Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.
Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.
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DOI：
10.1016/j.xcrm.2023.101037
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发表时间：
2023-05-16
影响因子：
14.3
通讯作者：
Jain, Piyush K.
中科院分区：
文献类型：
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作者：
Nguyen, Long T.;Rananaware, Santosh R.;Yang, Lilia G.;Macaluso, Nicolas C.;Ocana-Ortiz, Julio E.;Meister, Katelyn S.;Pizzano, Brianna L. M.;Sandoval, Luke Samuel W.;Hautamaki, Raymond C.;Fang, Zoe R.;Joseph, Sara M.;Shoemaker, Grace M.;Carman, Dylan R.;Chang, Liwei;Rakestraw, Noah R.;Zachary, Jon F.;Guerra, Sebastian;Perez, Alberto;Jain, Piyush K.
基于CRISPR-Cas的诊断有可能提高核酸检测水平。CRISPR-Cas系统可以与一锅反应中的预扩增步骤相结合，以简化工作流程并减少遗留污染。在这里，我们报告了一种具有改进的热稳定性的工程化Cas 12 b，其福尔斯逆转录环介导等温扩增（RT-LAMP）的最佳温度范围（60°C-65°C）内。使用从头结构分析，我们将突变引入野生型BrCas 12 b以收紧其疏水核心，从而增强热稳定性。利用工程化BrCas 12 b的一锅法检测测定，称为SPLENDID（用于感染性疾病的核酸检测的单锅法LAMP介导的工程化BrCas 12 b），在一锅法设置中在高达67°C下表现出稳健的反式切割活性。我们在80份丙型肝炎病毒（HCV）血清样本和66份严重急性呼吸综合征冠状病毒2（SARS-CoV-2）唾液样本中对SPLENDID进行了临床验证，具有较高的特异性和准确性。我们可以在短短20分钟内获得结果，并且通过提取过程，整个分析可以在一小时内进行。机器学习工具使Cas的工程化能够提高其热稳定性eBrCas 12 b允许RT-LAMP和CRISPR-Cas在一个锅中组合，创建SPLENDID SPLENDID能够以高准确度临床检测血清中的HCV RNA SPLENDID能够以高准确度临床检测唾液中的SARS-CoV-2 RNA Nguyen et al.改造CRISPR相关的Cas蛋白以提高其热稳定性。工程化Cas允许在简单的一锅法测试中结合靶扩增和检测步骤，该测试证明了检测HCV和SARS-CoV-2 RNA的高临床准确性，并且可以容易地扩展到其他靶点。
CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C–65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour. Machine learning tools enable engineering of Cas to improve its thermostability eBrCas12b allows RT-LAMP and CRISPR-Cas to be combined in one pot, creating SPLENDID SPLENDID enables clinical detection of HCV RNA in serum with high accuracy SPLENDID enables clinical detection of SARS-CoV-2 RNA in saliva with high accuracy Nguyen et al. engineer the CRISPR-associated Cas protein to improve its thermostability. Engineered Cas allows combining target amplification and detection steps in a simple one-pot test that demonstrates high clinical accuracy for detecting HCV and SARS-CoV-2 RNAs and can be easily extended to other targets.
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影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
64.5
作者：
Fozouni P;Son S;Díaz de León Derby M;Knott GJ;Gray CN;D'Ambrosio MV;Zhao C;Switz NA;Kumar GR;Stephens SI;Boehm D;Tsou CL;Shu J;Bhuiya A;Armstrong M;Harris AR;Chen PY;Osterloh JM;Meyer-Franke A;Joehnk B;Walcott K;Sil A;Langelier C;Pollard KS;Crawford ED;Puschnik AS;Phelps M;Kistler A;DeRisi JL;Doudna JA;Fletcher DA;Ott M
通讯作者：
Ott M
影响因子：
56.9
作者：
Chen, Janice S.;Ma, Enbo;Doudna, Jennifer A.
通讯作者：
Doudna, Jennifer A.
使用逆转录环介导的等温扩增测定法快速目视检测丙型肝炎病毒。
DOI：
10.1016/j.ijid.2020.10.082
发表时间：
2021-01
期刊：
International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases
影响因子：
--
作者：
Hongjaisee S;Doungjinda N;Khamduang W;Carraway TS;Wipasa J;Debes JD;Supparatpinyo K
通讯作者：
Supparatpinyo K
DOI：
10.1186/s10020-021-00289-0
发表时间：
2021-03-26
期刊：
Molecular medicine (Cambridge, Mass.)
影响因子：
--
作者：
de Oliveira Coelho B;Sanchuki HBS;Zanette DL;Nardin JM;Morales HMP;Fornazari B;Aoki MN;Blanes L
通讯作者：
Blanes L