Genome characterization and CRISPR-Cas9 editing of a human neocentromere

Abstract

The maintenance of genome integrity is ensured by proper chromosome inheritance during mitotic and meiotic cell divisions. The chromosomal counterpart responsible for chromosome segregation to daughter cells is the centromere, at which the spindle apparatus attaches through the kinetochore. Although all mammalian centromeres are primarily composed of megabase-long repetitive sequences, satellite-free human neocentromeres have been described. Neocentromeres and evolutionary new centromeres have revolutionized traditional knowledge about centromeres. Over the past 20 years, insights have been gained into their organization, but in spite of these advancements, the mechanisms underlying their formation and evolution are still unclear. Today, through modern and increasingly accessible genome editing and long-read sequencing techniques, research in this area is undergoing a sudden acceleration. In this article, we describe the primary sequence of a previously described human chromosome 3 neocentromere and observe its possible evolution and repair results after a chromosome breakage induced through CRISPR-Cas9 technologies. Our data represent an exciting advancement in the field of centromere/neocentromere evolution and chromosome stability.

Keywords: CRISPR-Cas9; Isochromosome; Long-read sequencing; Neocentromere.

Fig. 1

Partial view of the ChIP-on-chip analysis data on chromosome 3, using anti-CENP-A and anti-CENP-C antibodies. The results are presented as the log2 ratio between the hybridization signals obtained with immunoprecipitated DNA using anti-CENP-A (A) and anti-CENP-C (B) antibodies and that from the input DNA sample. The X-axis shows the genomic position of each oligo on chromosome 3. The data are visualized through the SignalMap software (NimbleGene Systems, Inc.). The shaded regions indicate the mapping of the CENP-A and CENP-C domains as identified by the Tamalpais statistical analysis: a major domain of about 163.6 kb (chr3:147,497,420–147,661,009, GRCh38/hg38) and two minor domains of about 21.5 and 6.7 kb (mapping at chr3:147,869,478–147,890,981 and chr3:148,079,102–148,085,846, GRCh38/hg38)

Fig. 2

FISH and immuno-FISH results. a Hybridization result obtained by using as a probe a chromosome 3 WCP. A small acrocentric chromosome (green arrow) and a small metacentric chromosome (blue arrow) are visible in the panel. b Probe selection for the cytogenetic characterization through BAC clone hybridization (blue probes: RP11-151A4 and RP11-655A17; green probes: RP11-454H13 and RP11-21N8; red probes: RP11-498P15 and RP11-693H4). c An exemplifying result of the hybridization performed using the probes described in b. d Probe selection for the immuno-FISH characterization (red probe: RP11-21N8; green: anti-CENPC antibody; blue probe: RP11-498P15). e FISH result of the hybridization performed using probes described in d. f Probe selection for the FISH characterization of the region flanking the break site (RP11-426P6 in red and RP11-1077E14 in green). g Interphase FISH result of the experiment described in f showing the inverted duplication of the probes in one of the derived chromosomes. Extracted and enlarged chromosomes are shown on the bottom left of c, e, and g for better clarity

Fig. 3

Schematic showing the editing with CRISPR-Cas 9. The white asterisk indicates small InDels obtained by NHEJ repair. The shown percentages illustrate the editing detectable events (37% of InDels and 35% of chromosomal rearrangements). Consequently, the remaining 28% consists either of non-edited cells or of undetectable mutations

Fig. 4

Model of isochromosome formation. Shown is a hypothetical model of the creation of the isochromosome containing two copies of the region 3q24 to qter, after the induced breakage of Neo3 by CRISPR-Cas9 methods