The CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

中文翻译：

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通过TRAP-seq对细胞中CRISPR编辑进行大规模并行定量分析，可以更好地设计Cas9，ABE，CBE gRNA，从而实现高效率和准确性

CRISPR RNA引导的核酸内切酶Cas9和Cas9衍生的腺嘌呤/胞嘧啶碱基编辑器（ABE / CBE）已用于研究和治疗应用中。但是，由于不同靶位点之间效率的巨大差异，阻碍了该基因编辑工具箱的广泛使用。在这里，我们介绍了TRAP-seq，这是一种通用且可扩展的方法，其中CRISPR gRNA表达盒和相应的替代位点被Targeted Reporter锚定位置测序在细胞中捕获。TRAP-seq可以忠实地概述引入相应内源基因组位点的CRISPR基因编辑结果，最重要的是，可以在细胞中大规模并行地定量CRISPR基因编辑。我们证明了该技术在人类胚胎肾细胞中12,000 gRNA的SpCas9编辑效率和插入缺失结果的高通量定量中的实用性。使用这种方法，我们还显示出TRAP-seq可以对细胞中12,000个位点的ABE和CBE效率进行高通量定量。大量的ABE / CBE结果数据使我们能够揭示影响碱基编辑效率的几种新颖核苷酸特征（例如，侧翼碱基的偏爱，核苷酸基序，STOP编码类型），以及设计用于设计SpCas9的改进的基于机器学习的预测工具，高效率和准确性（> 70％）的ABE和CBE gRNA。我们已经将SpCas9，ABE和CBE的所有12,000个CRISPR基因编辑结果整合到了以CRISPR为中心的门户网站中：The Human CRISPR Atlas。