Specific Capture of Large DNA Fragment by Coupling CRISPR/Cas9 Targeting and Matrix-Free Separation and Enrichment

Despite the sequencing of long reads with Third Generation Sequencing Technologies, the study of numerous plant genomes remains challenging due to their complexity in terms of size, polyploidy or high percentage of repetitive elements. Moreover, the current state-of-the-art for large DNA separation and preparation is labor-intensive, if not resolution inappropriate for fragments over 20kb. New strategies for targeting large regions of interest are really needed.

We here report a new sequence capture approach for large DNA fragments. We used the first steps of the CATCH method (Cas9-Assisted Targeting of CHromosomal segments as described by Jiang and Zhu, 2016; and Jiang et al., 2015) based on the endonuclease function of the CRISPR/Cas9 system to cut a specific locus of 30kb from a Medicago truncatulaBAC clone of 100kb.

Then, we adapted the µLAS technology (described in Ranchon et al., 2016) on capillary electrophoresis to perform on-line and automatically the separation, concentration and isolation of the target fragment from the bulk of the BAC. This DNA processing technology does not require a separation matrix, but rather an electrohydrodynamic actuation that allows us sorting DNA molecules according to their sizes. We have set up conditions to sort the specific 30kb fragments from 70 and 100kb molecules. We’ll describe the various steps used and present the collection yield obtained, the amount and purity of the target fraction recovered.

This technology allowed us to capture specifically large DNA molecules (> 30kb), a new approach that will be useful to analyze large genomic regions of interest from complex plant genomes.