Marker Recycling in Candida albicans through CRISPR-Cas9-Induced Marker Excision.
Marker Recycling in Candida albicans through CRISPR-Cas9-Induced Marker Excision.
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DOI：
10.1128/msphere.00050-17
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发表时间：
2017-03
期刊：
影响因子：
4.8
通讯作者：
Mitchell AP
中科院分区：
文献类型：
--
作者：
Huang MY;Mitchell AP
关键是要能够改变基因，以阐明其功能。这些改变通常依赖于标记，这些标记允许在已经整合了一段DNA的群体中选择稀有细胞。因此，可以实现的改变的数量受到可用的选择标记的数量的限制。这种限制是规避标记回收战略，其中一个标记是消除后，其首次使用。然后，标记可以再次使用。在这份报告中，我们描述了一种新的标记回收策略，该策略由最近开发的CRISPR-Cas9技术实现。我们在这里描述了一种新的方法来标记回收，一个控制序列的步骤中，遗传标记被选择，然后丢失。标记再循环对于遗传操作很重要，因为它允许重复使用单个选择标记。我们的方法依赖于CRISPR-Cas9系统在DNA中产生靶向双链断裂的能力，以及选择标记内的双链断裂可以促进标记侧翼的直接重复序列之间的重组的预期。我们称这种方法为CRISPR-Cas9诱导的标记切除（CRIME）。我们测试了这种方法的实用性与真菌病原体白色念珠菌，这是典型的二倍体。我们使用了两个选择标记，修饰为包括侧翼直接重复。在原理验证研究中，我们通过使用两个标记在三个基因中创建了连续的纯合缺失，并且在最终菌株中具有可用于进一步选择和回收的标记之一。这一策略将加速白色念珠菌中多突变株的产生。CRISPR-Cas9系统已经应用于许多生物体，因此本文所述的遗传设计原理可能具有广泛的适用性。重要性能够改变基因以阐明其功能是至关重要的。这些改变通常依赖于标记，这些标记允许在已经整合了一段DNA的群体中选择稀有细胞。因此，可以实现的改变的数量受到可用的选择标记的数量的限制。这种限制是规避标记回收战略，其中一个标记是消除后，其首次使用。然后，标记可以再次使用。在这份报告中，我们描述了一种新的标记回收策略，该策略由最近开发的CRISPR-Cas9技术实现。
It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology. We describe here a new approach to marker recycling, a controlled sequence of steps in which a genetic marker is selected and then lost. Marker recycling is important for genetic manipulation, because it allows a single selection marker to be used repeatedly. Our approach relies upon the ability of the CRISPR-Cas9 system to make a targeted double-strand break in DNA and the expectation that a double-strand break within a selection marker may promote recombination between directly repeated sequences that flank the marker. We call the approach CRISPR-Cas9-induced marker excision (CRIME). We tested the utility of this approach with the fungal pathogen Candida albicans, which is typically diploid. We used two selection markers, modified to include flanking direct repeats. In a proof-of-principle study, we created successive homozygous deletions in three genes through use of the two markers and had one of the markers available in the final strain for further selection and recycling. This strategy will accelerate the creation of multiple-mutant strains in C. albicans. CRISPR-Cas9 systems have been applied to many organisms, so the genetic design principles described here may be broadly applicable. IMPORTANCE It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology.