Customized optical mapping by CRISPR-Cas9 mediated DNA labeling with multiple sgRNAs

Abstract

Whole-genome mapping technologies have been developed as a complementary tool to provide scaffolds for genome assembly and structural variation analysis (1,2). We recently introduced a novel DNA labeling strategy based on a CRISPR-Cas9 genome editing system, which can target any 20bp sequences. The labeling strategy is specifically useful in targeting repetitive sequences, and sequences not accessible to other labeling methods. In this report, we present customized mapping strategies that extend the applications of CRISPR-Cas9 DNA labeling. We first design a CRISPR-Cas9 labeling strategy to interrogate and differentiate the single allele differences in NGG protospacer adjacent motifs (PAM sequence). Combined with sequence motif labeling, we can pinpoint the single-base differences in highly conserved sequences. In the second strategy, we design mapping patterns across a genome by selecting sets of specific single-guide RNAs (sgRNAs) for labeling multiple loci of a genomic region or a whole genome. By developing and optimizing a single tube synthesis of multiple sgRNAs, we demonstrate the utility of CRISPR-Cas9 mapping with 162 sgRNAs targeting the 2Mb Haemophilus influenzae chromosome. These CRISPR-Cas9 mapping approaches could be particularly useful for applications in defining long-distance haplotypes and pinpointing the breakpoints in large structural variants in complex genomes and microbial mixtures.

Figure 1.

Interrogation of individual bases with CRISPR–Cas9 labeling. Yellow lines indicate single molecules. The thick blue bars represent Nt.BSPQI reference map. The narrower blue bar represent consensus map of combined Nt.BSPQI CRISPR–Cas9 labeling. Red arrows and bases indicate the single base differences between the two strains. Additional details can be found in the Table 1.

Figure 2.

The workflow of sgRNA synthesis. The multiple oligos with a promoter sequence (red) and an overlap sequence (green) on either side of the target sequence are hybridized with a single complementary oligo that shares the overlap sequence.

Figure 3.

(A) Mapping results of RR722 molecules labeled with the 48 sgRNAs (Supplementary Table S1). The lines in the blue bar (designed reference map of RR722) represent the locations of the 48 sgRNAs on RR722. The yellow lines below the reference are labels with dark green dots representing where labels matched to the reference and light green dots representing labels not found in the reference. (B) Mapping results of RR3131 molecules labeled with the set of 48 sgRNAs (Supplementary Table S1). The lines in the blue bar (designed reference map of RR3131) represent the locations of the 48 sgRNAs on RR3131. The yellow lines below the reference are labels with dark green dots representing where labels matched to the reference map and light green dots representing labels not found in the reference map. The red arrows indicate the off-target labeling.

Figure 4.

sgRNA design flow-chart.

Figure 5.

Mapping results of RR722 molecules labeled with the 162 sgRNAs (Supplementary Table S2). (A) The lines in the blue bar (designed reference map of RR722) represent the locations of the 162 sgRNAs on RR722. The yellow lines below the reference are labels with dark green dots representing where labels matched to the reference and light green dots representing labels not found in the reference. (B) Alignment results to RR3131.