Lulu and Nana, humanity’s first gene-edited babies, both have some CRISPR edits in the CCR5 gene, as He Jiankui (JK) attempted. His goal was to confer HIV resistance.
Unfortunately, the editing turned out quite badly, as shown by *his own data* in *his own manuscript*. (thread)
Unfortunately, the editing turned out quite badly, as shown by *his own data* in *his own manuscript*. (thread)
2/
Here’s the first piece of evidence.
This is a Sanger sequencing chromatogram of a PCR amplicon of the CCR5 target site, using DNA from cells extracted from Lulu’s embryo prior to pregnancy.
In the manuscript, JK claims there’s a wild-type allele and a 15bp deletion allele.
Here’s the first piece of evidence.
This is a Sanger sequencing chromatogram of a PCR amplicon of the CCR5 target site, using DNA from cells extracted from Lulu’s embryo prior to pregnancy.
In the manuscript, JK claims there’s a wild-type allele and a 15bp deletion allele.
3/
Strangely, in his single public talk about Lulu and Nana in Hong Kong, JK showed the same exact data—but he mislabeled Lulu’s embryo’s edits, mixing them up with Nana’s.
JK’s manuscript (and the chromatograms) makes it clear:
Lulu = wild-type, -15bp
Nana = +1bp, -4bp
Strangely, in his single public talk about Lulu and Nana in Hong Kong, JK showed the same exact data—but he mislabeled Lulu’s embryo’s edits, mixing them up with Nana’s.
JK’s manuscript (and the chromatograms) makes it clear:
Lulu = wild-type, -15bp
Nana = +1bp, -4bp
4/
(This is puzzling. Surely JK understood that his talk at the Second International Summit on Human Genome Editing in Hong Kong would be the most important talk of his career to date. How did he mix up such critical information when making his slide?)
(This is puzzling. Surely JK understood that his talk at the Second International Summit on Human Genome Editing in Hong Kong would be the most important talk of his career to date. How did he mix up such critical information when making his slide?)
5/
If you know what to look for, you can immediately tell there’s something wrong with this picture.
If there are only 2 alleles—one wild-type, one with a deletion—there should be at most 2 peaks in any 1 position of the chromatogram.
Do you see any positions with >2 peaks?
If you know what to look for, you can immediately tell there’s something wrong with this picture.
If there are only 2 alleles—one wild-type, one with a deletion—there should be at most 2 peaks in any 1 position of the chromatogram.
Do you see any positions with >2 peaks?
6/
There are many positions with 3 peaks. This means there are 3 different alleles in play.
Normal cells have a maximum of 2 alleles at 1 genomic position (chromosome pair).
The best (only?) explanation: different cells in Lulu’s embryo had different CRISPR edits of CCR5.
There are many positions with 3 peaks. This means there are 3 different alleles in play.
Normal cells have a maximum of 2 alleles at 1 genomic position (chromosome pair).
The best (only?) explanation: different cells in Lulu’s embryo had different CRISPR edits of CCR5.
7/
Another problem: the peaks for the wild-type allele are so much taller than the other 2 alleles. The wild-type allele is very over-represented in the DNA from Lulu’s embryo.
This suggests that some cells sampled from Lulu’s embryo had 2 wild-type alleles—no edits.
Another problem: the peaks for the wild-type allele are so much taller than the other 2 alleles. The wild-type allele is very over-represented in the DNA from Lulu’s embryo.
This suggests that some cells sampled from Lulu’s embryo had 2 wild-type alleles—no edits.
8/
Conclusion: Lulu’s embryo was mosaic—a patchwork of cells with different edits, possibly some with no edits.
Is the same true of Lulu’s body?
I’ll explain tomorrow why mosaicism in the context of human germline editing could have serious, adverse health consequences.
Conclusion: Lulu’s embryo was mosaic—a patchwork of cells with different edits, possibly some with no edits.
Is the same true of Lulu’s body?
I’ll explain tomorrow why mosaicism in the context of human germline editing could have serious, adverse health consequences.
