Department of Plant Breeding, Swedish University of Agricultural Sciences, P.O. Box 101, SE 23053 Alnarp, Sweden.
Concejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
Physiol Plant. 2018 Dec;164(4):378-384. doi: 10.1111/ppl.12731. Epub 2018 Apr 27.
Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein-9 (CRISPR-Cas9) can be used as an efficient tool for genome editing in potato (Solanum tuberosum). From both a scientific and a regulatory perspective, it is beneficial if integration of DNA in the potato genome is avoided. We have implemented a DNA-free genome editing method, using delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to potato protoplasts, by targeting the gene encoding a granule bound starch synthase (GBSS, EC 2.4.1.242). The RNP method was directly implemented using previously developed protoplast isolation, transfection and regeneration protocols without further adjustments. Cas9 protein was preassembled with RNA produced either synthetically or by in vitro transcription. RNP with synthetically produced RNA (cr-RNP) induced mutations, i.e. indels, at a frequency of up to 9%, with all mutated lines being transgene-free. A mutagenesis frequency of 25% of all regenerated shoots was found when using RNP with in vitro transcriptionally produced RNA (IVT-RNP). However, more than 80% of the shoots with confirmed mutations had unintended inserts in the cut site, which was in the same range as when using DNA delivery. The inserts originated both from DNA template remnants from the in vitro transcription, and from chromosomal potato DNA. In 2-3% of the regenerated shoots from the RNP-experiments, mutations were induced in all four alleles resulting in a complete knockout of the GBSS enzyme function.
成簇规律间隔短回文重复序列和 CRISPR 相关蛋白 9（CRISPR-Cas9）可作为马铃薯（Solanum tuberosum）基因组编辑的有效工具。从科学和监管的角度来看，如果能避免 DNA 整合到马铃薯基因组中，将是有益的。我们已经实施了一种无 DNA 的基因组编辑方法，通过将 CRISPR-Cas9 核糖核蛋白（RNP）递送至马铃薯原生质体，靶向编码颗粒结合淀粉合成酶（GBSS，EC 2.4.1.242）的基因。该 RNP 方法直接使用先前开发的原生质体分离、转染和再生方案实施，无需进一步调整。Cas9 蛋白与合成或体外转录产生的 RNA 预先组装。用合成 RNA 产生的 RNP（cr-RNP）诱导突变，即插入缺失，频率高达 9%，所有突变系均不含转基因。当使用体外转录产生的 RNA 的 RNP（IVT-RNP）时，发现所有再生芽的突变频率为 25%。然而，在确认发生突变的芽中，超过 80%的芽在切割部位有非预期的插入，这与使用 DNA 递送时的情况相同。插入物源自体外转录的 DNA 模板残留物，以及来自染色体马铃薯 DNA。在 RNP 实验中再生的 2-3%的芽中，四个等位基因都诱导了突变，导致 GBSS 酶功能完全缺失。
