Materials and Methods

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

MATERIALS AND METHODSHigh-molecular-weight DNA extractionTwo Haemophilus influenzae strains with complete genome sequences were used: the standard lab strain Rd KW20 (RR722, {"type":"entrez-nucleotide","attrs":{"text":"NC_000907","term_id":"16271976","term_text":"NC_000907"}}NC_000907) and a marked derivative of clinical isolate 86-028NP (RR3131, {"type":"entrez-nucleotide","attrs":{"text":"NC_007416.2","term_id":"189313712","term_text":"NC_007416.2"}}NC_007416.2, carrying novobiocin and nalidixic acid resistance alleles, NovR and NalR) (27,33,34). Bacterial culture followed standard protocols; cells were grown to stationary phase (OD600 nm = 1.2) in supplemented brain-heart infusion (10 μg/ml hemin 2 μg/ml NAD) shaking at 37°C, and then cells were harvested by centrifugation at 4000 rpm for 5 min before DNA extractions (35,36). Purification of ultra-high MW DNA fragments followed the Bionano Prep Cell Culture DNA Isolation Protocol. Briefly, cells were: (a) resuspended in cell buffer (∼5 × 109 CFU/ml); (b) embedded in 2% low-melt agarose (BioRad) plugs to minimize shearing forces; (c) lysed using Bionano cell lysis buffer supplemented with 167 μl Proteinase K (Qiagen) rocking overnight at 50°C; (d) RNase treatment by adding 50 μl of RNase A solution and incubating the plugs for 1 h at 37°C (Qiagen) and (e) washing in TE buffer with intermittent mixing. Finally, DNA was purified from low-melt agarose plugs by drop dialysis. Plugs were melted at 72°C, then incubated with 2 μl Agarase (Thermo Fisher Scientific) for 45 min. Melted plugs were dialyzed into TE buffer using 0.1 μm Millipore membrane filters for 45 min at a ratio of 15 ml buffer per ∼200 μl sample. DNA was allowed to homogenize overnight at room temperature before fluorometric quantification using the Qbit dsDNA BR kit (Thermo Fisher Scientific).dsDNA synthesissgRNA oligos sgRNAs were encoded on 55 nt DNA oligos with a 5′ T7 promoter sequence (5′-TTCTAATACGACTCACTATAG-3′), followed by the target 20mer sequence, complementary to the target gDNA sequence, and finally an overlap sequence (5′-GTTTTAGAGCTAGA-3′). Individually synthesized sgRNA oligos were then pooled into an equimolar mixture. sgRNA complementary oligo: An 80 nt long oligo was designed with the 3′ end complementary to the overlap sequence and remainder encoded the Cas9 binding sequence (5′-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3′). All oligos were obtained from Integrated DNA Technology. The sgRNA oligo mix was hybridized to the sgRNA complementary oligo (at 10 μM each) in 1× NEBuffer2 (New England BioLabs, NEB) with 2 mM dNTPs at 90°C for 15 s followed by 43°C for 5 min. To complete dsDNA synthesis, the hybridization mixture was incubated at 37°C for 1hr with 5 U of Klenow Fragment 3′→5′ exo- (NEB). To degrade linear ssDNA remaining, the dsDNA was then treated with Exonuclease I in 1× Exonuclease I reaction buffer (NEB) for 1 h at 37°C. Finally, dsDNA was purified using QIAquick Nucleotide Removal Kit (Qiagen) and eluted in 30 ul elution buffer. Quality and concentration were assessed using agarose gel electrophoresis and the Synergy H1Hybrid Multi-Mode Reader (Bio Tek).sgRNA synthesissgRNA was synthesized using HiScribe T7 High Yield RNA Synthesis Kit (NEB) following the Standard RNA Synthesis protocol. In summary, 1 μg dsDNA was incubated with 1× reaction buffer, 10 mM NTPs and T7 RNA polymerase enzyme mix at 37°C for 2 h followed by DNase I treatment at 37°C for 15 min to remove dsDNA from the reaction. sgRNA was then purified using RNA Clean & Concentrator Kits (Zymo Research). The concentration of the purified sgRNA was assessed using Synergy H1Hybrid Multi-Mode Reader (Bio Tek).CRISPR–Cas9 labeling of chromosomal DNAFor DNA nicking using the 48 and 162 sgRNA mix (supplementary Tables S1 and S2),1.25 μM of the synthesized sgRNA was first incubated with 125 nM of Cas9 D10A (NEB) in 1× NEBuffer 3.1 (NEB) at 37°C for 15 min to form a sgRNA-Cas9 complex. 300 ng of the DNA sample was then added to the sgRNA–Cas9 complex mixture and incubated at 37°C for 60 min. For DNA nicking with both Cas9 and Nt.BspQI, 2.5 μM gRNA was first incubated with 63 nM of Cas9 D10A in 1X NEBuffer 3.1 at 37°C for 15min. After that, 300 ng of DNA and 5 U of Nt.BspQI (NEB) were added to the sample mixture and incubated at 37°C for 2 h. The nicked DNA samples were then labeled using 5 U Taq DNA Polymerase (NEB), 1× thermopol buffer (NEB), 266 nM free nucleotides mix (dATP, dCTP, dGTP (NEB) and Atto-532-dUTP (Jena Bioscience)) at 72°C for 60 min. The labeled sample was then treated with Proteinase K at 56°C for 30min and 1uM IrysPrep stop solution (BioNano Genomics) was added to the reaction.DNA loading and imagingLabeled DNA samples were stained and prepared for loading on an Irys Chip (BioNano Genomics) following manufacturer instructions. The sample was then linearized and imaged. The stained samples were loaded and imaged inside the nanochannels following the established protocol. Each Irys Chip contains two nanochannel devices, which can generate data from >60 Gb of long chromosomal DNA fragments (>150 kb). The image analysis was done using BioNano Genomics commercial software (IrysView 2.5) for segmenting and detecting DNA backbone YOYO-1 staining, similar to early optical mapping methods, and localizing the green labels by fitting the point-spread functions.Data analysisSingle-molecule maps were de novo assembled and aligned to the reference as described in previous work (37). Briefly, the assembler is a custom implementation of the overlap-layout-consensus paradigm with a maximum likelihood model. An overlap graph was generated based on the pairwise comparison of all molecules as input. Redundant and spurious edges were removed. The assembler outputs the longest path in the graph and consensus maps were derived. Consensus maps are further refined by mapping single-molecule maps to the consensus maps and label positions are recalculated. Refined consensus maps are extended by mapping single molecules to the ends of the consensus and calculating label positions beyond the initial maps. After the merging of overlapping maps, a final set of consensus maps was output and used for subsequent analysis. RefAligner works similarly but compares molecules directly to an in silico nicked reference instead of first forming contigs. These maps were then opened in Irsyview visualization software from BioNano Genomics.

Article TitleCustomized optical mapping by CRISPR–Cas9 mediated DNA labeling with multiple sgRNAs

Abstract

All additional data is available in the supplementary section.


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