μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome

Abstract

Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.

Figure 1.

Mechanism of DNA separation by μLAS. (A) The panel shows the parabolic profile of the Poiseuille flow in blue and the counter electrophoretic force in red, which are responsible for DNA transport. In a viscoelastic fluid, a transverse force oriented toward the capillary walls occurs. (B) The amplitude of transverse viscoelastic forces is size-dependent and DNA molecules are transported along different streamlines according to their molecular weight.

Figure 2.

HMW DNA separation by μLAS. (A) The four chromatograms correspond to the kb extend DNA ladder separated by μLAS using four different concentrations of PVP 40 kDa, as indicated in inset. The flow velocity v_m is set to 1.5 mm/s. (B) Using 5% PVP 40 kDa, the graph presents the separation of the same sample for four different flow velocities v_m ranging from 0.6 to 10 mm/s as indicated in inset. (C) The contour plot shows the resolution R₅₀ as a function of the electric field and flow velocity with a buffer containing 5% PVP 40 kDa. (D) Chromatogram of the kb extend DNA ladder with the custom-synthetized PVP 43 kDa dissolved at a volumic concentration of 3%. (E) Separation of a custom DNA ladder containing four bands of 50, 100, 150 and 210 kb at 0.5 mm/s and 9 V/cm with the custom-synthetized PVP 43 kDa dissolved at a volumic concentration of 3%. (F) Resolution of the separation in the size range 3–210 kb.

Figure 3.

μLAS for online concentration and separation. (A) Schematics of the capillary system 2 composed of (i) an inlet capillary of 50 μm in inner diameter and 5 cm in length, (ii) a loading chamber of 320 μm in inner diameter and 5 cm in length and (iii) an outlet capillary of 50 μm in inner diameter and 19 cm in length. The laser-induced fluorescence (LIF) detector is placed 7 cm downstream of the interconnection between the loading chamber and the outlet capillary. Collection is performed by isolating a target volume at the outlet during a calibrated time period. (B) The black curve shows the intensity versus time for the kb extend ladder at a concentration of 100 pg/μl and the red curve is the temporal evolution of the electric field. The sample is concentrated during the first 10 min of the process (dashed blue line).

Figure 4.

Capture of a 31 kb DNA fragment from a BAC clone. (A) The chromatogram shows a broadly distributed DNA sample (black curve), and the result of its fractionation with different collection times, as indicated in the legend (red, green and blue curves). The dashed black curve corresponds to the reference DNA ladder. (B) The plot presents the DNA mass as a function of the collection time (black data points and corresponding linear fit), as inferred from the analysis of the three curves shown in panel (A). The blue line and symbol graph shows the full width at half maximum as a function of the collection time. (C) The chromatograms show the Cas9-digested BAC clone sample (black curve) with two main peaks of 31 and 65 kb, and the purified sample with one single peak of 31 kb (red curve). The dashed blue line corresponds to the end of the concentration phase. (D) The graph presents the read depth as a function of genomic position on the BAC clone. The two vertical gray lines represent the position of the sgRNA.

Figure 5.

Workflow of the CATCH method for plant genomic DNA purification and sequencing: the cartoons represent the consecutive steps for the isolation of a target region from plant genomes. We used three different strategies: (1) PFGE alone; (2) PFGE and μLAS isolation; (3) μLAS isolation only.

Figure 6.

Capture of a 30 kb DNA fragment from genomic DNA. (A) The blue chromatogram corresponds to the Cas9-digested gDNA obtained according to method 3 without purification by PFGE. The black curve is the Cas9-digested BAC reaction product shown as reference. The dashed vertical line corresponds to the end of the concentration phase. (B) The red chromatogram presents the Cas9-digested gDNA sample obtained by PFGE, band excision and electro-elution (following the process flow of methods 1 and 2). The green curve is the control without Cas9 digestion, and the black one corresponds to the kb extend ladder. The dashed vertical line indicates the migration of the 31 kb fragment. (C) The blue chromatogram corresponds to the Cas9-digested sample as shown in panel (A), and the red one to the final product purified with μLAS following method 3. (D) The red chromatogram corresponds to the Cas9-digested sample as shown in panel (B), and the black one to the final product purified with μLAS (method 2).

Figure 7.

Sequencing of the 31 kb DNA fragment purified by methods 1 and 2. (A) The graph presents the read depth as a function of genomic position on the first 500 kb of the Medicago truncatula chromosome V, as purified with method 1. (B) Same plot as in A based on the purified material obtained by method 2. The vertical dashed grey lines represent the position of the sgRNA.