Search-and-replace genome editing without double-strand breaks or donor DNA
Search-and-replace genome editing without double-strand breaks or donor DNA
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DOI：
10.1038/s41586-019-1711-4
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发表时间：
2019-12-05
期刊：
影响因子：
64.8
通讯作者：
Liu, David R.
中科院分区：
文献类型：
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作者：
Anzalone, Andrew V.;Randolph, Peyton B.;Liu, David R.
导致疾病的大多数遗传变异（1）具有挑战性，难以有效纠正，并且没有过量的副产物（2-5）。在这里，我们描述了引物编辑，一种通用且精确的基因组编辑方法，其使用与工程逆转录酶融合的催化受损Cas9内切核酸酶将新的遗传信息直接写入指定的DNA位点，该逆转录酶用引物编辑指导RNA（pegRNA）编程，该引物编辑指导RNA既指定靶位点又编码所需的编辑。我们在人类细胞中进行了超过175次编辑，包括有针对性的插入、缺失和所有12种类型的点突变，而不需要双链断裂或供体DNA模板。我们在人类细胞中使用引物编辑来有效地纠正镰状细胞病（需要HBB中的颠换）和Tay-Sachs病（需要HEXA中的缺失）的主要遗传原因，并且副产物很少;在PRNP中安装保护性颠换;并将各种标签和表位精确地插入靶基因座。四种人类细胞系和原代有丝分裂后小鼠皮质神经元支持具有不同效率的引物编辑。引物编辑显示出比同源定向修复更高或相似的效率和更少的副产物，与碱基编辑相比具有互补的优势和弱点，并且在已知的Cas9脱靶位点处诱导比Cas9核酸酶低得多的脱靶编辑。引物编辑大大扩展了基因组编辑的范围和能力，原则上可以纠正高达89%的与人类疾病相关的已知遗传变异。
Most genetic variants that contribute to disease(1) are challenging to correct efficiently and without excess by products(2-5). Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells, including targeted insertions, deletions, and all 12 types of point mutation, without requiring double-strand breaks or donor DNA templates. We used prime editing in human cells to correct, efficiently and with few by products, the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA); to install a protective transversion in PRNP; and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer by products than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expandsthe scope and capabilities of genome editing, and in principle could correct up to 89% of known genetic variants associated with human diseases.