Enzymatic exome sequencing and its application to triaged genotyping of multiplexed CRISPR-edited plants

Whole-genome sequencing has become a common strategy to genotype individual plants of interest. Although a limited number of genomic regions usually need to be surveyed with this strategy, excess sequencing information is almost always generated at an appreciable financial cost for the users. For example, heterochromatic sequences, which can account for more than 80% of the genome size of some plants, are oftentimes not required in these genotyping projects. Therefore, strategies that enrich euchromatic DNA coding for the protein-coding genes prior to sequencing can provide far more useful sequence information and drastically lower sequencing costs. Here, we present the development and application of MsRR-Seq, which relies on the cytosine methylation-sensitive restriction enzyme MspJI to deplete heterochromatic DNA before library construction and sequencing. By applying MsRR-Seq to citrus and maize, we show that protein-coding genes can be enriched in sequencing datasets. We then describe the application of MsRR-Seq to facilitate the identification of complex mutants from populations of citrus plants resulting from multiplex CRISPR/Cas9 editing of four genes. Overall, this work demonstrates an easy and low-cost method to enrich euchromatic DNA in whole-genome sequencing projects, an approach that is especially useful for large plant genomes with an excessively high proportion of methylated repetitive sequences.