TY - JOUR
T1 - The comparison of ZFNs, TALENs, and SpCas9 by GUIDE-seq in HPV-targeted gene therapy
AU - Cui, Zifeng
AU - Liu, Hui
AU - Zhang, Hongfeng
AU - Huang, Zhaoyue
AU - Tian, Rui
AU - Li, Lifang
AU - Fan, Weiwen
AU - Chen, Yili
AU - Chen, Lijie
AU - Zhang, Sen
AU - Das, Bhudev C.
AU - Severinov, Konstantin
AU - Hitzeroth, Inga Isabel
AU - Debata, Priya Ranjan
AU - Jin, Zhuang
AU - Liu, Jiashuo
AU - Huang, Zheying
AU - Xie, Weiling
AU - Xie, Hongxian
AU - Lang, Bin
AU - Ma, Ji
AU - Weng, Haiyan
AU - Tian, Xun
AU - Hu, Zheng
N1 - Publisher Copyright:
© 2021 The Authors
PY - 2021/12/3
Y1 - 2021/12/3
N2 - 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.
AB - 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.
KW - CRISPR
KW - GUIDE-seq
KW - HPV gene therapy
KW - TALEN
KW - ZFN
UR - http://www.scopus.com/inward/record.url?scp=85116332770&partnerID=8YFLogxK
U2 - 10.1016/j.omtn.2021.08.008
DO - 10.1016/j.omtn.2021.08.008
M3 - Article
AN - SCOPUS:85116332770
SN - 2162-2531
VL - 26
SP - 1466
EP - 1478
JO - Molecular Therapy - Nucleic Acids
JF - Molecular Therapy - Nucleic Acids
ER -