Structural basis for the activity of the type VII CRISPR-Cas system.

The newly identified type VII CRISPR-Cas candidate system uses a CRISPR RNA-guided ribonucleoprotein complex formed by Cas5 and Cas7 proteins to target RNA1. However, the RNA cleavage is executed by a dedicated Cas14 nuclease, which is distinct from the effector nucleases of the other CRISPR-Cas systems. Here we report seven cryo-electron microscopy structures of the Cas14-bound interference complex at different functional states. Cas14, a tetrameric protein in solution, is recruited to the Cas5-Cas7 complex in a target RNA-dependent manner. The N-terminal catalytic domain of Cas14 binds a stretch of the substrate RNA for cleavage, whereas the C-terminal domain is primarily responsible for tethering Cas14 to the Cas5-Cas7 complex. The biochemical cleavage assays corroborate the captured functional conformations, revealing that Cas14 binds to different sites on the Cas5-Cas7 complex to execute individual cleavage events. Notably, a plugged-in arginine of Cas7 sandwiched by a C-shaped clamp of C-terminal domain precisely modulates Cas14 binding. More interestingly, target RNA cleavage is altered by a complementary protospacer flanking sequence at the 5' end, but not at the 3' end. Altogether, our study elucidates critical molecular details underlying the assembly of the interference complex and substrate cleavage in the type VII CRISPR-Cas system, which may help rational engineering of the type VII CRISPR-Cas system for biotechnological applications.

Structural basis for pegRNA-guided reverse transcription by a prime editor.

The prime editor system composed of Streptococcus pyogenes Cas9 nickase (nSpCas9) and engineered Moloney murine leukaemia virus reverse transcriptase (M-MLV RT) collaborates with a prime editing guide RNA (pegRNA) to facilitate a wide variety of precise genome edits in living cells1. However, owing to a lack of structural information, the molecular mechanism of pegRNA-guided reverse transcription by the prime editor remains poorly understood. Here we present cryo-electron microscopy structures of the SpCas9-M-MLV RTΔRNaseH-pegRNA-target DNA complex in multiple states. The termination structure, along with our functional analysis, reveals that M-MLV RT extends reverse transcription beyond the expected site, resulting in scaffold-derived incorporations that cause undesired edits at the target loci. Furthermore, structural comparisons among the pre-initiation, initiation and elongation states show that M-MLV RT remains in a consistent position relative to SpCas9 during reverse transcription, whereas the pegRNA-synthesized DNA heteroduplex builds up along the surface of SpCas9. On the basis of our structural insights, we rationally engineered pegRNA variants and prime-editor variants in which M-MLV RT is fused within SpCas9. Collectively, our findings provide structural insights into the stepwise mechanism of prime editing, and will pave the way for the development of a versatile prime editing toolbox.

Cryo-EM structure of the transposon-associated TnpB enzyme.

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.