CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity.
CRISPR-STAT: an easy and reliable PCR-based method to evaluate target-specific sgRNA activity.
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CRISPR-stat：一种简单可靠的基于PCR的方法，用于评估目标特异性SGRNA活性。
DOI：
10.1093/nar/gkv802
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发表时间：
2015-12-15
影响因子：
14.9
通讯作者：
Sood R
中科院分区：
文献类型：
--
作者：
Carrington B;Varshney GK;Burgess SM;Sood R
CRISPR/Cas9 已成为一种多功能基因组工程工具，依赖于单引导 RNA (sgRNA) 和 Cas9 酶进行基因组编辑。简单、快速且经济的生成 sgRNA 的方法已在培养细胞、小鼠、斑马鱼和其他模型系统中成为常规的靶向诱变方法。预筛选 sgRNA 的靶标功效对于成功诱变和最大程度地减少活性较差靶标上的浪费畜牧业都是可取的。在这里，我们描述了一种简单、快速且经济高效的基于荧光聚合酶链式反应 (PCR) 的方法，即 CRISPR 体细胞组织活性测试 (CRISPR-STAT)，用于确定 sgRNA 的靶标特异性效率。作为原理证明，我们通过分析注射胚胎中的体细胞活性，使用斑马鱼中具有已知和不同水平种系传递效率的 28 种 sgRNA 验证了我们的方法。我们的数据显示注射胚胎的荧光 PCR 图谱与种系传播效率之间存在很强的正相关性。此外，该测定足够灵敏，可以评估多重基因靶向。这种方法很容易由具有毛细管测序仪的实验室实施。尽管我们使用 CRISPR/Cas9 和斑马鱼验证了该方法，但它可以应用于其他模型系统和其他基因组靶向核酸酶。
CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases.