5’ modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

Mullally, Grace; Aelst, Kara van; Naqvi, Mohsin M.; Diffin, Fiona M.; Karvelis, Tautvydas; Gasiūnas, Giedrius; Šikšnys, Virginijus; Szczelkun, Mark D.

doi:10.1101/2020.04.09.033399

Cited by 9 publications

(11 citation statements)

References 35 publications

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“…Each vial was counted in a Tri-Carb Trio 3100TR Liquid Scintillation Counter for 10 min. Where indicated, the cleavage data was fitted to the model described in Mullally et al 45 using numerical integration in Berkeley Madonna v8.3.18 (https://www.berkeleymadonna.com) and further analysed using Graph Pad Prism v8 (https://www.graphpad.com).…”

Section: Methodsmentioning

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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Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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“…Instead, the conformations of the guide RNAs remain largely unperturbed and the off-target TS DNAs "skip over" the unpaired RNA bases to resume productive base-pairing downstream (Figure 5A-B RNA polymerase (Figure S12C-D). This potentially explains the impact of the 5'-guanines on both R-loop stability and in vitro cleavage activity (Kulcsar et al, 2020;Mullally et al, 2020;Okafor et al, 2019).…”

Section: Cas9 Recognizes Off-targets With Single-nucleotide Deletions By Base Skipping or Via Multiple Mismatchesmentioning

confidence: 99%

Structural basis for Cas9 off-target activity

Pačesa

Lin

Cléry³

et al. 2021

Preprint

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The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of non-canonical base pairing interactions and preservation of base stacking within the guide–off-target heteroduplex. Off-target sites containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping rather than RNA bulge formation. Additionally, PAM-distal mismatches result in duplex unpairing and induce a conformational change of the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.

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“…[ 10 ] However, although the SGP has long been used to express a foreign gene in plants, the transcription initiation site of the SGP is still unclear. Because the additional nucleotides at the 5′‐end of gRNA reduce the cleavage activity of SpCas9, [ 12 ] we first examined the 5′‐end sequences of gRNAs derived by TRV‐SGP (tSGP) and Pea early‐browning virus SGP (pSGP). The A. tumefaciens containing TRV1 and TRV2 derivatives (tSGP‐gRNA and pSGP‐gRNA) were then co‐infiltrated into the leaves of SpCas9‐expressing N. attenuata , a wild tobacco species (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

Ribozyme‐processed guide RNA enhances virus‐mediated plant genome editing

Kim

Lee

et al. 2021

Biotechnology Journal

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In virus-induced gene-editing system, subgenomic promoters have been used to express guide RNAs (gRNAs). However, the transcription initiation site of the subgenomic promoters remains elusive. Here, we examined the sequence of gRNAs expressed by subgenomic promoters and found the variable length of overhangs at 5′-end of gRNAs. The overhangs at 5′-end of gRNA decrease the cleavage activity of SpCas9. To overcome this problem, we inserted hammerhead ribozyme between the subgenomic promoter and gRNA and confirmed that gRNAs with a precise 5′-end increase the editing efficacy in wild tobacco. This system will be widely used for editing target genes in plants with high efficiency.

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5’ modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

Cited by 9 publications

References 35 publications

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

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

Structural basis for Cas9 off-target activity

Ribozyme‐processed guide RNA enhances virus‐mediated plant genome editing

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