The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation
The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation
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DOI：
10.1111/pbi.12200
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发表时间：
2014-08-01
影响因子：
13.8
通讯作者：
Zhu, Jian-Kang
中科院分区：
文献类型：
--
作者：
Zhang, Hui;Zhang, Jinshan;Zhu, Jian-Kang
CRISPR/Cas9系统已被证明可以在包括植物在内的多种生物体中有效诱导靶向基因编辑。最近的工作表明，CRISPR/Cas9诱导的拟南芥基因突变在早期世代大多是体细胞突变，尽管有些突变可以在后代中稳定遗传。然而，目前还不清楚这种系统是否会在水稻等作物中发挥类似的作用。在这项研究中，我们在两个水稻亚种中测试了11个靶基因对CRISPR/Cas9诱导的编辑的适应性，并确定了基因修饰的模式，特异性和遗传性。对第一代转化植物（T0）中编辑基因的基因型和频率的分析表明，CRISPR/Cas9系统在水稻中是高效的，在第一次细胞分裂之前，近一半的转化胚性细胞中编辑了靶基因。在T0植物中容易发现编辑的靶基因的纯合子。基因突变按照经典的孟德尔定律传递给下一代（T1），没有任何可检测到的新突变或逆转。即使进行了包括全基因组重测序在内的广泛搜索，我们也没有发现任何证据表明本研究中测试的许多靶标中的任何一个在水稻中大规模脱靶。通过对大量T0植物的推定脱靶位点进行特异性测序，仅在一个脱靶位点中发现低频突变，该位点的序列与预期靶点有1 bp的差异。总的来说，这项研究中的数据表明CRISPR/Cas9系统是作物基因组工程的有力工具。
The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9-induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9-induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large-scale off-targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off-target sites of a large number of T0 plants, low-frequency mutations were found in only one off-target site where the sequence had 1-bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.