An optimised CRISPR/Cas9 protocol to create targeted mutations in homoeologous genes and an efficient genotyping protocol to identify edited events in wheat
An optimised CRISPR/Cas9 protocol to create targeted mutations in homoeologous genes and an efficient genotyping protocol to identify edited events in wheat
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DOI：
10.1186/s13007-019-0500-2
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发表时间：
2019-10-24
期刊：
影响因子：
5.1
通讯作者：
Ouellet, Therese
中科院分区：
文献类型：
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作者：
Cui, Xiucheng;Balcerzak, Margaret;Ouellet, Therese
背景使用规则间隔短回文重复序列（CRISPR）/Cas9系统的靶向基因组编辑已在大量植物物种中应用。使用基因特异性单向导RNA（sgRNA）和CRISPR/Cas9系统，可以获得小的编辑事件，例如几个碱基的缺失。然而，对于某些应用程序需要更大的删除。此外，编辑事件的鉴定和表征在具有复杂基因组的植物（例如小麦）中可能具有挑战性。结果在这项研究中，我们使用CRISPR/Cas9系统并开发了一种方案，该方案使用一对共表达的sgRNA靶向相同的基因，产生大量的大缺失。通过在小麦原生质体测定中靶向三个基因TaABCC 6、TaNFXL 1和TansLTP9.4来验证该方案。每个基因中位于两个sgRNA之间的序列缺失是三个基因中两个观察到的最常见的编辑事件。用于在藻类中表达的密码子优化的Cas9 crCas 9和植物密码子优化的Cas9 pcoCas 9之间的编辑频率的比较评估显示出与表达pcoCas 9的载体更一致的结果。在转基因小麦植物中研究了通过sgRNA对的共表达编辑TaNFXLl。鉴于面包小麦的倍性，一种快速、稳健和廉价的基因分型方案也适用于六倍体基因组，并被证明是鉴定小麦中同源特异性编辑事件的有用工具。结论靶向单个基因的sgRNA与CRISPR/Cas9系统联合共表达在小麦中产生了大的缺失。此外，成功地调整了鉴定TaNFXL 1同源物中编辑事件的基因分型方案。
Background Targeted genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been applied in a large number of plant species. Using a gene-specific single guide RNA (sgRNA) and the CRISPR/Cas9 system, small editing events such as deletions of few bases can be obtained. However larger deletions are required for some applications. In addition, identification and characterization of edited events can be challenging in plants with complex genomes, such as wheat. Results In this study, we used the CRISPR/Cas9 system and developed a protocol that yielded high number of large deletions employing a pair of co-expressed sgRNA to target the same gene. The protocol was validated by targeting three genes, TaABCC6, TaNFXL1 and TansLTP9.4 in a wheat protoplast assay. Deletions of sequences located between the two sgRNA in each gene were the most frequent editing events observed for two of the three genes. A comparative assessment of editing frequencies between a codon-optimized Cas9 for expression in algae, crCas9, and a plant codon-optimized Cas9, pcoCas9, showed more consistent results with the vector expressing pcoCas9. Editing of TaNFXL1 by co-expression of sgRNA pair was investigated in transgenic wheat plants. Given the ploidy of bread wheat, a rapid, robust and inexpensive genotyping protocol was also adapted for hexaploid genomes and shown to be a useful tool to identify homoeolog-specific editing events in wheat. Conclusions Co-expressed pairs of sgRNA targeting single genes in conjunction with the CRISPR/Cas9 system produced large deletions in wheat. In addition, a genotyping protocol to identify editing events in homoeologs of TaNFXL1 was successfully adapted.