A Rad51-independent pathway promotes single-strand template repair in gene editing.
A Rad51-independent pathway promotes single-strand template repair in gene editing.
复制标题
RAD51非依赖途径在基因编辑中促进单链模板修复。
DOI：
10.1371/journal.pgen.1008689
复制
发表时间：
2020-10
期刊：
影响因子：
4.5
通讯作者：
Haber JE
中科院分区：
文献类型：
--
作者：
Gallagher DN;Pham N;Tsai AM;Janto NV;Choi J;Ira G;Haber JE
重组酶的Rad 51/RecA家族在双链断裂（DSB）的典型修复中执行关键功能：切除的DSB末端进入同源双链DNA（dsDNA）模板序列的链侵入以启动修复。然而，使用单链DNA（ssDNA）作为模板的DSB的修复（CRISPR/Cas9介导的基因编辑的常见方法）是Rad 51不依赖的。我们通过用位点特异性HO内切核酸酶创建DSB并用80-nt单链寡核苷酸（ssODN）修复DSB，分析了酿酒酵母中这些Rad 51非依赖性事件的遗传要求，并通过Cas9介导的DSB与体内产生ssDNA模板的细菌retron系统的组合证实了这些结果。我们发现，单链模板修复（SSTR），是依赖于Rad 52，Rad 59，Srs 2和Mre 11-Rad 50-Xrs 2（MRX）复合物，但不像其他Rad 51独立的重组事件，独立于Rdh 54。我们表明，Rad 59的行为，以减轻抑制Rad 51对Rad 52的链退火活性在SSTR和单链退火（SSA）。当引入相同大小和序列的双链寡核苷酸作为模板时，基因编辑是Rad 51依赖性的。基因编辑期间错配的同化取决于Msh 2的活性，其在ssODN的3'侧的作用非常不同，与5'端相比，其可以直接退火至切除的DSB端。此外，DNA聚合酶Polδ的3'至5'校对活性经常切除非常接近模板3'末端的错配。我们进一步报告说，SSTR伴随着多达600倍的增加，在邻近区域的序列直接进行修复的突变。这些DNA聚合酶依赖性突变可能会损害基因编辑的准确性。DNA双链断裂（DSB）是染色体上最致命的损伤类型之一，无法修复此类损伤可能导致染色体不稳定，通常与人类癌症相关。DNA修复机制的知识在基因治疗的开发中也是至关重要的，这一过程包括故意破坏DNA以修改遗传序列。在这里，我们比较了在芽殖酵母中产生DSB的两种位点特异性方法（HO内切核酸酶和CRISPR/Cas9），以通过单链DNA模板修复（SSTR）修饰几种DNA靶标。我们表明，基因编辑使用DSB修复途径，该途径独立于经典修复蛋白Rad 51，并且与先前研究的Rad 51独立途径不同，因为它需要其他几种已知的重组蛋白。我们在基因编辑和单链退火中都表明，Rad 59可以抑制Rad 51对Rad 52链退火活性的调节。我们还确定了模板中的错配如何被整合到基因组中，并且这种同化反映了Msh 2介导的错配修复以及Polδ介导的校对的不同方面。这些见解通过这一重要的基因编辑途径深入了解了DSB修复的机制。
The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52’s strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3’ side of the ssODN which can anneal directly to the resected DSB end compared to the 5’ end. In addition DNA polymerase Polδ’s 3’ to 5’ proofreading activity frequently excises a mismatch very close to the 3’ end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing. DNA double strand breaks (DSBs) are one of the most lethal types of damage that can be inflicted on a chromosome and failure to repair such lesions can result in chromosome instability, commonly associated with human cancer. A knowledge of DNA repair mechanisms is also critical in the exploitation of gene therapy, a process that includes intentionally breaking the DNA to modify the genetic sequence. Here we compared two site-specific methods to create DSBs (HO endonuclease and CRISPR/Cas9) in budding yeast, to modify several DNA targets by single-strand DNA template repair (SSTR). We show that gene editing uses a DSB repair pathway that is independent of the canonical repair protein Rad51 and distinct from previously studied Rad51-independent pathways in its requirements for several other known recombination proteins. We show both in gene editing and in single-strand annealing that Rad59 acts to suppress the modulation of Rad52’s strand annealing activity by Rad51. We also determined how mismatches in the template are incorporated into the genome, and that this assimilation reflects different aspects of Msh2-mediated mismatch repair as well as Polδ-mediated proofreading. These insights provide insight into the mechanisms of DSB repair by this important gene-editing pathway.
登录
查看更多内容
影响因子：
4
作者：
Gallagher DN;Haber JE
通讯作者：
Haber JE
影响因子：
3.3
作者：
Cortés-Ledesma, F;Malagón, F;Aguilera, A
通讯作者：
Aguilera, A
影响因子：
64.8
作者：
Costelloe, Thomas;Louge, Raphael;Tomimatsu, Nozomi;Mukherjee, Bipasha;Martini, Emmanuelle;Khadaroo, Basheer;Dubois, Kenny;Wiegant, Wouter W.;Thierry, Agnes;Burma, Sandeep;van Attikum, Haico;Llorente, Bertrand
通讯作者：
Llorente, Bertrand
影响因子：
14.9
作者：
Cassani, Corinne;Gobbini, Elisa;Longhese, Maria Pia
通讯作者：
Longhese, Maria Pia
影响因子：
56.9
作者：
BARDWELL, AJ;BARDWELL, L;FRIEDBERG, EC
通讯作者：
FRIEDBERG, EC