CRISPR-mediated isolation of specific megabase segments of genomic DNA

Abstract

Megabase-sized, complex, repetitive regions of genomes are poorly studied, due to the technical and computational challenges inherent to both assembling precise reference sequences and accurately assessing structural variation across contiguous megabase DNA regions. Here we describe a strategy to overcome these challenges, CISMR (CRISPR-mediated isolation of specific megabase-sized regions of the genome), which enables us to perform targeted isolation of contiguous megabase-sized segments of the genome. Direct sequencing of the purified DNA segments can have >100-fold enrichment of the target region, thus enabling the exploration of both DNA sequence and structural diversity of complex genomic regions in any species.

Figure 1.

Large segments of the mouse X chromosome are specifically targeted with in vitro CRISPR. (A) The specific products of the in vitro Otc CRISPR digestion of both YAC ADK.A6 DNA and mouse genomic DNA are resolved by PFGE and detected with Diamond stain (Promega) (Supplementary Figure S1). Whereas the expected YAC digestion products are clearly detectable, digestion of the more complex mouse genomic DNA does not produce detectable products. Untreated and Cas9-only digestions of YAC and mouse genomic DNA serve as negative controls. (B) A Southern blot of the PFG shown in (A) is hybridized with a DIG-labeled Otc probe. The intact YAC chromosome (∼520 kb) and the expected 263 kb in vitro Otc CRISPR digestion product from both YAC and mouse genomic DNA are visible upon chemiluminescent detection of the probe and exposure of X-ray film. (C) A faint but, detectable ∼2.3 Mb in vitro Srsx CRISPR digestion product from mouse genomic DNA is resolved by PFGE along with the multi-megabase H. wingei (Hw) chromosomes ladder (Bio-Rad) labeled on the left. (D) The Southern blot of the PFG shown in (C) is hybridized with a DIG-labeled Srsx probe. The estimated ∼2.3 Mb in vitro Srsx CRISPR digestion product from mouse genomic DNA is visible upon chemiluminescent detection of the probe and exposure of X-ray film.

Figure 2.

Enrichment of Illumina sequencing reads of the isolated 263 kb and ∼2.3 Mb genomic segments. (A) Single-end sequencing read depth of the ∼2.3 Mb Srsx-gene array region and the 263 kb Mb Otc-gene region, both of which are flanked by 500 kb of non-targeted sequence. The five gaps present within the Srsx region are due to incomplete assembly of the reference sequence. Schematic of the mouse X chromosome shows the origin of the targeted and sequenced genomic DNA segments. (B) Higher resolution of Illumina sequencing read depth and coverage across 25 kb of sequence flanking the 3′ Otc-gene region sgRNA cut site (vertical dotted line). The sgRNA targets exon 5 of Otc, which is consistent with the alignment of multiple reads up to the sgRNA cut site.

Figure 3.

Combining CISMR with sequencing strategies to resolve megabase-sized structurally complex regions of the genome. CISMR isolated DNA segments containing low identity or high identity segmental duplications can be resolved with current long-read sequencing technologies or region-specific clone libraries, respectively. Both approaches are amenable to low inputs of DNA to generate an accurate assembly of the complex genomic region.