CRISPR–Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR–Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.
[...]
We show that extensive on-target genomic damage is a common outcome at all loci and in all cell lines tested. Moreover, the genetic consequences observed are not limited to the target locus, as events such as loss-of-heterozygosity will uncover recessive alleles, whereas translocations, inversions and deletions will elicit long-range transcriptional consequences. Given that a target locus would presumably be transcriptionally active, mutations that juxtapose this to one of the hundreds of cancer-driver genes may initiate neoplasia. In the clinical context of editing many billions of cells, the multitude of different mutations generated makes it likely that one or more edited cells in each protocol would be endowed with an important pathogenic lesion. Such lesions may constitute a first carcinogenic 'hit' in stem cells and progenitors, which have a long replicative lifespan and may become neoplastic with time. Such a circumstance would be similar to the activation of LMO2 by pro-viral insertion in some of the early gene-therapy trials, which caused cancer in these patients30. Results reported here also illustrate a need to thoroughly examine the genome when editing is conducted ex vivo. As genetic damage is frequent, extensive and undetectable by the short-range PCR assays that are commonly used, comprehensive genomic analysis is warranted to identify cells with normal genomes before patient administration.
https://www.nature.com/articles/nbt.4192
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One of the barriers to using CRISPR-Cas9 gene editing in the clinic is the possibility that the enzyme will clip DNA in the wrong spot. In a study published in Nature [DOI: 10.1038/s41586-018-0500-9] [DX] today (September 12), researchers describe a strategy to predict these off-target mutations throughout the genome and show in mice that a carefully designed guide RNA strand does not produce any detectable slip-ups.
The study confirms that "you'd better make sure that you've got a really accurate guide RNA," says Janet Rossant, a developmental biologist at the University of Toronto and the Hospital for Sick Children who did not participate in the work. "This [method] is a better way of testing for how specific that guide RNA will be before you go into animal models and, of course, into humans," she adds.
According to coauthor Marcello Maresca, a biologist at AstraZeneca in Sweden, one long-term goal of his company is to be able to use therapeutic gene editing to address a number of human diseases. "However, realizing the potential of CRISPR medicines requires the development of methods to enable the efficient modification of the target gene with no effects elsewhere in the genome," he writes in an email to The Scientist.
VIVO = "verification of in vivo off-targets".
Related: CRISPR Safer than Thought; Misleading Study Found Shared Mutations in Closely Related Mice
CRISPR Becomes More Precise
Paper That Found CRISPR "Off-Target Effects" Retracted
Repair of Double-Strand Breaks Induced by CRISPR Leads to Large Deletions and Complex Rearrangements
Did CRISPR Really Fix a Genetic Mutation in These Human Embryos?
(Score: 5, Funny) by Azuma Hazuki on Monday July 16 2018, @08:27PM (11 children)
Just throw a little frog DNA in there to patch up the missing bits and we'll have this theme park up and running in no time!
I am "that girl" your mother warned you about...
(Score: 2) by takyon on Monday July 16 2018, @08:30PM (9 children)
This has probably been made obsolete by previous studies anyway.
T Cells Instead of Viruses Used for CRISPR Gene Editing [soylentnews.org]
MAGESTIC: A More Efficient and Accurate Version of CRISPR [soylentnews.org]
[SIG] 10/28/2017: Soylent Upgrade v14 [soylentnews.org]
(Score: 0) by Anonymous Coward on Monday July 16 2018, @08:41PM (6 children)
Did they use "short-range PCR assays" or do "comprehensive genomic analysis" to check for damage in those other studies?
(Score: 3, Interesting) by sbgen on Monday July 16 2018, @10:31PM (5 children)
"Comprehensive genomic analysis" is not done that often. Generally the region that is being edited and some other regions that algorithms predict to be similar to this region are sequenced to confirm target/off-target events. Such an effort will miss rearrangements.
Warning: Not a computer expert, but got to use it. Yes, my kind does exist.
