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Researchers develop method to detect unintended on-target effects in CRISPR genome editing by DNA donors carrying diagnostic substitutions

NOMIS Awardee and Nobel laureate Svante Pääbo and colleagues have described a method to detect unintended on-target effects in CRISPR genome editing by DNA donors carrying diagnostic substitutions. Their findings were published in Nucleic Acid Research.

Svante Pääbo

The genome editing approach presented allows detection of copy number variants of the target site as well as loss of heterozygosity when the target site is sequenced, thus greatly reducing time and cost of genome editing experiments. In addition, it facilitates the identification of non-clonal cellular colonies and the generation of control cell lines that have demonstratively undergone editing but carry the wild-type state at the position of interest. The authors analyzed more than 850 human embryonic stem cell clones edited with SAFE donors and detected all copy number changes and almost all clones with gene conversion.

Abstract

CRISPR nucleases can introduce double-stranded DNA breaks in genomes at positions specified by guide RNAs. When repaired by the cell, this may result in the introduction of insertions and deletions or nucleotide substitutions provided by exogenous DNA donors. However, cellular repair can also result in unintended on-target effects, primarily larger deletions and loss of heterozygosity due to gene conversion. Here we present a strategy that allows easy and reliable detection of unintended on-target effects as well as the generation of control cells that carry wild-type alleles but have demonstratively undergone genome editing at the target site. Our ‘sequence-ascertained favorable editing’ (SAFE) donor approach relies on the use of DNA donor mixtures containing the desired nucleotide substitutions or the wild-type alleles together with combinations of additional ‘diagnostic’ substitutions unlikely to have any effects. Sequencing of the target sites then results in that two different sequences are seen when both chromosomes are edited with ‘SAFE’ donors containing different sets of substitutions, while a single sequence indicates unintended effects such as deletions or gene conversion. We analyzed more than 850 human embryonic stem cell clones edited with ‘SAFE’ donors and detect all copy number changes and almost all clones with gene conversion.

Read the Nucleic Acid Research publication: Detection of unintended on-target effects in CRISPR genome editing by DNA donors carrying diagnostic substitutions

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NOMIS Researchers

Director, Department of Genetics
Max Planck Institute for Evolutionary Anthropology
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