SPRINT: a Cas13a-based platform for detection of small molecules.
SPRINT: a Cas13a-based platform for detection of small molecules.
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DOI：
10.1093/nar/gkaa673
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发表时间：
2020-09-25
影响因子：
14.9
通讯作者：
Batey RT
中科院分区：
文献类型：
--
作者：
Iwasaki RS;Batey RT
最近在生物工程方面的努力使得使用基于crispr的方法检测样品中的核酸更加快速、廉价和敏感。我们扩展了其中一种基于cas13的方法，在一次检测中检测小分子。利用基于sherlock的体外转录分析（SPRINT），体外转录的RNA序列特异性地触发Cas13a的RNase活性。该事件激活其非特异性RNA酶活性，使具有猝灭剂/荧光团对标记的RNA寡核苷酸能够裂解，从而使荧光团去猝灭。这种荧光输出可以测量，以评估转录输出。利用核糖体开关或蛋白质通过特定的效应分子来调节转录，作为一种将效应浓度转化为荧光强度的耦合分析。通过这种方法，我们定量了样品中的8种不同的化合物，包括辅因子、核苷酸、氨基酸代谢物、四环素和单原子离子。用这种方法，几百个反应可以在几个小时内很容易地量化。这种增加的吞吐量也使转录调节剂，抑制转录的合成化合物或其他偶联酶反应的详细表征成为可能。这些SPRINT反应很容易适应便携式格式，因此可用于现场或护理点的分析物检测。
Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.
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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：
Ackerman, Cheri M.;Myhrvold, Cameron;Sabeti, Pardis C.
通讯作者：
Sabeti, Pardis C.
影响因子：
4.7
作者：
Chang HJ;Mayonove P;Zavala A;De Visch A;Minard P;Cohen-Gonsaud M;Bonnet J
通讯作者：
Bonnet J
影响因子：
64.8
作者：
Batey, RT;Gilbert, SD;Montange, RK
通讯作者：
Montange, RK
DOI：
10.1126/science.1215063
发表时间：
2012-01-13
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Baker JL;Sudarsan N;Weinberg Z;Roth A;Stockbridge RB;Breaker RR
通讯作者：
Breaker RR