(Score: 3, Interesting) by Immerman on Tuesday July 17 2018, @02:02AM (4 children)
I've now heard of only two experiments that have done comprehensive analysis of Cas9 CRISPR results - and both found serious unintended consequences.
Not that that's necessarily a problem in many contexts - if you're creating a new, modified organism, and can simply discard all but a few healthy organisms that your work results in, then no big deal.
If you're trying to perform therapeutic gene editing on an individual; however, this does not bode well.
It also makes me even more leery of gene drives - anything that boosts the mutation rate of its host species will boost that species rate of evolution as well, provided the species can survive the increased mutation rate. For something like the proposed gene drive that makes mosquitoes immune to malaria - that might work out wonderfully in the short term - but it would also permanently boost the evolutionary speed of that species, along with every species it's able to interbreed with (theory notwithstanding, interspecies breeding does occasionally result in fertile offspring who are capable of breeding with one or both parent species)
(Score: 0) by Anonymous Coward on Tuesday July 17 2018, @02:08AM
What was the earlier one?
(Score: 2) by sbgen on Tuesday July 17 2018, @02:23AM (2 children)
I am assuming you are referring to the Nature Methods' paper from last year that claimed unwanted editing by CRISPR. That has been refuted with data by multiple groups, and subsequently has been retracted. The current paper will go through the wringer too - there are billions in patents riding on the technology :-((
I share your concern about the problem with using this technology for therapeutic purpose. I wish hyping would slow a bit and work would progress.
Warning: Not a computer expert, but got to use it. Yes, my kind does exist.
(Score: 2) by Immerman on Tuesday July 17 2018, @02:59AM (1 child)
Quite possibly, I don't recall the details.
The fact that, at this point, *every* thorough analysis has found serious problems makes me inclined to err on the side of caution. Maybe both the studies are seriously flawed - but you won't get me anywhere near a therapeutic treatment based on them until at least one independent thorough analysis shows a conclusive lack of problems, and unless my life is in immediate danger I'll wait for several such studies with positive findings.
Of course when it comes to medical care I have that option. With gene drives already being released into the wild I have no such option - that gene drive will propagate in the wild until the carrier population goes extinct - it's not possible to delete a gene drive, at best you can replace it with a new one. And we'll all have to deal with the fallout from that, potentially for as long as our species exists.
(Score: 0) by Anonymous Coward on Tuesday July 17 2018, @07:34AM
Until the alternative is a certain and painful death in a rather short while. Such as is the prognosis for most genetic diseases.
(Score: 4, Informative) by sbgen on Monday July 16 2018, @10:27PM (1 child)
Sorry, those two papers do not address the findings of the present paper. They do advance the editing efficiency of the CRISPR system via double strand breaks caused by CRISPR enzyme. Therefore, the problem of rearrangements at double strand break still remains. The current paper provides data about the latter. The degree to which the rearrangements happen and their impact need to be studied a lot more in lots of clinically relevant cells and cell lines.
Warning: Not a computer expert, but got to use it. Yes, my kind does exist.
(Score: 0) by Anonymous Coward on Tuesday July 17 2018, @07:28AM
How the mutations at the site targeted by CRISPR, compare to mutations at the same site after affecting cells by CRISPR targeted to a different site, and after CRISPR "targeted" to a nonexistent sequence?
(Score: 2) by Snow on Monday July 16 2018, @08:57PM
Whatever... you just want a theme park only filled with female specimens.
(Score: 4, Funny) by MostCynical on Monday July 16 2018, @09:05PM (1 child)
thanks for curing my genetic disease.
Now, about this second head..
"I guess once you start doubting, there's no end to it." -Batou, Ghost in the Shell: Stand Alone Complex
(Score: 3, Touché) by choose another one on Tuesday July 17 2018, @08:56AM
> Now, about this second head..
We'll prescribe a bird cage and cloth to go over it, just say you're a pirate and your parrot is asleep, you'll be fine.
How's the extra arm working out?
(Score: 2) by Bot on Tuesday July 17 2018, @12:19PM
When you broke the error correction code of DNA with your crispr editing...
Account abandoned.