Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 121205, Russia.
Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, U.S.A.
Biochem Soc Trans. 2020 Feb 28;48(1):257-269. doi: 10.1042/BST20190662.
Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.
原核生物适应性免疫是通过将称为间隔区的短 DNA 片段获取到 CRISPR（成簇规律间隔短回文重复序列）阵列中而建立的。CRISPR 适应性是一个多步骤的过程，包括前间隔区的选择、产生和整合到阵列中。适应后，间隔区通过识别互补的核酸序列提供免疫，将其引导进行破坏。因此，为了防止有害的自身免疫，CRISPR 适应性必须是一个高度调控和不频繁的过程，至少在没有遗传入侵者的情况下是这样。多年来，已经开发出了许多巧妙的方法来研究 CRISPR 适应性。在本文中，我们讨论并比较了在体内检测 CRISPR 适应性及其中间产物的方法，并提出了抑制 PCR 作为一种简单的改进，以增加检测间隔区获取的敏感性来监测该过程。
