Shevidi Saba, Uchida Alicia, Schudrowitz Natalie, Wessel Gary M, Yajima Mamiko
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode, Island.
Dev Dyn. 2017 Dec;246(12):1036-1046. doi: 10.1002/dvdy.24586. Epub 2017 Oct 13.
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.
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.
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.
基因组中的单个碱基对突变可导致人类多种先天性疾病。最近使用CRISPR/Cas9的基因编辑方法迅速成为在基因组中复制或修复此类突变的强大工具。这些方法依赖于切割DNA，但存在意想不到的风险。
在本研究中，我们展示了一种与胞嘧啶脱氨酶融合的改良CRISPR/Cas9系统（Cas9-DA），它可在基因组中诱导单核苷酸转换。将Cas9-DA与靶向SpAlx1、SpDsh或SpPks的sgRNAs一起导入海胆卵，它们分别对骨骼发生、胚胎轴形成或色素形成至关重要。我们发现Cas9和Cas9-DA都能编辑基因组，并以相似的效率引起预测的表型变化。然而，Cas9导致以gRNA靶序列为中心的基因组显著缺失，而Cas9-DA导致gRNA靶序列内C到T转换的单核苷酸或双核苷酸编辑。
这些结果表明，Cas9-DA方法可能有助于在降低基因组畸变风险的情况下操纵基因活性。《发育动力学》246:1036 - 1046，2017年。©2017威利期刊公司。
