As widely reported last week, a group in China has published results of CRISPR editing experiments in human triponuclear embryos (Liang et al, Protein & Cell 2015). The news blurb in Nature is worth a read to get the context of the paper, which follows on the heels of a previous statement published in Science by leaders in the CRISPR field and others, in which they discourage CRISPR experiments in human embryos at this time pending further discussion of the implications of such research.
- Efficiency of HDR was low.
- Edited embryos were mosaic.
- Off-target mutations were evident.
- A separate, highly homologous gene (HBD) could serve as donor template for repair, thus introducing sequences inadvertently from the other gene into the target gene.
|UCSC screen grab; OT is black bar, indel variant is long red bar.|
|UCSC screen grab; OT in black, indel variant in blue.|
In summary, only 1 of the OTs holds up to scrutiny. Importantly, neither OT found by exome sequencing holds up. This flips their conclusion on its head: “Our whole-exome sequencing result only covered a fraction of the genome and likely underestimated the off- target effects in human 3PN zygotes.”. While it’s certainly possible that some more OTs could be found by whole genome sequencing, the exome data was essentially totally negative. Note that they chose a CRISPR to work with because it had a low apparent OT rate in 293T cells. In retrospect, it was just by luck that G1-OT5 has a negative T7 assay in 293T cells. It could have been heterozygous, but it's apparently not.
From Time, quoting Carl Zimmer from National Geographic: “The experiment “came out poorly,” Zimmer says; in some cases, DNA was placed in the wrong spot and “off-target” mutations were discovered in the DNA.”
From the Washington Post: “And in some of the embryos, the gene editing caused unintended mutations in other genes.”
From USA today (emphasis is mine): “The team also found that the complex used in the procedure was also acting on other parts of the genome, leading to other bits of it mutating. That happened much more than in previous experiments on adult human cells and animal embryos — and could happen yet more if the whole genome were used, as it would be if the embryo were to be implanted.”
(OK, note from this last article the specific comparison to the very observations that I have blogged about in more detail than most people probably ever wanted to hear about...My point is that, due to the technical problems in the Liang paper, I don’t think we can yet say the off-target effects were “much more than in previous experiments on adult human cells and animal embryos”. )
One final note - my analysis of this paper should not be interpreted to mean that I fully endorse CRISPR experimentation or applications in human embryos. I also applaud the authors' cautionary tone that the incomplete efficiency of CRISPR editing in humans is a problem that any therapeutic applications need to address.
Whew, this was the longest post yet.