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

Nucleic Acids Res. 2021 Jan 25;49(2):e8. doi: 10.1093/nar/gkaa1088.

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Alleles
  • Base Sequence
  • Benzoxazoles / analysis
  • CRISPR-Cas Systems*
  • Chromosome Mapping / methods*
  • Chromosomes, Bacterial / genetics*
  • Computer Simulation
  • Conserved Sequence / genetics
  • DNA-Directed RNA Polymerases
  • Drug Resistance, Bacterial / genetics
  • Fluorescent Dyes / analysis
  • Gene Editing / methods
  • Genome, Bacterial
  • Genome, Human
  • Haemophilus influenzae / drug effects
  • Haemophilus influenzae / genetics*
  • Haplotypes / genetics
  • Humans
  • Lab-On-A-Chip Devices
  • Nalidixic Acid / pharmacology
  • Novobiocin / pharmacology
  • Nucleotide Motifs / genetics
  • Polymorphism, Single Nucleotide
  • Quinolinium Compounds / analysis
  • RNA, Guide, CRISPR-Cas Systems / chemical synthesis
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • Repetitive Sequences, Nucleic Acid / genetics
  • Sequence Alignment
  • Staining and Labeling / methods
  • Viral Proteins

Substances

  • Benzoxazoles
  • Fluorescent Dyes
  • Quinolinium Compounds
  • RNA, Guide, CRISPR-Cas Systems
  • Viral Proteins
  • 1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium
  • Novobiocin
  • Nalidixic Acid
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases