Genome editing and targeted sequence insertion with CrispR/Cas9 to elevate disease resistance in potato and tomato (JONES_S17DTP)

可编程核酸酶，如CRISPR/Cas9，通过使特定的基因组序列成为突变或同源重组(HR)的靶标，正在给生物学带来革命性的变化。这种新的育种技术(NBT)可以导致功能丧失的突变，或者能够通过HR将有用的基因从一种植物精确替换到另一种植物的相应染色体位置。我们利用野生马铃薯和番茄近缘的抗晚疫病基因。本项目的主要目标是利用CRISPR/Cas9(并测试相关的Cpf1可编程核酸酶)来取代番茄和马铃薯二倍体自交系中非功能的RPI基因同源物，用功能RPI基因等位基因来替代。我们使用的是来自www.solynta.com的马铃薯近交系。在以Kie为靶标之前，将使用具有长PacBio读数的RenSeq来表征感兴趣的茄属基因型的抗性基因谱系(Witek等人2016年)。学生将产生KIE事件，这些事件(取决于NBT法规)可以在F1杂交产品中推向市场，并将与私营和公共部门的育种者互动。Cermak T(2015年)番茄基因组基因组生物学16：232DOI 10.1186/s13059-015-0796-9 Witek(2016)使用Renseq和SMRT测序进行RPI基因克隆的NAT Biot 34：656-660 www.ncbi.nlm.nih.gov/pubmed/27111721

Programmable nucleases such as CrispR/Cas9 are revolutionizing biology by enabling specific genome sequences to be targeted for mutation, or for homologous recombination (HR). Such New Breeding Technologies (NBTs) can result in loss-of-function mutations, or enable precise replacement (Knock-In Exchange, or KIE) of useful genes from one plant via HR into the corresponding chromosomal position of another plant. We work with Resistance to Phytophthora infestans (Rpi) genes from wild potato and tomato relatives. The main goal of this project is to use CrispR/Cas9 (and test also the related Cpf1 programmable nuclease) to replace non-functional Rpi gene homologs in tomato and potato diploid inbred lines, with functional Rpi gene alleles. We work with inbred potato lines from www.solynta.com. Prior to targeting with KIE, resistance gene repertoires of Solanum genotypes of interest will be characterized using RenSeq with long PacBio reads (Witek et al 2016). The student will generate KIE events that (depending on NBT regulation) could be brought to market in F1 hybrids, and will interact with private and public sector breeders.Cermak T (2015) High-frequency, precise modification of the tomato genome Genome Biology 16:232 DOI 10.1186/s13059-015-0796-9 Witek (2016) Rpi gene cloning using Renseq and sMrt sequencing Nat Biot 34:656-660 www.ncbi.nlm.nih.gov/pubmed/27111721