Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects
Viral DNA integrated in host cells is a major barrier to completely curing HIV-1. However, genome editing using the recently developed technique of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has the potential to eradicate HIV-1. The present study aimed to use a lentivira...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2021-05-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2021.629153/full |
| _version_ | 1818864283003387904 |
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| author | Yufan Xu Xiaorong Peng Yanghao Zheng Changzhong Jin Xiangyun Lu Dating Han Haijing Fu Chaoyu Chen Nanping Wu |
| author_facet | Yufan Xu Xiaorong Peng Yanghao Zheng Changzhong Jin Xiangyun Lu Dating Han Haijing Fu Chaoyu Chen Nanping Wu |
| author_sort | Yufan Xu |
| collection | DOAJ |
| description | Viral DNA integrated in host cells is a major barrier to completely curing HIV-1. However, genome editing using the recently developed technique of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has the potential to eradicate HIV-1. The present study aimed to use a lentiviral vector-based CRISPR/Cas9 system combined with dual-small/single guide RNAs (sgRNAs) to attack HIV-1 DNA in the latency reactivation model J-Lat 10.6 cell line and to assess off-target effects using whole-genome sequencing (WGS). We designed 12 sgRNAs targeting HIV-1 DNA, and selected high-efficiency sgRNAs for further pairwise combinations after a preliminary evaluation of the editing efficiency. Three combinations of dual-sgRNAs/Cas9 with high editing efficiency were screened successfully from multiple combinations. Among these combinations, the incidences of insertions and deletions in the sgRNA-targeted regions reached 76% and above, and no credible off-target sites were detected using WGS. The results provided comprehensive basic experimental evidence and methodological recommendations for future personalized HIV-1 treatment using CRISPR/Cas9 genome editing technology. |
| first_indexed | 2024-12-19T10:29:11Z |
| format | Article |
| id | doaj.art-4b42e66c80944714963c001d12bd9a44 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-19T10:29:11Z |
| publishDate | 2021-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-4b42e66c80944714963c001d12bd9a442022-12-21T20:25:48ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2021-05-011210.3389/fmicb.2021.629153629153Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target EffectsYufan XuXiaorong PengYanghao ZhengChangzhong JinXiangyun LuDating HanHaijing FuChaoyu ChenNanping WuViral DNA integrated in host cells is a major barrier to completely curing HIV-1. However, genome editing using the recently developed technique of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has the potential to eradicate HIV-1. The present study aimed to use a lentiviral vector-based CRISPR/Cas9 system combined with dual-small/single guide RNAs (sgRNAs) to attack HIV-1 DNA in the latency reactivation model J-Lat 10.6 cell line and to assess off-target effects using whole-genome sequencing (WGS). We designed 12 sgRNAs targeting HIV-1 DNA, and selected high-efficiency sgRNAs for further pairwise combinations after a preliminary evaluation of the editing efficiency. Three combinations of dual-sgRNAs/Cas9 with high editing efficiency were screened successfully from multiple combinations. Among these combinations, the incidences of insertions and deletions in the sgRNA-targeted regions reached 76% and above, and no credible off-target sites were detected using WGS. The results provided comprehensive basic experimental evidence and methodological recommendations for future personalized HIV-1 treatment using CRISPR/Cas9 genome editing technology.https://www.frontiersin.org/articles/10.3389/fmicb.2021.629153/fullCRISPR/Cas9HIV-1dual-sgRNAsoff-target effectgenome editing |
| spellingShingle | Yufan Xu Xiaorong Peng Yanghao Zheng Changzhong Jin Xiangyun Lu Dating Han Haijing Fu Chaoyu Chen Nanping Wu Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects Frontiers in Microbiology CRISPR/Cas9 HIV-1 dual-sgRNAs off-target effect genome editing |
| title | Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects |
| title_full | Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects |
| title_fullStr | Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects |
| title_full_unstemmed | Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects |
| title_short | Inactivation of Latent HIV-1 Proviral DNA Using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Treatment and the Assessment of Off-Target Effects |
| title_sort | inactivation of latent hiv 1 proviral dna using clustered regularly interspaced short palindromic repeats cas9 treatment and the assessment of off target effects |
| topic | CRISPR/Cas9 HIV-1 dual-sgRNAs off-target effect genome editing |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2021.629153/full |
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