Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles.
Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles.
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DOI：
10.1038/s41467-021-26989-z
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发表时间：
2021-11-15
影响因子：
16.6
通讯作者：
Gagnon KT
中科院分区：
文献类型：
--
作者：
Ageely EA;Chilamkurthy R;Jana S;Abdullahu L;O'Reilly D;Jensik PJ;Damha MJ;Gagnon KT
CRISPR-CAS12A是用于开发模型生物，治疗和诊断的领先技术，这些应用可以从稳定或调整酶特性的化学修饰中受益。介导与CAS12A的结合。预测的2'-羟基接触替换了这些RNA的核苷酸均保留了2'-羟基敏感性，而在磷酸盐连接中，有6个h羟基敏感性。基于鲁棒的细胞编辑，较高的伪动物通常可以使用CIS活性来保留5'。当设计以结构和化学兼容性为指导时，伪诺可以忍受几乎完全的修改。
CAS12A用于人类治疗和诊断的开发可能会大大受益于其指南RNA的化学修饰。细胞的活性和体外反式裂解活性。
CRISPR-Cas12a is a leading technology for development of model organisms, therapeutics, and diagnostics. These applications could benefit from chemical modifications that stabilize or tune enzyme properties. Here we chemically modify ribonucleotides of the AsCas12a CRISPR RNA 5′ handle, a pseudoknot structure that mediates binding to Cas12a. Gene editing in human cells required retention of several native RNA residues corresponding to predicted 2′-hydroxyl contacts. Replacing these RNA residues with a variety of ribose-modified nucleotides revealed 2′-hydroxyl sensitivity. Modified 5′ pseudoknots with as little as six out of nineteen RNA residues, with phosphorothioate linkages at remaining RNA positions, yielded heavily modified pseudoknots with robust cell-based editing. High trans activity was usually preserved with cis activity. We show that the 5′ pseudoknot can tolerate near complete modification when design is guided by structural and chemical compatibility. Rules for modification of the 5′ pseudoknot should accelerate therapeutic development and be valuable for CRISPR-Cas12a diagnostics. Development of Cas12a for human therapeutics and diagnostics may significantly benefit from, or even require, chemical modification of its guide RNA. Here the authors show that the noncanonical 5′ pseudoknot structure of the AsCas12a crRNA guide can be heavily modified and still retain very high editing activity in cells and trans cleavage activity in vitro.
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影响因子：
16.6
作者：
Cromwell CR;Sung K;Park J;Krysler AR;Jovel J;Kim SK;Hubbard BP
通讯作者：
Hubbard BP
DOI：
10.1126/science.aaf5573
发表时间：
2016-08-05
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Abudayyeh OO;Gootenberg JS;Konermann S;Joung J;Slaymaker IM;Cox DB;Shmakov S;Makarova KS;Semenova E;Minakhin L;Severinov K;Regev A;Lander ES;Koonin EV;Zhang F
通讯作者：
Zhang F
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
DOI：
10.1038/nrd.2016.238
发表时间：
2017-02
期刊：
Nature reviews. Drug discovery
影响因子：
--
作者：
Fellmann C;Gowen BG;Lin PC;Doudna JA;Corn JE
通讯作者：
Corn JE
影响因子：
14.9
作者：
AURUP, H;TUSCHL, T;ECKSTEIN, F
通讯作者：
ECKSTEIN, F