Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada
mSphere. 2019 Aug 21;4(4):e00408-19. doi: 10.1128/mSphere.00408-19.
CRISPR-Cas9 genome editing relies on an efficient double-strand DNA break (DSB) and repair. Contrary to mammalian cells, the protozoan parasite lacks the most efficient nonhomologous end-joining pathway and uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair to repair DSBs. Here, we reveal that predominantly uses single-strand annealing (SSA) (>90%) instead of MMEJ (<10%) for DSB repair (DSBR) following CRISPR targeting of the miltefosine transporter gene, resulting in 9-, 18-, 20-, and 29-kb sequence deletions and multiple gene codeletions. Strikingly, when targeting the LdBPK_241510 gene, SSA even occurred by using direct repeats 77 kb apart, resulting in the codeletion of 15 genes, though with a reduced frequency. These data strongly indicate that DSBR is not efficient in , which explains why more than half of DSBs led to cell death and why the CRISPR gene-targeting efficiency is low compared with that in other organisms. Since direct repeat sequences are widely distributed in the genome, we predict that many DSBs created by CRISPR are repaired by SSA. It is also revealed that DNA polymerase theta is involved in both MMEJ and SSA in Collectively, this study establishes that DSBR mechanisms and their competence in an organism play an important role in determining the outcome and efficacy of CRISPR gene targeting. These observations emphasize the use of donor DNA templates to improve gene editing specificity and efficiency in In addition, we developed a novel Cas9 constitutive expression vector (pLdSaCN) for gene targeting in Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in .
CRISPR-Cas9 基因组编辑依赖于高效的双链 DNA 断裂 (DSB) 和修复。与哺乳动物细胞不同，原生动物寄生虫 缺乏最有效的非同源末端连接途径，而是使用微同源介导的末端连接 (MMEJ)，偶尔使用同源定向修复来修复 DSB。在这里，我们揭示了 在 CRISPR 靶向 miltefosine 转运基因后，主要使用单链退火 (SSA) (>90%)而不是 MMEJ(<10%)进行 DSB 修复 (DSBR)，导致 9、18、20 和 29kb 序列缺失和多个基因缺失。引人注目的是，当靶向 LdBPK_241510 基因时，SSA 甚至发生在相距 77kb 的直接重复之间，导致 15 个基因的缺失，尽管频率降低。这些数据强烈表明， 在 中，DSBR 效率不高，这解释了为什么超过一半的 DSB 导致细胞死亡，以及为什么与其他生物体相比，CRISPR 基因靶向效率较低。由于直接重复序列广泛分布在 基因组中，我们预测由 CRISPR 产生的许多 DSB 通过 SSA 修复。研究还表明，DNA 聚合酶θ参与 中的 MMEJ 和 SSA。 综上所述，本研究确立了在生物体中，DSBR 机制及其能力在决定 CRISPR 基因靶向的结果和效果方面起着重要作用。这些观察结果强调了使用供体 DNA 模板来提高 中的基因编辑特异性和效率。此外，我们还为 中的基因靶向开发了一种新型的 Cas9 组成型表达载体 (pLdSaCN)。 由于双链 DNA 断裂 (DSB) 修复机制的差异，CRISPR-Cas9 基因编辑效率在不同生物体中可能有很大差异。与哺乳动物细胞不同，原生动物寄生虫 使用微同源介导的末端连接 (MMEJ)，偶尔使用同源定向修复 (HDR) 来修复 DSB，但缺乏非同源末端连接途径。在这里，我们表明 主要使用单链退火 (SSA) 而不是 MMEJ 进行 DSB 修复 (DSBR)，导致大的缺失，其中可能包括多个基因。这强烈表明 在 中整体的 DSBR 效率低下，因此会影响 CRISPR-Cas9 基因编辑的结果，突出了在 中使用供体 DNA 提高基因编辑保真度和效率的重要性。
