Unexpected: CRISPR gene editing causes heritable mutations in genome structure

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Unexpected: CRISPR gene editing causes heritable mutations in genome structure

CRISPR-Cas9 is a powerful tool for modifying microbial, animal and plant, and human genomes.

In healthcare, CRISPR-Cas9 gene editing offers unprecedented hope for curing major diseases such as genetic diseases, cancer, and even heart disease.

But all of this presupposes that the DNA is modified correctly without unexpected changes.

Off-targeting is a major concern when doing CRISPR gene editing, as it can disrupt other functional genes with serious consequences.

In addition, large structural mutations in the genome (including deletions, insertions, rearrangements, and even chromosomal fragmentation of large fragments) near the target site are also a worrying problem.

To date, many studies have been conducted on these potential risks of CRISPR gene editing at the cellular level, but their impact on individual organisms remains limited.

Recently, researchers from Uppsala University in Sweden published a research paper titled: CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations in Nature Communications , a sub-journal of Nature .

The study confirms that CRISPR-Cas9 gene editing causes unforeseen structural mutations in DNA that can be inherited by the next generation .

This study reminds us once again that CRISPR-Cas9 needs to be carefully and carefully validated before clinical application.

Unexpected: CRISPR gene editing causes heritable mutations in genome structure

In order to better understand the impact of unexpected CRISPR-Cas9 genome editing results on the individual level, especially genome structural variation, the research team gene-edited zebrafish zygotes by microinjection of RNP (gRNA+Cas9) .

The genomes of these edited first-generation zebrafish and their next generation were sequenced.

Unexpected: CRISPR gene editing causes heritable mutations in genome structure

Post-editing validation, usually performed using short-read or Sanger sequencing, these methods are able to detect small insertions or deletions, the most common result of CRISPR-Cas9 gene editing, but may not detect larger genomes Structural variation, while long-read nanopore sequencing can work better.

The research team used long-read nanopore sequencing to sequence the genomes of more than 1,100 zebrafish in two generations to verify the editing of CRISPR-Cas9 near the target site and other off-target sites, and found a large number of various types of Unexpected gene mutation.

Specifically, about 6% of the first generation of CRISPR-Cas9 gene-edited zebrafish have genomic structural variations (inserts or deletions ≥ 50 bp) , both at on-target and off-target sites.

In addition, the study found that these unforeseen genomic mutations in first-generation zebrafish were passed on to offspring, with 26 percent of second-generation zebrafish carrying off-target mutations and 9 percent carrying structural mutations .

Unexpected: CRISPR gene editing causes heritable mutations in genome structure

Another important finding of this study is that these first-generation zebrafish that have undergone CRISPR-Cas9 gene editing are chimeric, and the germ cells they produce are also chimeric , and they are in the single-cell stage of the fertilized egg The gene editing carried out should not theoretically be chimeric, which may be due to the continued gene editing in the subsequent division of the fertilized egg, or the different levels of DNA repair in the cells produced by the division, which led to this result.

Knowing that these unexpected mutations caused by CRISPR-Cas9 are heritable is important, the team said, because the mutations could have long-term effects on the growth of offspring.

However, it should be pointed out that this only occurs when gene editing is performed on embryos or germ cells.

Current CRISPR gene editing clinical trials are gene editing of specific tissues or organs, not involving embryos and germ cells.

CRISPR-Cas9 gene editing has great value in healthcare, but we need to minimize potential risks.

This study proposes a new strategy to validate gene editing results using long-read nanopore sequencing technology, an important step in reducing the risk of CRISPR-Cas9 clinical applications.

Reference :
https://www.nature.com/articles/s41467-022-28244-5

Unexpected: CRISPR gene editing causes heritable mutations in genome structure

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