A CRISPR/Cas9 library to map the HIV-1 provirus genetic fitness.
A CRISPR/Cas9 library to map the HIV-1 provirus genetic fitness.
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DOI：
10.4149/av_2019_201
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发表时间：
2019
期刊：
影响因子：
1.7
通讯作者：
K. Yoder
中科院分区：
文献类型：
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作者：
K. Yoder
整合的前病毒基因组是治愈HIV-1感染的主要障碍。基因组编辑技术，如CRISPR/Cas9，可以通过在病毒基因组的序列特定位点引入DNA双链断裂来禁用或去除HIV-1原病毒。宿主DNA修复通过易出错的非同源末端连接途径在断裂处产生诱变插入或缺失。CRISPR/Cas9编辑已被证明可以减少细胞培养中具有复制能力的病毒基因组，但仅对少数可能的基因组编辑靶标进行了分析。目前还没有HIV-1的双链断裂遗传适应度图谱来指导编辑靶标的选择。然而，CRISPR/Cas9基因组编辑使得沿着原病毒的长度靶向双链断裂成为可能，从而生成双链断裂遗传适应度图谱。我们用不同细菌种类的CRISPR/Cas9鉴定了所有可能的HIV-1靶点。对该引导rna文库的GC含量和人类基因组中潜在的脱靶位点进行了评估。通过消除HIV-1长末端重复序列和env基因中的重复引导RNA靶标，降低了文库的复杂性。尽管HIV-1基因组富含at，但具有原间隔邻近基序NGG的S. pyogenes CRISPR/Cas9提供了最多的HIV-1引导rna。该HIV-1引导rna库可用于生成双链断裂遗传脆弱性图谱，从而进一步应用于针对HIV-1原病毒设计的任何基因组编辑技术。关键词:hiv - 1;基因组编辑;CRISPR;基因健康;指导rna。
The integrated proviral genome is the major barrier to a cure for HIV-1 infection. Genome editing technologies, such as CRISPR/Cas9, may disable or remove the HIV-1 provirus by introducing DNA double strand breaks at sequence specific sites in the viral genome. Host DNA repair by the error-prone non-homologous end joining pathway generates mutagenic insertions or deletions at the break. CRISPR/Cas9 editing has been shown to reduce replication competent viral genomes in cell culture, but only a minority of possible genome editing targets have been assayed. Currently there is no map of double strand break genetic fitness for HIV-1 to inform the choice of editing targets. However, CRISPR/Cas9 genome editing makes it possible to target double strand breaks along the length of the provirus to generate a double strand break genetic fitness map. We identified all possible HIV-1 targets with different bacterial species of CRISPR/Cas9. This library of guide RNAs was evaluated for GC content and potential off-target sites in the human genome. Complexity of the library was reduced by eliminating duplicate guide RNA targets in the HIV-1 long terminal repeats and targets in the env gene. Although the HIV-1 genome is AT-rich, the S. pyogenes CRISPR/Cas9 with the proto-spacer adjacent motif NGG offers the most HIV-1 guide RNAs. This library of HIV-1 guide RNAs may be used to generate a double strand break genetic fragility map to be further applied to any genome editing technology designed for the HIV-1 provirus. Keywords: HIV-1; genome editing; CRISPR; genetic fitness; guide RNAs.