Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii.
Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii.
复制标题
DOI：
10.1038/s41467-021-27004-1
复制
发表时间：
2021-11-19
影响因子：
16.6
通讯作者：
Molnar A
中科院分区：
文献类型：
--
作者：
Ferenczi A;Chew YP;Kroll E;von Koppenfels C;Hudson A;Molnar A
在CRISPR/Cas精确基因组编辑中，单链寡核苷酸被广泛用作DNA修复模板。然而，单链模板DNA修复(SSTR)的潜在机制还没有被充分了解，限制了对精确编辑的合理改进。在这里，我们研究了CRISPR/Cas12a在真核模型绿色微藻Chlamydomonas rehardtii中诱导的DNA双链断裂(DSB)的SSTR。我们证明了在Cas12a诱导的DSB的SSTR过程中，ssODN物理地整合到基因组中。这一过程在遗传上独立于依赖RAD51的同源重组和Fanconi贫血途径，被非同源末端连接强烈拮抗，并且几乎完全由另一种末端连接酶聚合酶θ介导。这些发现表明，莱茵梭子蟹和动物之间的SSTR存在差异。我们的工作说明了莱茵梭菌作为研究核DNA修复的模式生物的前景。在基因组编辑过程中，单链寡核苷酸经常被用作DNA修复的模板。在这里，作者表明，与动物不同，莱茵衣藻的单链模板dna修复依赖于另一种末端连接酶聚合酶θ。
Single-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair. Single-stranded oligodeoxynucleotides are often used as templates for DNA repair during genome editing. Here the authors show that, unlike in animals, single-strand templated DNA repair in Chlamydomonas reinhardtii relies on the alternative end-joining enzyme polymerase θ.
登录
查看更多内容
影响因子：
14.9
作者：
Brinkman, Eva K.;Kousholt, Arne N.;van Steensel, Bas
通讯作者：
van Steensel, Bas
影响因子：
16.8
作者：
Kent, Tatiana;Chandramouly, Gurushankar;McDevitt, Shane Michael;Ozdemir, Ahmet Y.;Pomerantz, Richard T.
通讯作者：
Pomerantz, Richard T.
影响因子：
7
作者：
Kan Y;Ruis B;Takasugi T;Hendrickson EA
通讯作者：
Hendrickson EA
影响因子：
3
作者：
Gautam, Shweta D.;Chen, Jon K.;Murray, Vincent
通讯作者：
Murray, Vincent
影响因子：
11.4
作者：
DEBUCHY, R;PURTON, S;ROCHAIX, JD
通讯作者：
ROCHAIX, JD