Structural insights into Cas13b-guided CRISPR RNA maturation and recognition

Abstract

Dear Editor, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems are RNA-guided adaptive immune systems in prokaryotes. 1,2 Class 2 CRISPR-Cas systems (including type II, V, and VI) involve large single effector proteins in complex with crRNA for interference. 3,4 The type II and V effectors, such as Cas9 and Cas12a, have been engineered into powerful tools for genome editing. The type VI system encompasses RNA-guided RNases. Its effectors Cas13a, Cas13b and Cas13d are capable of both precursor CRISPR RNA (pre-crRNA) processing and target RNA cleavage, which protect the host from phage attacks. 5-7 Once bound to a target RNA, they are activated, switching on a non-specific RNase activity. Moreover, they have been utilized to target and edit RNA as programmable RNAbinding modules. 6,[8][9][10][11][12] Although related to Cas13a and Cas13d, Cas13b possesses many distinctive features. These include the lack of significant sequence similarity with Cas13a and Cas13d, disparate crRNA repeat region, double-sided protospacer flanking sequence (PFS)-dependent target RNA cleavage. [5][6][7][8]13 To investigate how Cas13b processes pre-crRNA, recognizes crRNA and settles the spacer nucleotides for target recognition, we solved the crystal structure of Bergeyella zoohelcum Cas13b (BzCas13b) in complex with its crRNA at 2.79 Å resolution (Supplementary information, Table S1). The binary complex was obtained by the SeMet-derived BzCas13b R1177A mutant co-expressed with CRISPR template in vivo. The architecture of BzCas13b assumes a triangular domain distribution around the central L-shaped crRNA ( Fig. 1a-e; Supplementary information, Movie S1). In the binary complex, Helical-1, HEPN-1 and HEPN-2 domains together form one side of the triangular structure. Helical-1 domain comprises six α-helices connected with random loops (Supplementary information, Fig. S1). The second side of the triangle is formed by RRI-1 (the repeat region interacting domain-1), RRI-2 domains and the linker region. RRI-1 domain can be subdivided into two separate motifs (RRI-1 I and II) that stack onto each other. Both motifs contain a short twostranded, antiparallel β-sheet flanked by five α-helices. RRI-2 domain includes a long central two-stranded, antiparallel β-sheet flanked by two α-helices, and a short central two-stranded, antiparallel β-sheet flanked by three α-helices. The linker region consists of random loops that connect two short α-helices, which shows multiple interactions with RRI-2 domain. Helical-2 domain is composed of nine α-helices and its rather long helix-23 extends in parallel with crRNA, thereby forming the third side of the triangle. Helix-8 of Helical-1 domain and helix-23 of Helical-2 domain protrude out of the complex in a crab claw-like manner to clamp the spacer region of crRNA (Supplementary information, Fig. S1). In addition, HEPN-1 domain bridges Helical-1 and Helical-2 domains.A mature 52-nt crRNA, originated from a co-expressed CRISPR encoding sequence and bein...

Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d

“…Current availability of the type VI CRISPR-effector structures allows us to further analyze structural and functional features of Cas13d by comparing similarities and differences between Cas13a, Cas13b, and Cas13d 25,26,31,38,39 . Overall, the domain architecture of Cas13d shares certain spatial consistency with Cas13a, whereas Cas13b displays a distinct arrangement (Fig.…”

“…The crRNA-target duplex is recognized by two HEPN and two Helical domains in Cas13d 31 ; in Cas13a, it is recognized inside the pocket formed by HEPN-1, HEPN-2, Helical-2, and Helical-3 domains 26 . As inferred from the charge distribution on the surface of protein, Cas13b may accommodate the crRNA-target duplex within the pocket formed by Helical-1, Helical-2, RRI-1, and HEPN-1 domains 38 .

Fig. 7Comparison of Cas13a, Cas13b, and Cas13d.

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“…While Helical-1 domain of Cas13a and RRI-2 domain of Cas13b are responsible for pre-crRNA processing 25,38,39 , their counterpart is lacking in the compact Cas13d. Instead, residues from HEPN-2 domain play a critical role in pre-crRNA processing by Cas13d.…”

It takes two (Las1 HEPN endoribonuclease domains) to cut RNA correctly

“…HEPN nucleases participate in a wide spectrum of RNA processing pathways and the recent identification of many new HEPN nucleases (3) has led to a surge in atomicresolution structures of different HEPN nuclease family members ( Fig. 7) (4,5,26, [28][29][30]35,[37][38][39][40][41][42][43][44]. The overall architecture of HEPN nucleases varies dramatically from the butterfly-shaped RNase PNK to bilobed Cas13 nucleases and intertwined RNase L. These diverse assemblies are important contributors to the activation and specialized functions of individual HEPN nucleases.…”

Applications of CRISPR technology in studying plant-pathogen interactions: overview and perspective

“…The type VI CRISPR/Cas13 systems function as a ribonuclease and have been divided into four subtypes, including type VI-A (Cas13a), VI-B (Cas13b), VI-C (Cas13c) and VI-D (Cas13d) (Abudayyeh et al 2016(Abudayyeh et al , 2017Cox et al 2017;Shmakov et al 2017;Konermann et al 2018;Zhang et al 2018aZhang et al , 2018b. Cas13 proteins target RNA rather than DNA molecules and are characterized by two distinct HEPN RNase domains and assembled with single crRNA to form a crRNA-guided protein complex for RNA targeting.…”