Genome editing through large insertion leads to the skipping of targeted exon.
Genome editing through large insertion leads to the skipping of targeted exon.
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DOI：
10.1186/s12864-015-2284-8
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发表时间：
2015-12-21
期刊：
影响因子：
4.4
通讯作者：
Schiebel E
中科院分区：
文献类型：
--
作者：
Uddin B;Chen NP;Panic M;Schiebel E
通过将核酸内切酶定向到特定的感兴趣基因位点，可以实现高效的基因组编辑。工程锌指核酸酶(ZFN)和CRISPR相关蛋白-9核酸酶(Cas9)为脊椎动物细胞的基因组编辑提供了这样一种优雅的方法。在这项研究中，我们利用ZFN和Cas9催化的双链断裂以及同源重组介导的提前终止密码子和选择标记的掺入来靶向人类细胞分裂周期14A(HCDC14A)和细胞分裂周期14B(HCDC14B)基因。Southern印迹杂交证实hCDC14A和hCDC14B在端粒酶永生化的人视网膜色素上皮(hTERT-RPE1)和人结肠癌(HCT116)细胞中具有靶向性。反转录-聚合酶链式反应(RT-PCR)产物的DNA序列分析证实，在所有的单/双等位基因消融中，目标外显子都被剪接掉了。外显子跳跃现象与所采用的基因组编辑方法Cas9或ZFN无关。因为外显子有一个可以被3整除的核苷酸编号，所以外显子缺失的读框保持不变。这表明可能是由于在目标外显子内插入了较大的DNA片段(1.7至2.5kb)而导致了外显子跳过事件。作为原则的证明，我们使用了基因断裂之后的非同源末端连接(NHEJ)方法。外显子的小变化(1到15个碱基)被转录成信使核糖核酸，而不跳过外显子。此外，loxP位点介导的选择标记的移除在目标外显子内留下了一条45bp的疤痕，可以在mRNA中追踪到，而不会跳过外显子。从这项研究中，我们得出结论，通过基因组编辑将大的DNA片段插入到外显子中可以导致从最终转录本中跳过它。因此，在设计靶点时需要采取更谨慎的方法，使靶向外显子的可能跳过导致框架移位介导的早熟终止密码子的整合。另一方面，跳过外显子可能是引入蛋白质缺失的一种有用的策略。本文的在线版本(doi：10.1186/s12864-0152284-8)包含补充材料，授权用户可以使用。
Highly efficient genome editing can be achieved through targeting an endonuclease to specific locus of interest. Engineered zinc-finger nuclease (ZFN) and CRISPR-associated protein-9 nuclease (Cas9) offer such an elegant approach for genome editing in vertebrate cells. In this study, we have utilized ZFN and Cas9-catalyzed double strand break followed by homologous recombination-mediated incorporation of premature stop codon and selection marker to target human cell division cycle 14A (hCDC14A) and cell division cycle 14B (hCDC14B) genes. Targeting of the hCDC14A and hCDC14B loci in telomerase immortalized human retinal pigment epithelium (hTERT-RPE1) and human colon cancer (HCT116) cells were confirmed by Southern blot hybridization. Nevertheless, DNA sequence analysis of reverse transcription polymerase chain reaction (RT-PCR) products confirmed that in all the single/double allele ablations, the targeted exon was spliced out. The phenomenon of exon skipping was independent of the genome editing approaches exploited, Cas9 or ZFN. Because the exons had a nucleotide number that could be divided by 3, the reading frame of the exon deletion was maintained. This indicates an exon-skipping event possibly due to the insertion of large DNA fragment (1.7 to 2.5 Kb) within the targeted exons. As a proof-of-principle, we have used gene disruption followed by non-homologous end joining (NHEJ) approach. Small alterations in the exon (one to fifteen bases) were transcribed to mRNA without exon skipping. Furthermore, loxP site-mediated removal of selection markers left a 45 bp scar within the targeted exon that can be traced in mRNA without exon skipping. From this study, we conclude that insertion of a large DNA fragment into an exon by genome editing can lead to its skipping from the final transcript. Hence, more cautious approach needs to be taken while designing target sites in such that the possible skipping of targeted exon causes a frame-shift mediated incorporation of pre-mature stop codon. On the other hand, exon skipping may be a useful strategy for the introduction of protein deletions. The online version of this article (doi:10.1186/s12864-015-2284-8) contains supplementary material, which is available to authorized users.