Carlsen Frida Meijer, Johansen Ida Elisabeth, Yang Zhang, Liu Ying, Westberg Ida Nøhr, Kieu Nam Phuong, Jørgensen Bodil, Lenman Marit, Andreasson Erik, Nielsen Kåre Lehmann, Blennow Andreas, Petersen Bent Larsen
Department of Plant and Environmental Sciences, Faculty of Science, The University of Copenhagen, Copenhagen, Denmark.
Kartoffel Mel Centralen Amba, Brande, Denmark.
Front Genome Ed. 2022 Jan 20;3:795644. doi: 10.3389/fgeed.2021.795644. eCollection 2021.
Potato, is a highly diverse tetraploid crop. Elite cultivars are extremely heterozygous with a high prevalence of small length polymorphisms (indels) and single nucleotide polymorphisms (SNPs) within and between cultivars, which must be considered in CRISPR/Cas gene editing strategies and designs to obtain successful gene editing. In the present study, in-depth sequencing of the gene encoding glucan water dikinase (GWD) 1 and the downy mildew resistant 6 (DMR6-1) genes in the potato cultivars Saturna and Wotan, respectively, revealed both indels and a 1.3-2.8 higher SNP prevalence when compared to the heterozygous diploid RH genome sequence as expected for a tetraploid compared to a diploid. This complicates guide RNA (gRNA) and diagnostic PCR designs. At the same time, high editing efficiencies at the cell pool (protoplast) level are pivotal for achieving full allelic knock-out in tetraploids. Furthermore, high editing efficiencies reduce the downstream cumbersome and delicate ex-plant regeneration. Here, CRISPR/Cas ribonucleoprotein particles (RNPs) were delivered transiently to protoplasts by polyethylene glycol (PEG) mediated transformation. For each of GWD1 and the DMR6-1, 6-10 gRNAs were designed to target regions comprising the 5' and the 3' end of the two genes. Similar to other studies including several organisms, editing efficiency of the individual RNPs varied significantly, and some generated specific indel patterns. RNP's targeting the 5' end of GWD1 yielded significantly higher editing efficiency as compared to targeting the 3' end. For DMR6-1, such an effect was not seen. Simultaneously targeting each of the two target regions with two RNPs (multiplexing) yielded a clear positive synergistic effect on the total editing when targeting the 3' end of the GWD1 gene only. Multiplexing of the two genes, residing on different chromosomes, yielded no or a slightly negative effect on editing from the single or combined gRNA/RNPs. These initial findings may instigate much larger studies needed for facilitating and optimizing precision breeding in plants.
马铃薯是一种高度多样化的四倍体作物。优良品种具有极高的杂合性，品种内和品种间存在大量短长度多态性（插入缺失）和单核苷酸多态性（SNP），在CRISPR/Cas基因编辑策略和设计中必须考虑这些因素，以实现成功的基因编辑。在本研究中，分别对马铃薯品种Saturna和Wotan中编码葡聚糖水二激酶（GWD）1和抗霜霉病6（DMR6-1）基因进行深度测序，结果显示，与杂合二倍体RH基因组序列相比，两者均存在插入缺失，且SNP发生率高出1.3至2.8倍，正如四倍体与二倍体相比所预期的那样。这使得向导RNA（gRNA）和诊断性PCR设计变得复杂。同时，细胞池（原生质体）水平的高编辑效率对于在四倍体中实现全等位基因敲除至关重要。此外，高编辑效率可减少下游繁琐且精细的外植体再生过程。在此，通过聚乙二醇（PEG）介导的转化将CRISPR/Cas核糖核蛋白颗粒（RNP）瞬时递送至原生质体。针对GWD1和DMR6-1中的每一个，设计了6至10个gRNA，靶向包含这两个基因5'端和3'端的区域。与包括多种生物的其他研究类似，单个RNP的编辑效率差异显著，有些产生了特定的插入缺失模式。靶向GWD1 5'端的RNP与靶向3'端相比，编辑效率显著更高。对于DMR6-1，未观察到这种效应。仅在靶向GWD1基因3'端时，用两个RNP同时靶向两个目标区域（多重编辑）对总编辑产生了明显的正向协同效应。位于不同染色体上的两个基因进行多重编辑，对单个或组合gRNA/RNP的编辑没有影响或产生轻微的负面影响。这些初步发现可能促使开展规模更大的研究，以促进和优化植物的精准育种。
