Tag-seq: a convenient and scalable method for genome-wide specificity assessment of CRISPR/Cas nucleases

Hongxin Huang,Guanjie Huang,Yongfei Hu,Jianqi Feng,Dong Wang,Ying Lin,Jiajian Zhou,Zhili Rong,Shufeng Ma

doi:10.1038/s42003-021-02351-3

Abstract

Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPR-associated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases. However, current methods for off-target discovery are tedious and costly, restricting their widespread applications. Here we present an easy alternative method for CRISPR off-target detection by tracing the integrated oligonucleotide Tag using next-generation-sequencing (CRISPR-Tag-seq, or Tag-seq). Tag-seq enables rapid and convenient profiling of nuclease-induced DSBs by incorporating the optimized double-stranded oligodeoxynucleotide sequence (termed Tag), adapters, and PCR primers. Moreover, we employ a one-step procedure for library preparation in Tag-seq, which can be applied in the routine workflow of a molecular biology laboratory. We further show that Tag-seq successfully determines the cleavage specificity of SpCas9 variants and Cas12a/Cpf1 in a large-scale manner, and discover the integration sites of exogenous genes introduced by the Sleeping Beauty transposon. Our results demonstrate that Tag-seq is an efficient and scalable approach to genome-wide identification of Cas-nuclease-induced off-targets.

Highlights

Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPRassociated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases
The cell-based strategies examine nuclease activity in vivo resulting in the detection of bona fide off-target sites, which improves the specificity of off-target events detection
Among the cell-based techniques, GUIDE-seq is the most widely used methods for identification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nuclease induced off-target sites, it achieves the high sensitivity through detecting the accumulation of integrated doublestranded oligodeoxynucleotide at break sites in living cells over time[15]

Summary

Introduction

Genome-wide identification of DNA double-strand breaks (DSBs) induced by CRISPRassociated protein (Cas) systems is vital for profiling the off-target events of Cas nucleases. The substantial improvements of the Tag-seq workflow are listed as following: 1) a single-tube reaction method is applied in molecular manipulation process, including DNA fragmentation, end repair, dA-tailing and adapter ligation, which greatly shorten the time for libraries preparation (Fig. 1a); 2) the sample barcodes and UMI used in Tagseq are compatible with commercial sequencing devices (Supplementary Fig. 1a); 3) a GC content balanced (45.7%) oligonucleotide Tag and a polyetherimide (PEI)-based transfection method enable high integration of Tag in DSB sites with costefficiency (Supplementary Fig. 1b–e and Supplementary Fig. 2); 4) Tag-specific primers with the “GAT” and “CA” nucleotide motifs ensure a high fidelity of PCR amplification (Supplementary Fig. 1a)16; 5) the all-in-one PCR strategy skipping the cleanup step[15] enables time-saving during libraries preparation (Supplementary Fig. f–h); 6) A state of art bioinformatic analysis pipeline is developed for Tag-seq analysis, it provides 3 visualization modules for inspecting the distribution of DSBs in a genome-wide level, the editing frequencies of a specific DSB and the comparison of a specific DSB among Tag-seq experiments (Fig. 1b, c).

Results

Conclusion