While CRISPR interference (CRISPRi) systems have been widely implemented in pooled lentiviral screening, there has been limited use with synthetic guide RNAs for the complex phenotypic readouts enabled by experiments in arrayed format. Here we describe a novel deactivated Cas9 fusion protein, dCas9-SALL1-SDS3, which produces greater target gene repression than first or second generation CRISPRi systems when used with chemically modified synthetic single guide RNAs (sgRNAs), while exhibiting high target specificity. We show that dCas9-SALL1-SDS3 interacts with key members of the histone deacetylase and Swi-independent three complexes, which are the endogenous functional effectors of SALL1 and SDS3. Synthetic sgRNAs can also be used with in vitro-transcribed dCas9-SALL1-SDS3 mRNA for short-term delivery into primary cells, including human induced pluripotent stem cells and primary T cells. Finally, we used dCas9-SALL1-SDS3 for functional gene characterization of DNA damage host factors, orthogonally to small interfering RNA, demonstrating the ability of the system to be used in arrayed-format screening.

中文翻译：

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一种新型 CRISPR 干扰效应器，可通过合成向导 RNA 进行功能基因表征

虽然 CRISPR 干扰 (CRISPRi) 系统已广泛应用于混合慢病毒筛选中，但合成指导 RNA 在阵列格式实验中实现复杂表型读数方面的应用有限。在这里，我们描述了一种新型失活 Cas9 融合蛋白 dCas9-SALL1-SDS3，当与化学修饰的合成单向导 RNA (sgRNA) 一起使用时，它比第一代或第二代 CRISPRi 系统产生更强的靶基因抑制，同时表现出高靶标特异性。我们发现 dCas9-SALL1-SDS3 与组蛋白脱乙酰酶和 Swi 独立的三个复合物的关键成员相互作用，这些复合物是 SALL1 和 SDS3 的内源功能效应子。合成的 sgRNA 还可与体外转录的 dCas9-SALL1-SDS3 mRNA 一起使用，用于短期递送至原代细胞，包括人诱导多能干细胞和原代 T 细胞。最后，我们使用 dCas9-SALL1-SDS3 进行 DNA 损伤宿主因子的功能基因表征，与小干扰 RNA 正交，证明了该系统用于阵列格式筛选的能力。