Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA
Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA
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DOI：
10.1073/pnas.1710597114
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发表时间：
2017-12-19
影响因子：
11.1
通讯作者：
Molnar, Attila
中科院分区：
文献类型：
--
作者：
Ferenczi, Aron;Pyott, Douglas Euan;Molnar, Attila
莱茵绿色是藻类、植物和纤毛生物学等研究领域的宝贵参考生物。因此，数十年来靶向核基因编辑的效率低下广泛阻碍了衣原体研究。在这里，我们报告了CRISPR/Cpf 1核糖核蛋白与单链DNA修复模板的单步共递送导致精确和靶向的DNA替换，在C中的效率高达10%。莱因哈德氏菌我们证明了它在转基因和选择自由生成的序列特异性突变和表位标记在内源性基因座的用途。由于基因编辑试剂的直接递送绕过了转基因的使用，这种方法可能适用于更广泛的物种，而无需开发稳定转化的方法。
The green alga Chlamydomonas reinhardtii is an invaluable reference organism to research fields including algal, plant, and ciliary biology. Accordingly, decades-long standing inefficiencies in targeted nuclear gene editing broadly hinder Chlamydomonas research. Here we report that single-step codelivery of CRISPR/Cpf1 ribonucleoproteins with single-stranded DNA repair templates results in precise and targeted DNA replacement with as much as similar to 10% efficiency in C. reinhardtii. We demonstrate its use in transgene-and selection-free generation of sequence-specific mutations and epitope tagging at an endogenous locus. As the direct delivery of gene-editing reagents bypasses the use of transgenes, this method is potentially applicable to a wider range of species without the need to develop methods for stable transformation.