Probing the stability of the SpCas9–DNA complex after cleavage

Aldag, Pierre; Welzel, Fabian; Jakob, Leonhard; Schmidbauer, Andreas; Rutkauskas, Marius; Fettes, Fergus; Grohmann, Dina; Seidel, Ralf

doi:10.1093/nar/gkab1072

Cited by 17 publications

(10 citation statements)

References 51 publications

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“…Our finding that the post-catalysis RNP-product interaction modulates Cas9 nuclease turnover points to the unique state of the RNP•product complex, in agreement with previous observations that the pre- and post-catalysis interactions of RNP and DNA were remarkably different ( 28 , 30 , 40 ). The continued rearrangements of RNP–DNA interaction in Cas9 reaction should bring attention to the consideration of the dwell time differences between active nuclease, catalytically dead nuclease, and the two types of nickases in development of new Cas9 technologies and their applications.…”

Section: Discussionsupporting

confidence: 92%

“…This process is facilitated by choreographed sequential conformational changes in the Cas9 nuclease to stabilize the R-loop structure and eventually aligns the active sites of the RuvC and HNH nuclease domains with their respective target phosphodiester bonds for DNA double-strand cleavage ( 22 , 26–28 ). After bond cleavage, SauCas9 deviates from the SpyCas9 paradigm as it has been shown to catalyze multiple-turnover reactions (Figure 1 ) ( 29 ), whereas SpyCas9 is essentially a single-turnover enzyme, with a reported half-life of 3–40 h ( 7 , 27 , 30 , 31 ). The turnover differences imply that interaction of SauCas9 with its product differ significantly from SpyCas9.…”

Section: Introductionmentioning

confidence: 99%

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DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage

Pan

Mabuchi

Robb

2023

Nucleic Acids Research

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The RNA-guided Cas9 endonuclease from Staphylococcus aureus (SauCas9) can catalyze multiple-turnover reactions whereas Cas9 from Streptococcus pyogenes (SpyCas9) is a single-turnover enzyme. Here we dissect the mechanism of multiple-turnover catalysis by SauCas9 and elucidate its molecular basis. We show that the multiple-turnover catalysis does not require more than stoichiometric RNA guides to Cas9 nuclease. Rather, the RNA-guide loaded ribonucleoprotein (RNP) is the reactive unity that is slowly released from product and recycled in the subsequent reaction. The mechanism that RNP is recycled for multiple-turnover reaction entails the unwinding of the RNA:DNA duplex in the R-loop. We argue that DNA rehybridization is required for RNP release by supplementing the energy cost in the process. Indeed, turnover is arrested when DNA rehybridization is suppressed. Further, under higher salt conditions, both SauCas9 and SpyCas9 showed increased turnover, and engineered SpyCas9 nucleases that form fewer direct or hydrogen bonding interactions with target DNA became multiple-turnover enzymes. Thus, these results indicate that for both SpyCas9 and SauCas9, turnover is determined by the energetic balance of the post-chemistry RNP-DNA interaction. Due to the conserved protein core folds, the mechanism underpinning turnover we establish here is likely operant in all Cas9 nucleases.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage

Pan

Mabuchi

Robb

2023

Nucleic Acids Research

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“…Engineering of TNB and/or REC2 subdomains may be a fertile ground for producing new type V enzymes with improved DNA cleavage properties, either by reducing off-target cleavage 7 by rejecting TS cleavage or by favouring more open states that are necessary for trans -cleavage. A recent publication from Aldag et al 40 demonstrated that type II Cas9 that lacks an equivalent TNB also produces torque-stable states during DNA cleavage, suggesting that this may be a general feature of coupling stabilising R-loop structures during DNA cleavage cycles.…”

Section: Discussionmentioning

confidence: 99%

CRISPR–Cas12a-mediated DNA clamping triggers target-strand cleavage

et al. 2022

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Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas12a is widely used for genome editing and diagnostics, so it is important to understand how RNA-guided DNA recognition activates the cleavage of the target strand (TS) following non-target-strand (NTS) cleavage. Here we used single-molecule magnetic tweezers, gel-based assays and nanopore sequencing to explore DNA unwinding and cleavage. In addition to dynamic and heterogenous R-loop formation, we also directly observed transient double-stranded DNA unwinding downstream of the 20-bp heteroduplex and, following NTS cleavage, formation of a hyperstable ‘clamped’ Cas12a–DNA intermediate necessary for TS cleavage. Annealing of a 4-nucleotide 3′ CRISPR RNA overhang to the unwound TS downstream of the heteroduplex inhibited clamping and slowed TS cleavage by ~16-fold. Alanine substitution of a conserved aromatic amino acid in the REC2 subdomain that normally caps the R-loop relieved this inhibition but favoured stabilisation of unwound states, suggesting that the REC2 subdomain regulates access of the 3′ CRISPR RNA to downstream DNA.

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“…Ca s9-based Ba ck g round E limination (CaBagE) is a non-size-based DNA-pre-enrichment method that leverages the high stability of Cas9/gRNA bound to DNA post cleavage to protect the two ends of the target from subsequent exonuclease digestion 33,34 . Meanwhile, (unprotected) non-target DNA is readily digested 33,34 . Using a modified CaBagE version, we sought to enrich and phase four additional targets.…”

Section: Resultsmentioning

confidence: 99%

“…We modified the CaBagE method 33,34 and designed a tiling strategy of spacing gRNAs as previously shown 37 . Five gRNAs were designed to isolate four ~20 kb adjacent targets (T1-T4) from the human MSH2 locus.…”

Section: Target Excision and Isolation With A Modified Cabage Methodsmentioning

confidence: 99%

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

Mikhaylova¹,

Rzepka²,

Kawamura³

et al. 2023

Preprint

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In the human genome, heterozygous sites are genomic positions with different alleles inherited from each parent. On average, there is a heterozygous site every 1-2 kilobases (kb). Resolving whether two alleles in neighboring heterozygous positions are physically linked—that is, phased—is possible with a short-read sequencer if the sequencing library captures long-range information. TELL-Seq is a library preparation method based on millions of barcoded micro-sized beads that enables instrument-free phasing of a whole human genome in a single PCR tube. TELL-Seq incorporates a unique molecular identifier (barcode) to the short reads generated from the same high-molecular-weight (HMW) DNA fragment (known as ‘linked-reads’). However, genome-scale TELL-Seq is not cost-effective for applications focusing on a single locus or a few loci. Here, we present an optimized TELL-Seq protocol that enables the cost-effective phasing of enriched loci (targets) of varying sizes, purity levels, and heterozygosity. Targeted TELL-Seq maximizes linked-read efficiency and library yield while minimizing input requirements, fragment collisions on microbeads, and sequencing burden. To validate the targeted protocol, we phased seven 180-200 kb loci enriched by CRISPR/Cas9-mediated excision coupled with pulse-field electrophoresis, four 20 kb loci enriched by CRISPR/Cas9-mediated protection from exonuclease digestion, and six 2-13 kb loci amplified by PCR. The selected targets have clinical and research relevance (BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, PMS2, SCN5A-SCN10A, andPKI3CA). These analyses reveal that targeted TELL-Seq provides a reliable way of phasing allelic variants within targets (2-200 kb in length) with the low cost and high accuracy of short-read sequencing.

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Probing the stability of the SpCas9–DNA complex after cleavage

Cited by 17 publications

References 51 publications

DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage

DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage

CRISPR–Cas12a-mediated DNA clamping triggers target-strand cleavage

Targeted Phasing of 2-200 Kilobase DNA Fragments with a Short-Read Sequencer and a Single-Tube Linked-Read Library Method

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