Materials and Methods

CRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system

MATERIALS AND METHODSCell culture and genomic DNA precipitationWe cultured E. coli EcNR2 and EcHB3 cells in Luria-Bertani (LB) media (BD Biosciences, USA) at 30°C in a shaking incubator. We harvested the cells by centrifugation and precipitated the genomic DNA using the GeneAll Exgene Cell SV Kit (GeneAll, Korea) according to the manufacturer's protocol. We purchased genomic DNA of NA12878 from the Coriell Institute (USA).SpCas9 protein purificationProfessor Hyongbum Kim's group donated the expression plasmid pET28a/Cas9-Cys, which has the SpCas9 protein-coding sequence appended with an N-terminal 6X -His tag and additional C-terminal cysteine for purification. The in vitro cleavage activity of the purified SpCas9 was previously confirmed (47). We transformed C2566 BL21-based cells (New England BioLabs, USA) with the plasmid and cultured them in LB-kanamycin (30 μg/ml) media to an optical density at 600 nm (OD600) of 0.5. We induced SpCas9 by treating the cultures with a 0.5 mM final concentration of isopropyl β-d-1-thiogalactopyranoside (IPTG) for 4 h at 30°C in a shaking incubator. We then harvested the cells and sonicated them 15 times with a 40% duty factor in 10 s bursts with a 10 s rest on ice between bursts. The lysis buffer was 20 mM Tris–HCl pH 8.0, 300 mM NaCl; 40 mL buffer/1 l cultured cells. After sonication, we centrifuged the crude extract and sonicated the supernatant overnight with Ni-NTA agarose resin (QIAGEN). We then loaded the resin-bound sample onto a column. We washed the loaded column three times with wash buffer (20 mM Tris–HCl pH 8.0, 300 mM NaCl, 20 mM imidazole), using a buffer volume corresponding to 3× the resin volume. We subsequently eluted the protein five times with elution buffer (20 mM Tris–HCl pH 8.0, 300 mM NaCl, 250 mM imidazole), using the same volume used for the Ni-NTA resin purification. Because the purified sample contained unwanted proteins, we filtered out small proteins using dialysis buffer 50 mM Tris–HCl pH 8.0, 200 mM KCl, 0.1 mM EDTA, 1 mM DTT, 20% glycerol, 1 tablet/50 ml cOmplete Protease Inhibitor Cocktail (Roche, USA) and a 100 kDa pore size Amicon® Ultra Centrifugal Filter (Merck Millipore, Germany).sgRNA library constructionTo cover all the target genes or exons, we designed sgRNA libraries to include 100 bp upstream and 100 bp downstream of the target regions. To construct sgRNA libraries that target various regions in the E. coli and human genomes, we performed in vitro transcription using a DNA microarray oligonucleotide (oligo) pool (CustomArray, USA) as a template. All of the microarray oligos contained a T7 promoter sequence upstream of the sgRNA sequence. First, we amplified the microarray oligos using real-time PCR with the KAPA SYBR® FAST Bio-Rad iCycler 2X qPCR Master Mix (Kapa Biosystems, USA) until the amplification was saturated. We then amplified the products again using the KAPA HiFi HotStart Ready Mix (Kapa Biosystems) to increase the amount of template DNA. We purified the double-amplified DNA product using the MinElute PCR Purification Kit (QIAGEN) and then transcribed it using the MAXIscript® T7 in vitro Transcription Kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol. We transcribed biotinylated sgRNAs using a UTP mixture containing 80% UTP and 20% biotin-16-dUTP (Thermo Fisher Scientific). We treated the transcribed RNAs with TURBO DNase (Thermo Fisher Scientific) and purified them using an Oligo Clean & Concentrator Kit column (Zymo Research, USA). Before using them, we subjected all the sgRNA libraries to a refolding step, in which the sample was heated to 95°C and cooled at −0.1°C/s until it reached 37°C.Cleavage step of CRISPR-CapWe performed CRISPR-Cap in vitro. With E. coli genomic DNA, we used 1 μg genomic DNA and a 100-fold excess molar ratio of refolded-sgRNA library and SpCas9. With human genomic DNA, we used 1 μg or 100 ng NA12878 genomic DNA and a 10 000-fold excess molar ratio of refolded sgRNA library and SpCas9. We calculated the amount of sgRNA library or SpCas9 to add to the reaction as: (molecules of genomic DNA) × number of sgRNAs in the sgRNA library (e.g. 550 with a 20-bp sgRNA library) × (excess molar ratio) × (molecular weight of sgRNA or SpCas9)/(Avogadro's constant). We incubated the final reaction (50 μl final volume) containing genomic DNA, sgRNA library, and SpCas9 in NEB3 buffer for 1 h at 37°C in a thermocycler. After the cleavage step, we performed the sorting step.Sorting step of CRISPR-Cap using streptavidin magnetic beadsTo sort out