Targeted Deletion of an Entire Chromosome Using CRISPR/Cas9

Abstract

CRISPR/Cas9 genome editing can facilitate efficient deletion of genomic region, but it has not been used to delete an entire chromosome. Here, Adikusuma et al. show proof-of-concept for efficient CRISPR-mediated selective chromosome deletion by removing the centromere or shredding the chromosome arm in mouse embryonic stem cells and zygotes.

Graphical abstract

Figure 1

Deletion of Y Chromosome Using CRISPR/Cas9 in Mouse ESCs and In Vivo Mouse Zygote Injection (A) Schematic showing the position of gRNA target sites in the long arm and centromere of the Y chromosome. (B) qPCR of genomic DNA to quantify Y chromosome dosage. Sox1 qPCR was used as the internal reference control. Data are presented as mean ± SEM from n ≥ 3 biological replicates. Statistical analysis using two-way ANOVA is presented in Table S3. (C) FISH analysis detection of Y chromosome loss. Y chromosome and DAPI staining was indicated by green and blue signals, respectively. Scale bar, 5 μm. (D) Xist genomic qPCR of phenotypically female mice generated through zygote injection of centro 41X gRNA. Asterisks indicate female candidates with single X. (E) Dmd and Sox3 genomic qPCR confirming single X chromosome in female XO candidates. (F) Genomic qPCR quantifying dosage of Y short and long arms. Sox1 qPCR was used as the internal reference control. Results are presented as mean ± SD from n ≥ 3 replicates.