A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells
CRISPR/Cas9 nucleases hold great potential for gene therapy, but they frequently induce unwanted off-target cleavage. We previously developed a GFP activation assay for detection of DNA cleavage in cells. Here, we demonstrate two novel applications of this assay. First, we use this assay to confirm...
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Frontiers Media S.A.
2021
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oai:doaj.org-article:ed723177ffba47588f6be8e71deee98e2021-11-11T10:19:22ZA Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells2296-634X10.3389/fcell.2021.771248https://doaj.org/article/ed723177ffba47588f6be8e71deee98e2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fcell.2021.771248/fullhttps://doaj.org/toc/2296-634XCRISPR/Cas9 nucleases hold great potential for gene therapy, but they frequently induce unwanted off-target cleavage. We previously developed a GFP activation assay for detection of DNA cleavage in cells. Here, we demonstrate two novel applications of this assay. First, we use this assay to confirm off-target cleavage that cannot be detected by targeted deep sequencing in cells before. Second, we use this approach to detect multiple alternative PAMs recognized by SpCas9. These noncanonical PAMs are associated with low cleavage activity, but targets associated with these PAMs must be considered as potential off-target sites. Taken together, the GFP activation assay is a powerful platform for DNA cleavage detection in cells.Ziying HuZiying HuChengdong ZhangDaqi WangSiqi GaoSang-Ging OngSang-Ging OngYongming WangYongming WangWei V. ZhengFrontiers Media S.A.articleCRISPRgenome editingDNA cleavageoff-targetPAMBiology (General)QH301-705.5ENFrontiers in Cell and Developmental Biology, Vol 9 (2021) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
CRISPR genome editing DNA cleavage off-target PAM Biology (General) QH301-705.5 |
| spellingShingle |
CRISPR genome editing DNA cleavage off-target PAM Biology (General) QH301-705.5 Ziying Hu Ziying Hu Chengdong Zhang Daqi Wang Siqi Gao Sang-Ging Ong Sang-Ging Ong Yongming Wang Yongming Wang Wei V. Zheng A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells |
| description |
CRISPR/Cas9 nucleases hold great potential for gene therapy, but they frequently induce unwanted off-target cleavage. We previously developed a GFP activation assay for detection of DNA cleavage in cells. Here, we demonstrate two novel applications of this assay. First, we use this assay to confirm off-target cleavage that cannot be detected by targeted deep sequencing in cells before. Second, we use this approach to detect multiple alternative PAMs recognized by SpCas9. These noncanonical PAMs are associated with low cleavage activity, but targets associated with these PAMs must be considered as potential off-target sites. Taken together, the GFP activation assay is a powerful platform for DNA cleavage detection in cells. |
| format |
article |
| author |
Ziying Hu Ziying Hu Chengdong Zhang Daqi Wang Siqi Gao Sang-Ging Ong Sang-Ging Ong Yongming Wang Yongming Wang Wei V. Zheng |
| author_facet |
Ziying Hu Ziying Hu Chengdong Zhang Daqi Wang Siqi Gao Sang-Ging Ong Sang-Ging Ong Yongming Wang Yongming Wang Wei V. Zheng |
| author_sort |
Ziying Hu |
| title |
A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells |
| title_short |
A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells |
| title_full |
A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells |
| title_fullStr |
A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells |
| title_full_unstemmed |
A Highly Sensitive GFP Activation Assay for Detection of DNA Cleavage in Cells |
| title_sort |
highly sensitive gfp activation assay for detection of dna cleavage in cells |
| publisher |
Frontiers Media S.A. |
| publishDate |
2021 |
| url |
https://doaj.org/article/ed723177ffba47588f6be8e71deee98e |
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