Abstract
Karyotype alterations have emerged as on-target complications from CRISPR-Cas9 genome editing. However, the events that lead to these karyotypic changes in embryos after Cas9-treatment remain unknown. Here, using imaging and single-cell genome sequencing of 8-cell stage embryos, we track both spontaneous and Cas9-induced karyotype aberrations through the first three divisions of embryonic development. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development.
Highlights
Karyotype alterations have emerged as on-target complications from CRISPR-Cas[9] genome editing
We recently showed that Cas9-induced double strand breaks (DSB) can generate micronuclei and chromosome bridges in human cell lines and primary blood stem and progenitor cells[6]
A gRNA targeting the second exon of Pou5f1 was selected because it has been extensively characterized in mouse embryo editing experiments[2,13]
Summary
Karyotype alterations have emerged as on-target complications from CRISPR-Cas[9] genome editing. We observe the generation of abnormal structures of the nucleus that arise as a consequence of errors in mitosis, including micronuclei and chromosome bridges, and determine their contribution to common karyotype aberrations including whole chromosome loss that has been recently reported after editing in embryos. Together, these data demonstrate that Cas9-mediated germline genome editing can lead to unwanted on-target side effects, including major chromosome structural alterations that can be propagated over several divisions of embryonic development. We show that CRISPR-Cas[9] DSBs lead to similar mitotic errors in the mouse pre-implantation embryo and elucidate how CRISPR-Cas[9] can lead to whole chromosome loss
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