scholarly journals Mechanisms of improved specificity of engineered Cas9s revealed by single molecule analysis

2017 ◽  
Author(s):  
Digvijay Singh ◽  
Yanbo Wang ◽  
John Mallon ◽  
Olivia Yang ◽  
Jingyi Fei ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Unwinding ◽  
Cleavage Reaction ◽  
Cleavage Rate ◽  
Guide Rna ◽  
Slowing Down ◽  
Rna Targets ◽  
Cleavage Reactions ◽  
Additional Base ◽  
Single Molecule Analysis

ABSTRACTIn microbes, CRISPR-Cas systems provide adaptive immunity against invading genetic elements. Cas9 in complex with a guide-RNA targets complementary DNA for cleavage and has been repurposed for wide-ranging biological applications. New Cas9s have been engineered (eCas9 and Cas9-HF1) to improve specificity, but how they help reduce off-target cleavage is not known. Here, we developed single molecule DNA unwinding assay to show that sequence mismatches affect cleavage reactions through rebalancing the internal unwinding/rewinding equilibrium. Increasing PAM-distal mismatches facilitate rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily upon mismatch detection. Intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. DNA interrogation experiments showed that engineered Cas9s require about one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity (1) by inhibiting stable DNA binding to partially matching sequences, (2) by making DNA unwinding more sensitive to mismatches, and (3) by slowing down intrinsic cleavage reaction.

scholarly journals Bulk and single-molecule analysis of a novel DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

2019 ◽  
Author(s):  
O.J. Wilkinson ◽  
C. Carrasco ◽  
C. Aicart-Ramos ◽  
F. Moreno-Herrero ◽  
M.S. Dillingham
Keyword(s):  
Single Molecule ◽  
Nuclease Activity ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Dna Looping ◽  
Dna Unwinding ◽  
Ssdna Binding ◽  
Dna Replication And Repair ◽  
Essential Enzyme ◽  
Single Molecule Analysis

ABSTRACTDNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a novel bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5’ to 3’ ssDNA translocase and 5’ to 3’ helicase activity. Single molecule analysis reveals that Bad is a highly processive DNA motor capable of moving along DNA for distances of more than 4 kbp at a rate of ∼200 base pairs per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments also show that the enzyme loops DNA while translocating, which is an emerging feature of highly processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.

scholarly journals Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex

2015 ◽  
Vol 59 (1) ◽  
pp. 125-132 ◽  
Author(s):  
Chunyan Yao ◽  
Hiroshi M. Sasaki ◽  
Takuya Ueda ◽  
Yukihide Tomari ◽  
Hisashi Tadakuma
Keyword(s):  
Single Molecule ◽  
Enzyme Complex ◽  
Cleavage Reaction ◽  
Single Molecule Analysis

scholarly journals Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

2019 ◽  
Author(s):  
Ikenna C Okafor ◽  
Digvijay Singh ◽  
Yanbo Wang ◽  
Minhee Jung ◽  
Haobo Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Engineering ◽  
Dna Unwinding ◽  
Dependent Manner ◽  
Guide Rnas ◽  
Single Molecule Fret ◽  
Wide Range ◽  
The Cost ◽  
Single Molecule Analysis ◽  
Target Activity

Abstract Cas9 has made a wide range of genomic manipulation possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity, but at the cost of the on-target activity. DNA unwinding is a checkpoint before cleavage by Cas9, and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in engineered Cas9s. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used, but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two non-matching guanines added to the 5′ end were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5′ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

scholarly journals Bulk and single-molecule analysis of a bacterial DNA2-like helicase–nuclease reveals a single-stranded DNA looping motor

2020 ◽  
Vol 48 (14) ◽  
pp. 7991-8005
Author(s):  
Oliver J Wilkinson ◽  
Carolina Carrasco ◽  
Clara Aicart-Ramos ◽  
Fernando Moreno-Herrero ◽  
Mark S Dillingham
Keyword(s):  
Single Molecule ◽  
Nuclease Activity ◽  
Biochemical Properties ◽  
Dna Helicase ◽  
Dna Looping ◽  
Dna Unwinding ◽  
Ssdna Binding ◽  
Dna Replication And Repair ◽  
Essential Enzyme ◽  
Single Molecule Analysis

Abstract DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase–nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5′ to 3′ ssDNA translocase and 5′ to 3′ helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase–nucleases and DNA looping motor proteins in general.

scholarly journals Single-molecule analysis of processive double-stranded RNA cleavage by Drosophila Dicer-2

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Masahiro Naganuma ◽  
Hisashi Tadakuma ◽  
Yukihide Tomari
Keyword(s):  
Single Molecule ◽  
Direct Evidence ◽  
Small Interfering Rnas ◽  
Cleavage Reaction ◽  
Double Stranded Rna ◽  
Dynamic Movement ◽  
Dsrna Binding ◽  
Dsrna Binding Protein ◽  
Biochemical Analyses ◽  
Single Molecule Analysis

AbstractDrosophila Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct—processive or distributive—modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2’s cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated.

scholarly journals Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

2019 ◽  
Author(s):  
Ikenna C. Okafor ◽  
Digvijay Singh ◽  
Yanbo Wang ◽  
Minhee Jung ◽  
Haobo Wang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Engineering ◽  
Dna Unwinding ◽  
Dependent Manner ◽  
Guide Rnas ◽  
Single Molecule Fret ◽  
Wide Range ◽  
The Cost ◽  
Single Molecule Analysis ◽  
Target Activity

ABSTRACTCas9 has made a wide range of genome engineering applications possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity but at the cost of the on-target activity. DNA unwinding is the primary checkpoint before cleavage by Cas9 and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in Cas9. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two guanines added were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5’ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

Single-molecule analysis of peroxisome proliferator-activated receptor [gamma]2 and [alpha] reveal subtype specific differences in chromatin binding dynamics

2018 ◽  
Author(s):  
Rikke AM Jensen ◽  
Ville Paakinaho ◽  
Diego M Presman ◽  
Erin E Swinstead ◽  
R Louis Schiltz ◽  
...  
Keyword(s):  
Single Molecule ◽  
Peroxisome Proliferator ◽  
Chromatin Binding ◽  
Peroxisome Proliferator Activated Receptor ◽  
Single Molecule Analysis
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