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Scientists Edit Beta-thalassemia Gene in Human Zygotes with CRISPR/Cas9

NEW YORK (GenomeWeb) – No longer a rumor, human germline editing with CRISPR/Cas9 is now the subject of a study published today in Protein & Cell. The results indicate that any therapeutic use of the technology in humans remains distant.

Scientists from Sun Yat-sen University in Guangzhou, China, led by Canquan Zhou and Junjiu Huang, used the CRISPR/Cas9 genome editing system in non-viable human zygotes to modify the gene that causes the hereditary blood disease beta-thalassemia.

"We found that CRISPR/Cas9 could effectively cleave the endogenous beta-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent," the authors wrote.

"Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing."

"Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing," they said.

The germline cells the scientists edited were tripronuclear zygotes, single cells that resulted from ooctyes fertilized by two sperm at the same time and thus carrying an extra set of chromosomes. Though they do not develop fully in vivo, tripronuclear zygotes may generate blastocysts in vitro, "providing an ideal model system" to examine efficiency and off-target effects of CRISPR/Cas9, the authors said.

The scientists directed the CRISPR/Cas9 editing system to the human beta-globin gene HBB, part of the beta-globin gene cluster and the gene that is mutated in beta-thalassemia, a blood disease that can be fatal, depending on the specific mutation. 

After testing three guide RNAs to target the gene, the scientists injected one of them along with Cas9 mRNA, GFP mRNA, and a single-stranded DNA oligonucleotide as a repair template.

Of the 86 zygotes injected, 71 survived; of those surviving, DNA from 54 was amplified with PCR; of those PCR-amplified zygotes 28 demonstrated that they were cleaved. Only four zygotes were edited with the single-stranded oligo as a repair template, while seven were repaired with the HBD gene, an endogenous homologous region, as a template.

"This high rate of repair using endogenous sequences presents obvious obstacles to gene therapy strategies using CRISPR/Cas9, as pseudogenes and paralogs may effectively compete with exogenous templates (or endogenous wild-type sequences) during [homology directed repair], leading to unwanted mutations," the authors said.

To examine off-target effects, the scientists examined the seven most likely off-target sites with a mismatch cleavage assay, and selected six cleaved embryos for whole-exome sequencing. They reported two notable sites within exons confirmed by the T7E1 assay.

The study comes less than a month after Science published an editorial on the topic of human germline engineering, co-authored by some of the leading scientists working with CRISPR/Cas9, including Jennifer Doudna of the University of California, Berkeley and George Church of Harvard Medical School. The editorial recommended that the research community "strongly discourage" attempts to engineer the human germline for clinical applications, a sentiment that is likely to be echoed given the results of the new study.

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