Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Cas13d

Abstract

Summary CRISPR-Cas endonucleases directed against foreign nucleic acids mediate prokaryotic adaptive immunity and have been tailored for broad genetic engineering applications. Type VI-D CRISPR systems contain the smallest known family of single effector Cas enzymes, and their signature Cas13d ribonuclease employs guide RNAs to cleave matching target RNAs. To understand the molecular basis for Cas13d function and explain its compact molecular architecture, we resolved cryo-electron microscopy structures of Cas13d-guide RNA binary complex and Cas13d-guide-target RNA ternary complex to 3.4 and 3.3 Å resolution, respectively. Furthermore, a 6.5 Å reconstruction of apo Cas13d combined with hydrogen-deuterium exchange revealed conformational dynamics that have implications for RNA scanning. These structures, together with biochemical and cellular characterization, provide insights into its RNA-guided, RNA-targeting mechanism and delineate a blueprint for the rational design of improved transcriptome engineering technologies.

The rapidly advancing Class 2 CRISPR‐Cas technologies: A customizable toolbox for molecular manipulations

“…Cas13d is a new RNA‐targeting effector whose crystal structure has recently been defined (Table and Figure C) . Similar to other Cas13, it has two HEPN domains responsible for pre‐crRNA processing (Figure C) .…”

“…B, The domain organizations of type V effectors, FnCas12a, LbCas12a, AsCas12a, AacCas12b, and BthCas12b . C, The domain organizations of type VI effectors, LshCas13a, PbCas13b, EsCas13d, and UrCas13d . BH, bridge helix; Hel, helical; IDL, inter‐domain linker; L, linker; LHD, looped‐out helical domain; NTD, N‐terminal domain; OBD, oligonucleotide‐binding domain; PI, PAM‐interacting; PLL, phosphate lock loop; WED, wedge.…”

RNA-Targeting CRISPR–Cas Systems and Their Applications

“…It has been used to knock-down RNA in bacteria [47], plant cells [71], and several types of mammalian cells [53,55,66]. Cas13d from Ruminococcus flavefaciens was truncated to generate its minimal functional variant [75]. The reason for that was to obtain a Cas13 was small enough to be delivered by viral vectors.…”

Development of CRISPR-Cas systems for genome editing and beyond

“…While exploration of natural Cas diversity provides one avenue for expanding and improving CRISPR-based tools, a complementary approach uses structure-guided engineering to modify and improve Cas effector function. Over the past several years a number of crystal structures have been solved for different members of Cas9 Jinek et al, 2014;Nishimasu et al, 2014Nishimasu et al, , 2015Hirano et al, 2016;Yamada et al, 2017), Cas12 (Dong et al, 2016;Yamano et al, 2016;Yang et al, 2016;Stella et al, 2017;Swarts et al, 2017;Wu et al, 2017;Liu et al, 2019), and Cas13 (Knott et al, 2017;Liu et al, 2017aLiu et al, , 2017bZhang et al, 2018aZhang et al, , 2018bSlaymaker et al, 2019) families. These structures include the apo forms with just the effector protein alone, or the effector in complex with its guide RNA alone or guide RNA in complex with target DNA or RNA, providing structural insights into target recognition and cleavage.…”