Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.
Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.
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DOI：
10.1186/s13059-021-02304-3
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发表时间：
2021-03-16
期刊：
影响因子：
12.3
通讯作者：
Miano JM
中科院分区：
文献类型：
--
作者：
Gao P;Lyu Q;Ghanam AR;Lazzarotto CR;Newby GA;Zhang W;Choi M;Slivano OJ;Holden K;Walker JA 2nd;Kadina AP;Munroe RJ;Abratte CM;Schimenti JC;Liu DR;Tsai SQ;Long X;Miano JM
大多数单核苷酸变异体(SNV)以非编码序列形式存在，其中有数百万个转录因子结合位点(TFBS)。在这里，CRISPR介导的同源定向修复(HDR)系统与最近报道的Prime Editing 2(PE2)系统在小鼠身上通过Tspan2启动子中称为Carg盒的TFBS进行了比较分析。定量RT-PCR显示，在Tspan2Carg盒中HDR介导的三个碱基对替换纯合子小鼠中，Tspan2mRNA在主动脉和膀胱中丢失，但心脏或大脑中没有。使用相同的Protspacer，在Tspan2Carg盒中进行PE2介导的单碱基替换的纯合子小鼠显示出类似的细胞特异性Tspan2mRNA的丢失；在主动脉和膀胱中，重叠的长非编码RNA的表达也几乎消失。免疫RNA荧光原位杂交证实Tspan2在HDR和PE2Carg box突变小鼠的血管平滑肌细胞中丢失。靶向测序显示，在所有PE2和HDR创始人中，靶向编辑的频率不同。然而，虽然在任何PE2创始人中都没有检测到靶上INDELs，但所有HDR创始人都显示出不同水平的靶向INDELs。靶向测序的非靶点分析显示，许多HDR创始人存在突变，但PE2创始人没有突变。PE2指导TFBS中单个碱基的高保真编辑，导致mRNA/长非编码RNA基因对表达的细胞特异性丢失。PE2平台扩展了基因组编辑工具箱，用于建模和纠正鼠标中相关的非编码SNV。网上版载有补充材料，可在10.1186/s13059-021-02304-3查阅。
Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse. The online version contains supplementary material available at 10.1186/s13059-021-02304-3.
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影响因子：
48
作者：
Anderson KR;Haeussler M;Watanabe C;Janakiraman V;Lund J;Modrusan Z;Stinson J;Bei Q;Buechler A;Yu C;Thamminana SR;Tam L;Sowick MA;Alcantar T;O'Neil N;Li J;Ta L;Lima L;Roose-Girma M;Rairdan X;Durinck S;Warming S
通讯作者：
Warming S
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
64.5
作者：
Arango, NA;Lovell-Badge, R;Behringer, RR
通讯作者：
Behringer, RR
DOI：
10.1073/pnas.2021996118
发表时间：
2021-01-05
影响因子：
11.1
作者：
Bosch, Justin A.;Birchak, Gabriel;Perrimon, Norbert
通讯作者：
Perrimon, Norbert
影响因子：
64.8
作者：
Anzalone, Andrew V.;Randolph, Peyton B.;Liu, David R.
通讯作者：
Liu, David R.