Single nucleotide editing without DNA cleavage using CRISPR/Cas9‐deaminase in the sea urchin embryo

Abstract

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. Conclusion These results suggest that the Cas9-DA approach may be useful for manipulating gene activity with decreased risks of genomic aberrations.

A tumor suppressor Retinoblastoma1 is essential for embryonic development in the sea urchin

“…These results further suggest the specificity of Rb1‐MO as well as the functionality of Rb1‐GFP mRNA in the embryo. The genome editing technology (e.g., CRISPR/Cas9 system) was not used in this study as a knockdown approach because of the maternal load of Rb1 mRNA during early embryogenesis of the sea urchin . This alternative approach will be, however, important to test these Rb1‐knockdown phenotypes in the larval stage in the future.…”

Establishment of knockout adult sea urchins by using a CRISPR‐Cas9 system

“…The preceding study using S. purpuratus introduced a combination of three sgRNAs targeting the Pks1 gene and resulted in efficient mutagenesis and larval albino phenotype, but only a slight portion of embryos showed pigmentation (Oulhen & Wessel, 2016). A similar albino phenotype was reported using CRISPR-Cas9 and CRISPR-Cas9-deaminase by introduction of mixture of 16 sgRNAs targeting the Pks1 gene, but the efficiency was not so high (Shevidi, Uchida, Schudrowitz, Wessel, & Yajima, 2017). Whereas, in our study, we introduced single sgRNA, and the introduction of sgRNA#2 achieved 100% of mutagenesis efficiency and all injected embryos showed the complete loss of pigmentation.…”

Acceleration of cancer science with genome editing and related technologies

“…Note that deaminase catalyzes single‐strand DNA; therefore, deaminase fused with ZF or TALE was likely to show less activity compared to that fused with dCas9 or Cas9n . The applicability of base editing systems has been initially proven in yeasts and mammalian cells, followed by various organisms including plants, mice, sea urchins, and bacteria . The high specificity of the system was confirmed by genome‐wide assessment .…”