Breaking News: Germline Editing
with CRISPR As this article was set to go to press, a team of Chinese research- ers published the first report of human germline editing (1). Led by Junjiu Huang at Sun Yat-sen University in Guangzhou,
China, the team used CRISPR/Cas9 to edit the human beta-glo- bin (HBB) gene in tripronuclear embryos (fertilized by two sperm) produced by in vitro fertilization. These embryos, the authors say, could never be viable.
The team injected 86 tripronuclear embryos with mRNAs en-
coding Cas9 and GFP, a guide RNA targeting HBB, and a sin- gle-stranded oligonucleotide encoding six “silent” mutations as a repair template. Forty-eight hours later, 71 embryos remained viable, of which 59 were GFP-positive. Analysis of 54 of those embryos revealed the HBB gene had been targeted success- fully in 28 of them, but only 4 were modified through homology- dependent repair using the supplied template, and those yielded genetic mosaics. Seven had used an endogenous gene related to HBB as the template, while 17 were repaired using non-ho- mologous end-joining. Exome sequencing analysis of six suc- cessfully targeted embryos identified off-target mutations in the transthyretin and C1QC coding sequences.
“[O]ur data underscore the need to more comprehensively un-
derstand the mechanisms of CRISPR/Cas9-mediated genome editing in human cells, and support the notion that clinical ap- plications of the CRISPR/Cas9 system may be premature at this stage,” the authors conclude.
In a statement emailed to BioTechniques, Jennifer Doudna,
writes, “Although it has attracted a lot of attention, the study sim- ply underscores the point that the technology is not ready for clin- ical application in the human germline. And that type of use of the technology needs to be on hold pending a broader societal dis- cussion of the scientific and ethical issues surrounding such use.”
From a technical standpoint, George Church says in an email
that his group has published methods to minimize off-target ef- fects, which Huang’s group cited in their article but did not em- ploy. The authors, he says, argue that off-target effects seem to be less-prevalent in embryonic cells than cancer cells, and for that reason, this kind of experiment could help more accurately assess safety and efficacy without endangering clinically viable embryos. “While it would be nice if they had tried the best tech- nology, the results are still quite relevant to the discussion.”
But he also notes that this study focused on an application for
which germline editing may be ideally suited. “[T]he HBB gene is representative of many diseases in which both parents are homo- zygous for deleterious alleles … and hence other (non-genome editing) methods would be considered inapplicable (like IVF-PGD [in vitro fertilization-preimplantation genetic diagnosis]) or possibly less safe and/or less effective (somatic gene therapy with mosaic off-target).” --J.P.
1. Liang, P., et al. 2015. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes,” Protein Cell. doi:10.1007/s13238-015-0153-5 Apr 18. [Epub ahead of print].
Vol. 58 | No. 5 | 2015
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