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Global detection of DNA repair outcomes induced by CRISPR-Cas9.

ABSTRACT: CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.

SUBMITTER: Liu M

PROVIDER: S-EPMC8421148 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Global detection of DNA repair outcomes induced by CRISPR-Cas9.

Liu Mengzhu M Zhang Weiwei W Xin Changchang C Yin Jianhang J Shang Yafang Y Ai Chen C Li Jiaxin J Meng Fei-Long FL Hu Jiazhi J

Nucleic acids research 20210901 15

CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to ...[more]

PMID: 34365511

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Project description:BackgroundAspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.ResultsHere we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13-20% of the transformants while 80-87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35-61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85-90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.ConclusionThe awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

Dataset Information

Global detection of DNA repair outcomes induced by CRISPR-Cas9.

Publications

Global detection of DNA repair outcomes induced by CRISPR-Cas9.

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