Odipio John, Alicai Titus, Ingelbrecht Ivan, Nusinow Dmitri A, Bart Rebecca, Taylor Nigel J
Donald Danforth Plant Science Center, St. Louis, MO, United States.
National Crops Resources Research Institute, Kampala, Uganda.
Front Plant Sci. 2017 Oct 18;8:1780. doi: 10.3389/fpls.2017.01780. eCollection 2017.
CRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava (), the () gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within exon 13. After -mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analyzed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90 to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence analysis revealed that 38/38 (100%) of the plant lines examined carried mutations at the targeted site, with insertions, deletions, and substitutions recorded. One putatively mono-allelic homozygous line (1/19) was found from cv. 60444, while 1 (1/19) and 4 (4/19) putatively bi-allelic homozygous lines were found in 60444 and TME204, respectively. The remaining plant lines, comprised mostly of the chimeras, were found to be putatively heterozygous. We observed minor (1 bp) nucleotide substitutions and or deletions upstream of the 5' and or downstream of the 3' targeted region. The data reported demonstrates that CRISPR/Cas9-mediated genome editing of cassava is highly efficient and relatively simple, generating multi-allelic mutations in both cultivars studied. Modification of described here generates visually detectable mutated events in a relatively short time frame of 6-8 weeks, and does not require sequencing to confirm editing at the target. It therefore provides a valuable platform to facilitate rapid assessment and optimization of CRISPR/Cas9 and other genome-editing technologies in cassava.
CRISPR/Cas9已成为一种强大的基因组编辑工具，可用于在作物物种中引入基因变化。为了开发热带主要作物木薯的CRISPR/Cas9技术能力，使用携带靶向第13外显子内两个序列的gRNA的构建体，在两个品种中靶向（）基因。在通过农杆菌介导将CRISPR/Cas9试剂导入木薯细胞后，两种构建体在选择培养基上再生的子叶期体细胞胚中诱导出可见的白化表型，以及由此再生的植株。共获得了58株（品种60444）和25株（品种TME 204）植株系，其中对38株植株系（每个品种19株）进行了诱变分析。表现出白化表型的植株系频率很高，在品种TME 204中为90%至100%。观察到的白化表型包括完全没有绿色组织的白化苗和含有白色和绿色组织混合物的嵌合体。序列分析表明，所检测的38/38（100%）植株系在靶向（）位点携带突变，记录到插入、缺失和替换。从品种60444中发现了1个推定的单等位基因纯合系（1/19），而在60444和TME204中分别发现了1个（1/19）和4个（4/19）推定的双等位基因纯合系。其余植株系大多为嵌合体，被发现为推定的杂合子。我们在5'端上游和/或3'端靶向（）区域下游观察到小的（1 bp）核苷酸替换和/或缺失。所报道的数据表明，CRISPR/Cas9介导的木薯基因组编辑效率高且相对简单，在所研究的两个品种中均产生了多等位基因突变。本文所述的修饰在6至8周的相对短时间内产生了视觉上可检测的突变事件，并且不需要测序来确认靶点处的编辑。因此，它提供了一个有价值的平台，便于快速评估和优化木薯中的CRISPR/Cas9和其他基因组编辑技术。
