Template-jumping prime editing enables large insertion and exon rewriting in vivo.
Template-jumping prime editing enables large insertion and exon rewriting in vivo.
复制标题
DOI：
10.1038/s41467-023-39137-6
复制
发表时间：
2023-06-08
影响因子：
16.6
通讯作者：
Xue, Wen
中科院分区：
文献类型：
--
作者：
Zheng, Chunwei;Liu, Bin;Dong, Xiaolong;Gaston, Nicholas;Sontheimer, Erik J.;Xue, Wen
大DNA片段的靶向插入为基因组工程和基因治疗带来了希望。引物编辑（PE）有效地插入短（<50 bp）序列。采用成对的引物编辑指导RNA（pegRNA）已经使得PE能够在体外更好地介导相对大的插入，但是较大插入（>400 bp）的效率仍然较低，并且体内应用尚未得到证实。受反转录转座子的高效基因组插入机制的启发，我们开发了一种使用单个pegRNA插入大DNA片段的模板跳跃（TJ）PE方法。TJ-pegRNA具有插入序列以及两个引物结合位点（PBS），其中一个PBS匹配切口sgRNA位点。TJ-PE精确插入200 bp和500 bp片段，效率分别高达50.5%和11.4%，并使GFP（~800 bp）插入和表达在细胞中。我们在体外转录分裂的环状TJ-petRNA通过置换组I催化内含子在细胞中的非病毒递送。最后，我们证明了TJ-PE可以重写酪氨酸血症I小鼠肝脏中的外显子以逆转疾病表型。TJ-PE具有插入大的DNA片段而没有双链DNA断裂的潜力，并有助于体内突变热点外显子重写。反转录转座子通过靶向启动逆转录（TPRT）复制其遗传信息。在这里，作者报告了一种模板跳跃引物编辑器（TJ-PE），其作用类似于TPRT，并在内源性位点插入大的DNA片段：它们重写了小鼠肝脏中的突变外显子。
Targeted insertion of large DNA fragments holds promise for genome engineering and gene therapy. Prime editing (PE) effectively inserts short (<50 bp) sequences. Employing paired prime editing guide RNAs (pegRNAs) has enabled PE to better mediate relatively large insertions in vitro, but the efficiency of larger insertions (>400 bp) remains low and in vivo application has not been demonstrated. Inspired by the efficient genomic insertion mechanism of retrotransposons, we develop a template-jumping (TJ) PE approach for the insertion of large DNA fragments using a single pegRNA. TJ-pegRNA harbors the insertion sequence as well as two primer binding sites (PBSs), with one PBS matching a nicking sgRNA site. TJ-PE precisely inserts 200 bp and 500 bp fragments with up to 50.5 and 11.4% efficiency, respectively, and enables GFP (~800 bp) insertion and expression in cells. We transcribe split circular TJ-petRNA in vitro via a permuted group I catalytic intron for non-viral delivery in cells. Finally, we demonstrate that TJ-PE can rewrite an exon in the liver of tyrosinemia I mice to reverse the disease phenotype. TJ-PE has the potential to insert large DNA fragments without double-stranded DNA breaks and facilitate mutation hotspot exon rewriting in vivo. Retrotransposons replicate their genetic information through target-primed reverse transcription (TPRT). Here the authors report a template-jumping prime editor (TJ-PE) to act similarly to TPRT and achieve insertions of large DNA fragments at endogenous sites: they rewrite a mutated exon in the mouse liver.
登录
查看更多内容
影响因子：
64.5
作者：
Raguram, Aditya;Banskota, Samagya;Liu, David R.
通讯作者：
Liu, David R.
影响因子：
16.6
作者：
Liu P;Liang SQ;Zheng C;Mintzer E;Zhao YG;Ponnienselvan K;Mir A;Sontheimer EJ;Gao G;Flotte TR;Wolfe SA;Xue W
通讯作者：
Xue W
影响因子：
16.6
作者：
Song M;Lim JM;Min S;Oh JS;Kim DY;Woo JS;Nishimasu H;Cho SR;Yoon S;Kim HH
通讯作者：
Kim HH
影响因子：
64.8
作者：
Anzalone, Andrew V.;Randolph, Peyton B.;Liu, David R.
通讯作者：
Liu, David R.
影响因子：
64.5
作者：
McClellan, Jon;King, Mary-Claire
通讯作者：
King, Mary-Claire