Resolving the 22q11.2 deletion using CTLR-Seq reveals chromosomal rearrangement mechanisms and individual variance in breakpoints

Abstract

We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.

Keywords: chromosome interactions; copy number variation; genome repeats; microdeletion syndromes; targeted genome assembly.

Fig. 1.

CTLR-Seq workflow and its application to the 22q11 deletion region. CTLR-Seq workflow for intact endogenous isolation of the 22q11.2 A–D deletion haplotype from patient cells and nanopore sequencing of target (endogenous patient DNA). Step 1: Live cells from a 22q11DS patient are dispensed into the sample well of a 0.75% agarose cassette, where they are lysed via the delivery of a 3% SDS lysis buffer by electrophoresis, exposing their genomic DNA (while immobilized on the wall of the agarose well) to in vitro CRISPR/Cas9-cutting. Step 2: The CRISPR-guide-RNA/Cas9 complexes (assembled prior) are added to the sample well and delivered to the exposed patient genomic DNA via electrophoresis, and CRISPR cuts take place. Step 3: Pulsed-gel electrophoresis is carried out, the deletion haplotype (Haplotype B) containing the 22q11.2 SegDup LCR22A/D rearrangement (i.e., target) migrates into the agarose gel at a much faster rate and is thus isolated away from the rest of the genome, including from the chromosome 22 haplotype without deletion (Haplotype A). Switching the direction of the current leads to the elution of the target into an elution module, followed by Step 4: Nanopore long-read DNA sequencing and haplotype-specific de novo sequencing assembly and other assays, such as linked-read sequencing, where the reads can be used to polish the nanopore-based de novo assembly.

Fig. 2.

Illustration of different intrachromosomal SegDup rearrangements resulting in 22q11DS. (A) The recombination of different 22q11.2 SegDup regions LCR22A, B, C, and D results in different subtypes of deletions at 22q11.2 such as A–D (i.e., typical deletion, 90% of cases), A–C, and A–B. (B) Genome coverage (y-axis; window size 25 bp, zoom level 7 on IGV) with nanopore long-reads from CTLR-Seq (five 22q11DS patients with the typical A–D deletion) aligned to hg38 (x-axis: chr22:18100000–21600000) between where the two CRISPR guide RNAs used for each patient sample are designed to cut. (Different CRISPR guide RNAs for the LCR22D end were tested and used; see Dataset S2.)

Fig. 3.

Haplotype-specific de novo sequence assemblies of 22q11.2 A–D deletion SegDup rearrangements using CTLR-Seq. (A–D) SafFire visualizations, with respect to the 22q11.2 locus (spanning SegDup regions LCR22A–LCR22D, chr22:18000000-21600000, hg38 annotations), of haplotype-specific de novo sequence assemblies of 22q11.2 A–D deletion SegDup/LCR rearrangements using CTLR-Seq. Light blue indicates region of direct sequence alignment. Orange indicates inverted alignment relative to reference.

Fig. 4.

Breakpoints identified in five 22q11.2 A–D deletion patients inside the NAHR region. X-axis: LCR22A and LCR22D coordinates (blue and red, respectively) are with respect to chromosome 22 (hg38). (Scale bar: 10 kb.) Boxed regions zoomed out via dashed lines indicate breakpoint location at base-pair resolution and hg38 annotations within. Red and blue dots represent 31 bp k-mers identified only in the 22q11DS patient (deletion haplotype) and LCR22A or LCR22D haplotypes, respectively. (A and B) The contributing LCR22A and LCR22D haplotypes from the available parent of origin were assembled and used for breakpoint analysis. For 22q11DS patients where the parent of origin was not available (C–E), the closest matching LCR22A and LCR22D haplotypes (e.g., HG00733 paternal for 04C27536) from the human pangenome assemblies were used as the “parent of origin” for breakpoint identification. Visualization for 2172 (D) is inverted relative to hg38, corresponding to patient assembly (Fig. 3C).

Fig. 5.

Breakpoints of 22q11.2DS across nine patients. Overview of the deletion breakpoint locations inside LCR/SegDups and the entire deletion intervals (green bars) with respective to hg38 (chr22:18100000-21600000) that were identified across the nine 22q11DS patients (Table 1). The A–D breakpoints (n = 4) were identified using CTLR-Seq; A–B (n = 2), A–C (n = 1), B–D (n = 1) can be identified using standard-coverage ultra-long nanopore WGS. The breakpoints of all nine patients overlap with transposon sequences residing within the 22q11.2 LCR/SegDups.

Fig. 6.

HiChIP 3D chromosome interaction analysis of the LCR22A–D NAHR region in neurons and astrocytes of two 22q11.2DS patients. (A) From 22q11DS patients, dermal fibroblasts were obtained and reprogrammed to iPSCs, from which cortical organoids were generated. On day 150 of differentiation, organoids were dissociated, and cells were separated into neurons and astrocytes. From these cell populations, HiChIP libraries were generated using an H3K27ac antibody. (B–E) HiChIP intrachromosomal interaction maps (red) of the approximately 160 kb region of NAHR for LCR22A and LCR22D (green). The typical 22q11DS A–D deletion region (approximately 3 Mb) is indicated in light gray.