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takyon writes:

A new study has contradicted a finding of CRISPR-Cas9 off-target activity, instead finding that "unexpected mutations" were due to two closely related mice:

In May, a study claimed that the revolutionary CRISPR gene editing technique can cause thousands of unwanted and potentially dangerous mutations. The authors called for regulators to reassess the safety of the technique. But doubts were raised about these claims from the very beginning, not least because it was a tiny study involving just three mice. Some critics have called for the paper to be withdrawn. Now a paper posted online on 5 July has proposed a simple and more plausible explanation for the controversial results. If it's right, the authors of the original study were wrong.

[...] When Stephen Tsang of Columbia University Medical Center and colleagues compared the entire genomes of two CRISPR-edited mice with a third one, they found thousands of shared mutations in the two edited mice. Tsang and co attributed to these mutations to CRISPR, and issued a widely-covered press release that suggested CRISPR is far riskier than dozens of other studies had suggested.

[...] But there is a much simpler explanation, says the latest study: the two CRISPR-edited mice just happened to be more closely related and thus shared more mutations. [...] "I agree the two mice are indeed more likely to be closely related," says geneticist Gaetan Burgio of the Australian National University, one of the many critics of the original paper. He says its publication in a prominent journal was a failure of peer review.

"Unexpected mutations after CRISPR-Cas9 editing in vivo" are most likely pre-existing sequence variants and not nuclease-induced mutations (open, DOI: 10.1101/159707) (DX)

The disputed press release from May, and the study it was based on:

Unexpected mutations after CRISPR–Cas9 editing in vivo (DOI: 10.1038/nmeth.4293) (DX)

Original Submission

takyon writes:

CRISPR 'gone wild' has made stocks swoon, but studies show how to limit off-target editing

The fear that CRISPR-based genome repair for preventing or treating genetic diseases will be derailed by "editing gone wild" has begun to abate, scientists who are developing the technique say. Although there are still concerns that CRISPR might run amok inside patients and cause dangerous DNA changes, recent advances suggest that the risk is not as high as earlier research suggested and that clever molecular engineering can minimize it.

"Progress is being made at a pretty stunning rate," said biochemist David Liu, of Harvard University and the Broad Institute, who has developed several versions of CRISPR. A parade of new discoveries, he said, "suggests that it's possible to use these genome-editing tools and not make unintended edits."

Upgrade makes genome editor CRISPR more muscular, precise

Many groups are trying to do better, and now, a team led by chemist David Liu at Harvard University has engineered a version of CRISPR that potentially is both more dexterous and more precise.

[...] The new work, reported online in the 28 February issue of Nature, modifies the Cas9 enzyme, creating at least four times as many potential docking sites. In theory, this could allow researchers to, say, cripple or replace many parts of genes associated with human disease that CRISPR currently cannot touch.

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity (DOI: 10.1038/nature26155) (DX)

Preprints:

• NmeCas9 is an intrinsically high-fidelity genome editing platform (open, DOI: 10.1101/172650) (DX)
• No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice (open, DOI: 10.1101/263129) (DX)
• In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations (open, DOI: 10.1101/272724) (DX)
• High-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations (open, DOI: 10.1101/273938) (DX)

Original Submission

takyon writes:

A disputed paper that raised questions about the safety of CRISPR has been retracted:

A scientific paper that purported to lay bare serious flaws in the gene-editing tool known as CRISPR and briefly tanked shares of genome-editing companies has been retracted by its publisher.

The paper [DOI: 10.1038/nmeth.4293] [DX], published last year in Nature Methods, claimed that CRISPR wreaked havoc on the genome, causing hundreds of unintended mutations in mice — and that the algorithms typically used to detect these changes were routinely missing them.

[...] Two months after publication of the paper, Nature Methods published an "an expression of concern" about the paper in July. The retraction notice, appended Friday, goes further, saying the authors did not use mice that had been bred in their own laboratory — so they could not know if any genetic mutations were the result of their intervention with CRISPR editing, or if it stemmed from variations in the mice's own genomes.

Nature Methods editorial discussing the retraction: CRISPR off-targets: a reassessment (open, DOI: 10.1038/nmeth.4664) (DX)

Previously: CRISPR Safer than Thought; Misleading Study Found Shared Mutations in Closely Related Mice

Original Submission

An Anonymous Coward writes:

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

Original Submission

An Anonymous Coward writes:

Biologists who last year made a blockbuster — but controversial — claim that they had fixed a disease-causing mutation in human embryos using CRISPR gene editing have released fresh evidence in support of their work. Critics argued that the researchers’ evidence wasn’t persuasive and that the feat did not seem biologically plausible, intensifying the existing controversy surrounding the use of gene editing in human embryos to prevent diseases.

Now, a year after the study was published in Nature¹, its authors, led by reproductive biologist Shoukhrat Mitalipov at the Oregon Health & Science University in Portland, have backed up their claims with new data², published on 8 August alongside a pair of letters critiquing the original results.³,⁴.

Whatever happens next, it is likely that questions about whether it is possible to repair mutations in human embryos will persist until other researchers can repeat the feat — no easy task in a field that is strictly regulated, and even illegal in some countries.

Did CRISPR really fix a genetic mutation in these human embryos?

Original Submission