Skip to main content
Premium Trial:

Request an Annual Quote

CRISPR Used to Eliminate Targeted Chromosomes in New Study

NEW YORK (GenomeWeb) – Researchers at the Chinese Academy of Sciences and their collaborators have developed a CRISPR-Cas9 system to eliminate targeted chromosomes from the human genome.

As they reported on Friday in Genome Biology, the researchers developed an approach that used either multiple cleavages induced by one or two single-guide RNAs (sgRNA) that targets multiple chromosome-specific sites or a combination of 14 sgRNAs, each targeting a specific site, to selectively eliminate a sex chromosome in cultured cells, embryos, and tissues in vivo. They were also able to produce a targeted autosome loss in aneuploid mouse embryonic stem cells with an extra human chromosome, human induced pluripotent stem cells with trisomy 21, and cancer cells.

The researchers began by examining whether they could completely eliminate the mouse Y chromosome by using CRISPR-Cas9-mediated multiple cuts at chromosome-specific sites. They designed sgRNAs to target the Rmby1a1, Ssty1, and Ssty2 loci on the chromosome's short and long arms, and found it could be selectively eliminated, both in cultured cells, and in vivo in male mice.

They then examined whether this method could be applied to generate animal models for aneuploidy, such as Turner syndrome. The researchers injected Cas9 mRNA and two specific sgRNAs that targeted the Rmby1a1, Ssty1, or Ssty2 locus into individual mouse zygotes, and injected zygotes were then cultured to the blastocyst stage. Gene-edited embryos showed normal development compared to untreated embryos or embryos treated with two sgRNAs targeting only a single-copy gene on the Y chromosome, with a similar blastocyst rate, the authors said.

When they performed DNA-FISH analysis on injected embryos at the four- to 16-cell stage, focusing only on male embryos, the green fluorescent signal for the Y chromosome probe was absent in some blastomeres of injected male embryos, suggesting the Y chromosome had been eliminated at an efficiency of 40 percent to 90 percent from the experiments targeted at the three different gene loci. The team further determined that this approach could also selectively eliminate the X chromosome in mice.

The researchers next looked at whether they could eliminate an extra chromosome in aneuploid cells using CRISPR-Cas9 editing. They focused on an ES cell line with an extra human chromosome 14 (hChr14). "Using sgRNAs targeted at repeated sequence sites, we were able to achieve complete elimination of hChr14 in up to 15 percent of cells," the authors wrote. "Further genotype evidence of the hChr14 deletion was confirmed by WGS, as well as the expression profile of genes unique to hChr14." They were also able to apply this method to promote human chromosome 7 loss in human cancer cell line HT-29, which contains four hChr7s in most cells, and extra human chromosome 21 loss in aneuploid mouse ES cell lines derived from mice with Down syndrome.

When they examined the potential for off-target effects of these experiments, the researchers found that mutations "rarely occurred" in seven to nine female mice obtained by Y chromosome elimination. Among 2,186 to 26,469 potential off-target sites for each sgRNA used in the experiments that included eight mice with Y or X chromosome deletion, and four cell lines with Y chromosome deletion or hChr14 deletion, the researchers found only two off-target sites in only one XO mouse. "The rest of the mice and cell lines contained no off-target mutations," they wrote. "Together, these results indicate that CRISPR-Cas9-mediated chromosome elimination did not induce significant off-target alteration in chromosome-deleted mice and cell lines beyond that expected for CRISPR-Cas9-mediated editing in general."

The authors concluded that this is the first study that they know of to use CRISPR to eliminate X and autosome chromosomes, and as such, "it paves the way for a potential genetic approach to chromosome therapy in vivo." It also offers "a new approach to develop animal models with chromosome deletions, and a potential therapeutic strategy for human aneuploidy diseases involving additional chromosomes," they added.