Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
CRISPR J. 2021 Dec;4(6):836-845. doi: 10.1089/crispr.2021.0062. Epub 2021 Nov 23.
Cas9 and a guide RNA (gRNA) function to target specific genomic loci for generation of a double-stranded break. Catalytic dead versions of Cas9 (dCas9) no longer cause double-stranded breaks and instead can serve as molecular scaffolds to target additional enzymatic proteins to specific genomic loci. To generate mutations in selected genomic residues, dCas9 can be used for genomic base editing by fusing a cytidine deaminase (CD) to induce C > T (or G>A) mutations at targeted sites. In this study, we test base editing in by expressing a transgenic base editor (based on the mammalian BE2) that consists of a fusion protein of CD, dCas9, and uracil glycosylase inhibitor. We utilized transgenic lines expressing gRNAs along with pan-tissue expression of the base editor () and found high rates of base editing at multiple targeted loci in the 20 bp target sequence. Highest rates of conversion of C > T were found in positions 3-9 of the gRNA-targeted site, with conversion reaching ∼100% of targeted DNA in somatic tissues. Surprisingly, the simultaneous use of two gRNAs targeting a genomic region spaced ∼50 bp apart led to mutations between the two gRNA targets, implicating a method to broaden the available sites accessible to targeting. These results indicate base editing is efficient in , and could be used to induce point mutations at select loci.
Cas9 和向导 RNA（gRNA）可靶向特定基因组位点以产生双链断裂。无催化活性的 Cas9（dCas9）不再引起双链断裂，而是可以作为分子支架将额外的酶蛋白靶向特定的基因组位点。为了在选定的基因组残基中产生突变，可以通过将胞嘧啶脱氨酶（CD）融合到 dCas9 上来进行基因组碱基编辑，从而在靶向位点诱导 C>T（或 G>A）突变。在这项研究中，我们通过表达一种基于哺乳动物 BE2 的转基因碱基编辑器（由 CD、dCas9 和尿嘧啶糖基化酶抑制剂的融合蛋白组成）来测试碱基编辑在 中的作用。我们利用表达 gRNA 的转基因系和 的泛组织表达（），并在 20bp 靶序列的多个靶向位点发现了高频率的碱基编辑。在 gRNA 靶向位点的 3-9 位发现了最高的 C>T 转化率，在体细胞组织中转化达到靶向 DNA 的约 100%。令人惊讶的是，同时使用两个靶向基因组区域相隔约 50bp 的 gRNA 导致两个 gRNA 靶之间的突变，这暗示了一种拓宽靶向可用位点的方法。这些结果表明碱基编辑在 中是有效的，并且可以用于在选定的位点诱导点突变。
