Japanese researchers have successfully developed a novel method to remove the extra copy of chromosome 21 in the cells of patients with Down syndrome, according to a study.
In the study, published in PNAS Nexus, researchers, led by Ryotaro Hashizume, MD, PhD, of the Mie University Graduate School of Medicine, and colleagues demonstrated that allele-specific multiple chromosome cleavage using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 can achieve "trisomy rescue" by eliminating the target chromosome from human trisomy 21 induced pluripotent stem cells and fibroblasts. The researchers specifically targeted the M2 allele of chromosome 21, a key technical advancement that enabled precise removal of only the extra chromosome copy.
The researchers achieved a chromosome elimination rate of 13.1% using their optimized approach, which involved making multiple precise cuts in one specific copy of chromosome 21. This rate increased to approximately 30.6% when combined with temporary suppression of DNA repair genes.
"Unlike previously reported allele-nonspecific strategies, we have developed a comprehensive allele-specific (AS) Cas9 target sequence extraction method that efficiently removes the target chromosome," the study authors wrote.
The technique proved effective in both dividing and nondividing cells, with chromosome elimination rates of 13.9% observed in differentiated skin fibroblasts. Importantly, the researchers found no evidence of unintended chromosome gains during their study. However, the results were obtained under controlled laboratory conditions using specific cell types—induced pluripotent stem cells and skin fibroblasts—and further research may be needed to evaluate the technique's effectiveness in other cell types relevant to clinical applications.
Gene expression analysis revealed that successful chromosome removal restored normal gene signatures and improved cellular phenotypes.
"GO terms associated with downregulated genes exhibit substantially higher false discovery rates (FDRs) compared with those of upregulated genes, indicating that the GO analysis results for downregulated genes should be interpreted with caution," the study authors underscored.
While promising, the researchers identified several challenges that need to be addressed prior to potential clinical application, including protecting nontarget alleles from Cas9-induced double-strand breaks, simplifying the phasing method, and evaluating the approach in clinically relevant cell types like neurons and glial cells.
"Although a nonchromosome-breaking elimination method is desirable, the findings of this study can be used to rescue somatic cells with trisomy," the study authors concluded.
The research was supported by the Japan Society for the Promotion of Science KAKENHI Grant Numbers JP16K09964, JP16K15242, and JP21K06835.
The authors declared no competing interests.