9/
Wondering if the chromatogram even shows the claimed -15bp allele?
With a little patience, you can pick it out of the peaks.
What’s the 3rd allele, then?
I can’t figure it out—as best I can tell, it’s a large insertion of random DNA. If you figure it out, I’d love to know.
Wondering if the chromatogram even shows the claimed -15bp allele?
With a little patience, you can pick it out of the peaks.
What’s the 3rd allele, then?
I can’t figure it out—as best I can tell, it’s a large insertion of random DNA. If you figure it out, I’d love to know.
10/
On to Nana’s embryo. Here’s the chromatogram. In the manuscript, JK claims there are two alleles: +1bp (insertion), -4bp (deletion). No wild-type allele.
Do you see any positions with >2 peaks?
On to Nana’s embryo. Here’s the chromatogram. In the manuscript, JK claims there are two alleles: +1bp (insertion), -4bp (deletion). No wild-type allele.
Do you see any positions with >2 peaks?
11/
Oh yes. Nana’s embryo was mosaic too.
I can see the +1bp and -4bp alleles marching through the chromatogram, but I can’t figure out the 3rd allele. Can you?
Oh yes. Nana’s embryo was mosaic too.
I can see the +1bp and -4bp alleles marching through the chromatogram, but I can’t figure out the 3rd allele. Can you?
12/
JK showed the chromatograms in his Summit talk. Did he not understand his own data, either before or after the pregnancy?
A savvy viewer could have picked up on the problems (if JK hadn’t rushed through his slides). @GaetanBurgio was one of the first to note the mosaicism.
JK showed the chromatograms in his Summit talk. Did he not understand his own data, either before or after the pregnancy?
A savvy viewer could have picked up on the problems (if JK hadn’t rushed through his slides). @GaetanBurgio was one of the first to note the mosaicism.
13/
It gets worse.
Here are the chromatograms from umbilical cord and placenta samples, after Lulu was born.
Do you see any problems?
It gets worse.
Here are the chromatograms from umbilical cord and placenta samples, after Lulu was born.
Do you see any problems?
14/
Lulu’s umbilical cord samples have 2 co-equal alleles, corresponding to wild-type and -15bp.
The placenta has 3 alleles. Mosaic! The 3rd allele looks different from the 3rd allele seen in Lulu’s embryo.
Conclusion: it’s likely that Lulu and Nana are mosaic in their bodies.
Lulu’s umbilical cord samples have 2 co-equal alleles, corresponding to wild-type and -15bp.
The placenta has 3 alleles. Mosaic! The 3rd allele looks different from the 3rd allele seen in Lulu’s embryo.
Conclusion: it’s likely that Lulu and Nana are mosaic in their bodies.
15/
We don’t know for sure, because JK’s team never had the chance to evaluate the babies themselves.
Maybe that’s for the best.
We don’t know for sure, because JK’s team never had the chance to evaluate the babies themselves.
Maybe that’s for the best.
16/
If you want tips on reading chromatograms like these, sign up to access a recording of a CRISPR workshop by yours truly at the @GeneticsSociety 2019 Annual Meeting—membership not required:
pathlms.com/ashg/events/17…
If you want tips on reading chromatograms like these, sign up to access a recording of a CRISPR workshop by yours truly at the @GeneticsSociety 2019 Annual Meeting—membership not required:
pathlms.com/ashg/events/17…
17/
Tomorrow, I’ll tackle the other major safety concern with germline editing—off-target mutations.
Spoiler alert: there were off-target mutations in JK’s embryos.
For more details/explanation for today’s thread, refer to #TheCRISPRGeneration at bit.ly/TheCRISPRGener…
Tomorrow, I’ll tackle the other major safety concern with germline editing—off-target mutations.
Spoiler alert: there were off-target mutations in JK’s embryos.
For more details/explanation for today’s thread, refer to #TheCRISPRGeneration at bit.ly/TheCRISPRGener…
