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posted by chromas on Monday July 16 2018, @08:21PM   Printer-friendly

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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  • (Score: 2) by Bot on Tuesday July 17 2018, @12:19PM

    by Bot (3902) on Tuesday July 17 2018, @12:19PM (#708284) Journal

    When you broke the error correction code of DNA with your crispr editing...

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