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Single nucleotide editing without DNA cleavage using CRISPR/Cas9‐deaminase in the sea urchin embryo
Author(s) -
Shevidi Saba,
Uchida Alicia,
Schudrowitz Natalie,
Wessel Gary M.,
Yajima Mamiko
Publication year - 2017
Publication title -
developmental dynamics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.634
H-Index - 141
eISSN - 1097-0177
pISSN - 1058-8388
DOI - 10.1002/dvdy.24586
Subject(s) - crispr , biology , cas9 , genome editing , genetics , genome , gene , guide rna , dna , computational biology
Background: A single base pair mutation in the genome can result in many congenital disorders in humans. The recent gene editing approach using CRISPR/Cas9 has rapidly become a powerful tool to replicate or repair such mutations in the genome. These approaches rely on cleaving DNA, while presenting unexpected risks. Results: In this study, we demonstrate a modified CRISPR/Cas9 system fused to cytosine deaminase (Cas9‐DA), which induces a single nucleotide conversion in the genome. Cas9‐DA was introduced into sea urchin eggs with sgRNAs targeted for SpAlx1, SpDsh , or SpPks , each of which is critical for skeletogenesis, embryonic axis formation, or pigment formation, respectively. We found that both Cas9 and Cas9‐DA edit the genome, and cause predicted phenotypic changes at a similar efficiency. Cas9, however, resulted in significant deletions in the genome centered on the gRNA target sequence, whereas Cas9‐DA resulted in single or double nucleotide editing of C to T conversions within the gRNA target sequence. Conclusions: These results suggest that the Cas9‐DA approach may be useful for manipulating gene activity with decreased risks of genomic aberrations. Developmental Dynamics 246:1036–1046, 2017 . © 2017 Wiley Periodicals, Inc.