The comparison of ZFNs, TALENs, and SpCas9 by GUIDE-seq in HPV-targeted gene therapy

Zifeng Cui, Hui Liu, Hongfeng Zhang, Zhaoyue Huang, Rui Tian, Lifang Li, Weiwen Fan, Yili Chen, Lijie Chen, Sen Zhang, Bhudev C. Das, Konstantin Severinov, Inga Isabel Hitzeroth, Priya Ranjan Debata, Zhuang Jin, Jiashuo Liu, Zheying Huang, Weiling Xie, Hongxian Xie, Bin LangJi Ma, Haiyan Weng, Xun Tian, Zheng Hu

Research output: Contribution to journalArticlepeer-review

Abstract

Zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR-associated Cas9 endonucleases are three major generations of genome editing tools. However, no parallel comparison about the efficiencies and off-target activity of the three nucleases has been reported, which is critical for the final clinical decision. We for the first time developed the genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) method in ZFNs and TALENs with novel bioinformatics algorithms to evaluate the off-targets. By targeting human papillomavirus 16 (HPV16), we compared the performance of ZFNs, TALENs, and SpCas9 in vivo. Our data showed that ZFNs with similar targets could generate distinct massive off-targets (287–1,856), and the specificity could be reversely correlated with the counts of middle “G” in zinc finger proteins (ZFPs). We also compared the TALENs with different N-terminal domains (wild-type [WT]/αN/βN) and G recognition modules (NN/NH) and found the design (αN or NN) to improve the efficiency of TALEN inevitably increased off-targets. Finally, our results showed that SpCas9 was more efficient and specific than ZFNs and TALENs. Specifically, SpCas9 had fewer off-target counts in URR (SpCas9, n = 0; TALEN, n = 1; ZFN, n = 287), E6 (SpCas9, n = 0; TALEN, n = 7), and E7 (SpCas9, n = 4; TALEN, n = 36). Taken together, we suggest that for HPV gene therapies, SpCas9 is a more efficient and safer genome editing tool. Our off-target data could be used to improve the design of ZFNs and TALENs, and the universal in vivo off-target detection pipeline for three generations of artificial nucleases provided useful tools for genome engineering-based gene therapy.

Original languageEnglish (US)
Pages (from-to)1466-1478
Number of pages13
JournalMolecular Therapy - Nucleic Acids
Volume26
DOIs
StatePublished - Dec 3 2021
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Drug Discovery

Keywords

  • CRISPR
  • GUIDE-seq
  • HPV gene therapy
  • TALEN
  • ZFN

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