TP53-dependent toxicity of CRISPR/Cas9 cuts is differential across genomic loci and can confound genetic screening.
TP53-dependent toxicity of CRISPR/Cas9 cuts is differential across genomic loci and can confound genetic screening.
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CRISPR/Cas9切割的TP 53依赖性毒性在基因组基因座之间是不同的，并且可以混淆遗传筛选。
DOI：
10.1038/s41467-022-32285-1
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发表时间：
2022-08-04
影响因子：
16.6
通讯作者：
中科院分区：
文献类型：
--
作者：
CRISPR/Cas9基因编辑可以以精确的方式对基因进行测序。这个过程涉及DNA双链断裂（DSB），这可能导致细胞适应性的丧失。我们假设DSB毒性可能是可变的，这取决于靶位点的染色质环境。在这里，通过分析同基因细胞系对CRISPR实验以及来自约900个细胞系的先前筛选数据，我们表明TP 53相关的断裂毒性在含有活性染色质的基因组区域中更高，例如基因调控元件或转录延伸组蛋白标记。DSB修复途径的选择和DNA序列环境也与毒性有关。我们还表明，由于sgRNA靶向位点的差异毒性引入的噪声，在TP 53野生型细胞中检测条件必要性的遗传筛选的能力降低。了解Cas9切割毒性的决定因素将有助于改进CRISPR试剂的设计，以避免TP 53缺陷和/或DNA修复缺陷细胞的偶然选择。CRISPR/Cas9诱导的DNA断裂的毒性取决于它们的修复机制和切割位点的染色质环境。在这里，作者表明，活性基因或调控元件的编辑可以通过TP 53依赖性机制引起更高的毒性。
CRISPR/Cas9 gene editing can inactivate genes in a precise manner. This process involves DNA double-strand breaks (DSB), which may incur a loss of cell fitness. We hypothesize that DSB toxicity may be variable depending on the chromatin environment in the targeted locus. Here, by analyzing isogenic cell line pair CRISPR experiments jointly with previous screening data from across ~900 cell lines, we show that TP53-associated break toxicity is higher in genomic regions that harbor active chromatin, such as gene regulatory elements or transcription elongation histone marks. DSB repair pathway choice and DNA sequence context also associate with toxicity. We also show that, due to noise introduced by differential toxicity of sgRNA-targeted sites, the power of genetic screens to detect conditional essentiality is reduced in TP53 wild-type cells. Understanding the determinants of Cas9 cut toxicity will help improve design of CRISPR reagents to avoid incidental selection of TP53-deficient and/or DNA repair deficient cells. Toxicity of CRISPR/Cas9 induced DNA breaks depends on their repair mechanism, and on the chromatin environment at the cut site. Here the authors show that edits in active genes or regulatory elements can incur a higher toxicity via a TP53-dependent mechanism.
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影响因子：
7.3
作者：
Gao J;Aksoy BA;Dogrusoz U;Dresdner G;Gross B;Sumer SO;Sun Y;Jacobsen A;Sinha R;Larsson E;Cerami E;Sander C;Schultz N
通讯作者：
Schultz N
影响因子：
7
作者：
Duttke, Sascha H.;Chang, Max W.;Benner, Christopher
通讯作者：
Benner, Christopher
影响因子：
11.4
作者：
Godfrey L;Crump NT;O'Byrne S;Lau IJ;Rice S;Harman JR;Jackson T;Elliott N;Buck G;Connor C;Thorne R;Knapp DJHF;Heidenreich O;Vyas P;Menendez P;Inglott S;Ancliff P;Geng H;Roberts I;Roy A;Milne TA
通讯作者：
Milne TA
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
7.7
作者：
Bowden, Anne Ramsay;Morales-Juarez, David A.;Jackson, Stephen P.
通讯作者：
Jackson, Stephen P.