical details in Table S1.

Fig. 2

PAMs modify the efficiency and orientation of spacer acquisition. (A) Genome- (count/10 kb) and plasmid- (coverage/base) derived spacers mapped to their protospacer location on the forward (purple) or reverse (green) strands. (B) Direction of oligo-derived spacers in the forward (purple) or reverse (green) orientation, mean ±SEM. (C) Representative sequence pLOGO (44) generated based on 896 unique genome- and plasmid-derived protospacers. Five bases of the protospacer are included at each end of the spacer. (D) Plot of the summed spacer coverage mapped to the plasmid among three replicates at each nucleotide for a 553 nucleotide stretch. Carrots demarcate canonical PAMs on the forward (purple) or reverse (green) strand. Scale bar is 33 bases. Individual replicates are shown below. (E) Percent of arrays expanded by spacer source for different oligo protospacers, mean ±SEM. (F) Ratio of oligo-derived spacers acquired in the forward vs reverse orientation for different oligo protospacers, mean ±SEM. (G–J) Normalized representation of oligo-derived spacers by base acquired in the forward and reverse direction for each oligo. Bars in I and J are 33 bases long to show dominant and minority spacers drawn from the oligo protospacers. For all panels, * indicates p<0.05, additional statistical details in Table S1.

Fig. 3

A molecular recording over time. (A) Experimental outline of the 3 × 5 recording. Over five days, three sets of five oligo protospacers (fifteen elements) were electroporated (one protospacer from each of the three sets each day) into cells expressing Cas1-Cas2. Time points at which cells were sampled for sequencing are numbered 1–6. (B) Schematic illustrating all possible pairwise ordering of new spacers. G/P denotes a spacer derived from the genome or plasmid. Ordering rules are shown below. In the case of y=z, * indicates a tolerance within ± 20% of the mean of both values. (C) At each of the six sample points (marked in A), percent of all arrays expanded with synthetic spacers from each of the indicated rounds, mean ±SEM. (D) Single, double, and triple expansions for each round, mean ±SEM. (E) Percent of all expansions at sample point six, broken down by electroporation round and set. Open circles are individual replicates, filled bars are mean ±SEM. (F) Results of ordering rule analysis for one replicate across each set. For all 120 permutations, results of the tested rule are shown (green indicates pass, red indicates fail). For all sets, only one permutation passed all rules and in every case that permutation matched the actual order in which the oligos were electroporated (as indicated by check mark). Additional statistical details in Table S1.

Fig. 4

Directed evolution of PAM recognition. (A) Schematic of the directed evolution. (B) Testing of selected mutants, plotting 5′ AAG versus non-AAG PAM protospacers normalized to count per 100,000 sequences. Scatter plot shows 65 induced mutants (open black circles), three induced wild-type replicates (open green circles), an uninduced wild-type (open red circle), the average of the induced mutants (filled black circle), and the average of the induced wild-types (filled green circle) ±SEM. Scatter plot to the right is an inset of the larger plot. (C) Heatmap of protospacer PAM frequency over the entire sequence space for wild type Cas1-Cas2 (wt), mutants that increase or maintain AAG PAM specificity (m-27 and m-24), and mutants that lose AAG PAM specificity (m-74, m-80, m-89). Numbers in the upper right correlate to numbers in B. (D) A subset of selected mutants re-assayed in triplicate as well as a subset of single point mutants chosen from the original selection. All points are the average of three replicates ±SEM. (E) Crystal structure of Cas1-Cas2 complex bound to a protospacers (38). Inset highlights, in magenta, residues in the Cas1 active site that (when mutated) decrease PAM specificity. The protospacer PAM complementary sequence (T30 T29 C28, numbering as in PDB ID 5DQZ) is also noted. Additional statistical details in Table S1.

Fig. 5

Recording in an additional mode. (A) Outline of the recording process. Three different synthetic protospacers (each containing a 5′ AAG PAM on the forward strand, and a 5′ TCG PAM on the reverse) were electroporated over three days (one protospacer each day) into two bacterial cultures under different induction conditions (shown below timeline). Sampling time points are numbered 1–3. (B) Schematic of the plasmid construct used, showing wild-type and PAM^NC mutant (m-89) Cas1-Cas2 driven by independently inducible promoters (T7lac and pLtetO, respectively). The heatmap shows 5′ PAM specificity for wild-type (boxed in yellow) and mutant m-89 (boxed in red). (C) At each of the three sample points (marked in B), percent of expanded arrays with spacers from each of the indicated rounds for the two conditions, mean ±SEM. (D–F) Ratio of synthetic spacers acquired in the forward versus reverse orientation for each round under each condition, mean ±SEM. (G) Ratio of forward to reverse integrations normalized to the sum of both possible orientations for each of the two conditions, mean ±SEM. For all panels, * indicates p<0.05, additional statistical details in Table S1.