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科学家开发对天然和工程CRISPR核酸酶的大规模并行动力学分析平台
作者:小柯机器人 发布时间:2020/9/9 17:48:08

美国德克萨斯大学奥斯汀分校Ilya J. Finkelstein研究组开发出大规模并行动力学分析自然和改造CRISPR核酸酶的平台。该项研究成果发表在2020年9月7日的《自然—生物技术》上。

该工作中,课题组研究人员提出了NucleaSeq(nuclease digestion and deep sequencing,核酸酶消化及深度测序),是一个大规模并行平台,针对超过10000个目标DNA(包括相对于指导RNA存在不匹配、插入或者删除的)测量了切割动力学以及时间分辨的切割产物。结合在同一目标库上的切割速率和结合特异性,课题组对五个SpCas9变体和AsCas12a进行了基准测试。基于这些数据集,课题组建立生物物理学模型,揭示了脱靶切割的机理性信息。

工程改造的Cas9s,尤其是Cas9-HF1,相比于野生型(wt)Cas9,切割特异性而不是结合特异性极大地提高了。令人惊讶的是,AsCas12a切割特异性与wtCas9相差无几。最初DNA切割位点和末端修饰对于不同核酸酶、指导RNA和错配核苷酸的位置各异。更广泛地说,NucleaSeq实现了对在工程和天然核酸酶的快速、定量和系统性地比较特异性和切割结果。

据了解,工程改造的SpCas9s和AsCas12a裂解的脱靶基因组位点比野生型Cas9少。然而,了解它们的保真度、机制和切割结果需要对错配目标DNA的系统性分析。

附:英文原文

Title: Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

Author: Stephen K. Jones, John A. Hawkins, Nicole V. Johnson, Cheulhee Jung, Kuang Hu, James R. Rybarski, Janice S. Chen, Jennifer A. Doudna, William H. Press, Ilya J. Finkelstein

Issue&Volume: 2020-09-07

Abstract: Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq—nuclease digestion and deep sequencing—a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.

DOI: 10.1038/s41587-020-0646-5

Source: https://www.nature.com/articles/s41587-020-0646-5

期刊信息

Nature Biotechnology:《自然—生物技术》,创刊于1996年。隶属于施普林格·自然出版集团,最新IF:31.864
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex