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ECB-ART-45725
Dev Dyn 2017 Dec 01;24612:1036-1046. doi: 10.1002/dvdy.24586.
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Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo.

Shevidi S , Uchida A , Schudrowitz N , Wessel GM , Yajima M .


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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.

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Genes referenced: LOC100887844 LOC105447179 stk36
gRNAs referenced: LOC584189 gRNA1 LOC584189 gRNA2 LOC588806 gRNA10 LOC588806 gRNA11 LOC588806 gRNA12 LOC588806 gRNA13 LOC588806 gRNA14 LOC588806 gRNA15 LOC588806 gRNA16 LOC588806 gRNA17 LOC588806 gRNA18 LOC588806 gRNA19 LOC588806 gRNA4 LOC588806 gRNA5 LOC588806 gRNA6 LOC588806 gRNA7 LOC588806 gRNA8 LOC588806 gRNA9 alx1 gRNA1 alx1 gRNA2

References [+] :
Barrangou, CRISPR provides acquired resistance against viruses in prokaryotes. 2007, Pubmed