the Cas9–DNA complexes containing the cleaved DNA, we used magnet-coated streptavidin C1 beads (Thermo Fisher Scientific). Before using them, we washed the streptavidin C1 beads three times with bead washing buffer (BWB; 5 mM Tris–HCl pH 7.5, 1 M NaCl) and then resuspended them in 50 μl 2× BWB. We mixed the washed beads directly with the product from the cleavage step and incubated the mixture for 10 min at room temperature to allow binding of the Cas9–DNA complexes to the streptavidin. We then isolated the beads using a magnetic stand and discarded the supernatant. We washed the bead pellet three times with BWB and then released the cleaved target DNA using a mixture of 50 μl nuclease-free water and 12.5 μl 0.2% SDS solution. We incubated the solution containing the released DNA for 5 min at room temperature and then purified the DNA using the MinElute PCR Purification Kit (QIAGEN) to remove any remaining SDS. Alternatively, after adding 50 μl of nuclease-free water without 0.2% SDS to the bead pellet, we could release the enriched DNA from the Cas9–DNA complexes by incubating the solution for 10 min at 65°C to inactivate the SpCas9. We used the column-purified DNA or heat-released DNA for NGS sample preparation.Sorting step for early-release CRISPR-CapTo perform early-release CRISPR-Cap, we mixed a 20% volume of 0.2% SDS solution directly with the product of the CRISPR-Cap cleavage step and incubated the mixture for 20 min at 37°C to promote the release of the cleaved target DNA. After SDS treatment, we purified the product using the MinElute PCR Purification Kit (QIAGEN) and used the purified DNA for NGS sample preparation.CRISPR-Cap cleavage in a cell lysateTo perform the cleavage step in a whole-cell lysate, we harvested 1 mL saturated overnight E. coli culture by centrifugation at 16 100 rcf for 1 min. We discarded the supernatant and resuspended the pellet in 50 μl Cas9 working buffer (48). We sonicated the sample for 1 min with a 20% duty factor in 3 s bursts with 3 s rests on ice between bursts to minimize random shearing. We then mixed the cell lysate with the 20-bp sgRNA library and SpCas9. Because the amount of genomic DNA in a cell lysate is difficult to quantify, we used a 100-fold molar excess of sgRNA library and SpCas9 with 1 μg genomic DNA.Next generation sequencing sample preparationWe prepared samples of enriched DNA for NGS with the SPARK™ DNA Sample Prep Kit for the Illumina® Sequencing Platform (Enzymatics, USA). We performed end repair and dA-tailing according to the manufacturer's protocol. We then added 10 μl NEBNext Adaptors (New England BioLabs) for Illumina to the dA-tailed DNA and performed a ligation step. We performed USER cleavage (New England BioLabs) on the adaptor-ligated sample for 15 min at 37°C and then purified the DNA using the MinElute PCR Purification Kit (QIAGEN). We performed index PCR with 16 cycles of limited PCR amplification to attach the Illumina sequencing index to the CRISPR-Cap product. We gel purified the index PCR products at a size of 250–600 bp with the MinElute Gel Extraction Kit (QIAGEN) and sequenced the purified samples using the Illumina Hiseq 4000, NextSeq, or Miseq platform.Sequencing analysisWe used the AdapterRemoval (49) software to remove the adapter sequences from the sequencing data. We used the Burrows-Wheeler alignment (BWA) tool (50) to align the data to the reference sequence. We used SAMtools (51) with default parameters and in-house python scripts to count the sequencing depth at each position. Before aligning the adapter-trimmed sequencing data, we excluded the sgRNA template sequences that remained after in vitro transcription using an in-house python script.Quantitative PCRWe performed quantitative PCR (qPCR) with custom primer pairs (Macrogen, Korea), the KAPA SYBR® FAST Bio-Rad iCycler 2× qPCR Master Mix, and a MyiQ Real-Time PCR machine (Bio-Rad, USA). The PCR conditions were: 3 min at 95°C, followed by 40 cycles of 20 s at 98°C, 15 s at 60°C, and 30 s at 72°C. We used E. coli EcHB3 samples as a control. We calculated fold changes in gene expression using the ΔCt value of each sample.

Article TitleCRISPR-Cap: multiplexed double-stranded DNA enrichment based on the CRISPR system

Abstract

Raw sequencing data are available under Sequence Read Archive: SRP096854 and SRP140115.


Login or Signup to leave a comment
Find your community. Ask questions. Science is better when we troubleshoot together.
Find your community. Ask questions. Science is better when we troubleshoot together.

Have a question?

Contact support@scifind.net or check out our support page.