scholarly journals Cas12a is a dynamic and precise RNA-guided nuclease without off-target activity on λ-DNA

2021 ◽  
Author(s):  
Bijoya Paul ◽  
Loic Chaubet ◽  
Emma Verver ◽  
Guillermo Montoya
Keyword(s):  
Dna Binding ◽  
Genome Editing ◽  
Optical Tweezers ◽  
Target Site ◽  
Target Dna ◽  
Multiple Binding ◽  
Target Sites ◽  
Dna Binding And Cleavage ◽  
Target Activity ◽  
Insight Into

Cas12a is an RNA-guided endonuclease that is emerging as a powerful genome-editing tool. Here we combined optical tweezers with fluorescence to monitor Cas12a binding onto λ-DNA, providing insight into its DNA binding and cleavage mechanisms. At low forces Cas12a binds DNA specifically with two off-target sites, while at higher forces numerous binding events appear driven by the mechanical distortion of the DNA and partial matches to the crRNA. Despite the multiple binding events, cleavage is only observed on the target site at low forces, when the DNA is flexible. Activity assays show that the preferential off-target sites are not cleaved, and the λ-DNA is severed at the target site. This precision is also observed in Cas12a variants where the specific dsDNA and the unspecific ssDNA cleavage are dissociated or nick the target DNA. We propose that Cas12a and its variants are precise endonucleases that efficiently scan the DNA for its target but only cleave the selected site in the λ-DNA.

scholarly journals CRISPR/Cas “non-target” sites inhibit on-target cutting rates

2020 ◽  
Author(s):  
Eirik A. Moreb ◽  
Mitchell Hutmacher ◽  
Michael D. Lynch
Keyword(s):  
Genome Editing ◽  
Target Site ◽  
High Fidelity ◽  
List Type ◽  
Dependent Manner ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Target Sites ◽  
Dose Dependent ◽  
Target Activity

AbstractCRISPR/Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 associated with a guide RNA (gRNA) searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM) and once found, cuts the DNA. The number of PAM sites in the genome are effectively a non-target pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity towards a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates. Decreasing the non-target pool by increasing PAM specificity provides a path towards improving on-target activity for slower high fidelity Cas9 variants. These results demonstrate the importance of competitive non-target sites on Cas9 activity and, in part, may help to explain sequence and context dependent activities of gRNAs. Engineering improved PAM specificity to reduce the competitive non-target pool offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.HighlightsThe pool of non-target PAM sites inhibit Cas9/gRNA on-target activitynon-target PAM inhibition is dose dependentnon-target PAM inhibition is a function of gRNA sequencenon-target PAM inhibition is a function of Cas9 levels

scholarly journals A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

2017 ◽  
Author(s):  
Yavuz S. Dagdas ◽  
Janice S. Chen ◽  
Samuel H. Sternberg ◽  
Jennifer A. Doudna ◽  
Ahmet Yildiz
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Divalent Cations ◽  
Guide Rna ◽  
Single Molecule Fret ◽  
Target Sites ◽  
Multiple Conformations ◽  
Dna Binding And Cleavage

AbstractThe Cas9 endonuclease is widely utilized for genome engineering applications by programming its single-guide RNA and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule FRET, we identified an intermediate state of S. pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

scholarly journals Structural insight into multistage inhibition of CRISPR-Cas12a by AcrVA4

2019 ◽  
Vol 116 (38) ◽  
pp. 18928-18936 ◽  
Author(s):  
Ruchao Peng ◽  
Zhiteng Li ◽  
Ying Xu ◽  
Shaoshuai He ◽  
Qi Peng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mobile Genetic Elements ◽  
Human Cells ◽  
Type I ◽  
Enzyme Recycling ◽  
Target Dna ◽  
Regulatory Genome ◽  
Structural Insight ◽  
Underlying Mechanisms ◽  
Insight Into

Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Lachnospiraceae bacterium Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.

scholarly journals CRISPR-Cas9 in gene therapy: much control on breaking, little control on repairing

2015 ◽  
Author(s):  
Kaveh Daneshvar
Keyword(s):  
Gene Therapy ◽  
Dna Repair ◽  
Immune Responses ◽  
Genome Editing ◽  
Target Cells ◽  
Target Dna ◽  
Target Activity ◽  
In Vivo Delivery ◽  
Target Effects

Recent advances in CRISPR-Cas9 genome editing tool have made great promises to basic and biomedical research as well as gene therapy. Efforts to make the CRISPR-Cas9 system applicable in gene therapy are largely focused on two aspects: 1) increasing the specificity of this system by eliminating off-target effects, and 2) optimizing in vivo delivery of the CRISPR-Cas9 DNA constructs to target cells and limiting the expression of Cas9 and gRNA to prevent immune responses. Moreover, there is an unnoted but crucial consideration about the mode of DNA repair at the lesion caused by CRISPR-Cas9. In this commentary, I briefly highlight recent publications on in vivo use of the CRISPR-Cas9 system in gene therapy. I then discuss concerns about the off-target activity and immune responses triggered by the use CRISPR-Cas9 in gene therapy. Following this, I focus on the undesired on-target DNA repair events that can occur as a result of the activity of CRISPR-Cas9. This concise commentary sets itself apart from previous perspectives by focusing on the modes of DNA repair employed following a CRISPR-Cas9 induced genomic insult, and by carefully weighing the benefits of the outcomes. In particular, the present manuscript underscores the need for more study on controlled DNA repair in systems targeted with CRISPR-Cas9 genome editing tools.

scholarly journals Systematic in vitro profiling of off-target affinity, cleavage and efficiency for CRISPR enzymes

2020 ◽  
Vol 48 (9) ◽  
pp. 5037-5053 ◽  
Author(s):  
Liyang Zhang ◽  
H Tomas Rube ◽  
Christopher A Vakulskas ◽  
Mark A Behlke ◽  
Harmen J Bussemaker ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Specificity ◽  
In Vitro Assays ◽  
Target Discrimination ◽  
Dna Binding Specificity ◽  
Affinity Cleavage ◽  
Target Activity ◽  
Insight Into

Abstract CRISPR RNA-guided endonucleases (RGEs) cut or direct activities to specific genomic loci, yet each has off-target activities that are often unpredictable. We developed a pair of simple in vitro assays to systematically measure the DNA-binding specificity (Spec-seq), catalytic activity specificity (SEAM-seq) and cleavage efficiency of RGEs. By separately quantifying binding and cleavage specificity, Spec/SEAM-seq provides detailed mechanistic insight into off-target activity. Feature-based models generated from Spec/SEAM-seq data for SpCas9 were consistent with previous reports of its in vitro and in vivo specificity, validating the approach. Spec/SEAM-seq is also useful for profiling less-well characterized RGEs. Application to an engineered SpCas9, HiFi-SpCas9, indicated that its enhanced target discrimination can be attributed to cleavage rather than binding specificity. The ortholog ScCas9, on the other hand, derives specificity from binding to an extended PAM. The decreased off-target activity of AsCas12a (Cpf1) appears to be primarily driven by DNA-binding specificity. Finally, we performed the first characterization of CasX specificity, revealing an all-or-nothing mechanism where mismatches can be bound, but not cleaved. Together, these applications establish Spec/SEAM-seq as an accessible method to rapidly and reliably evaluate the specificity of RGEs, Cas::gRNA pairs, and gain insight into the mechanism and thermodynamics of target discrimination.

scholarly journals Engineered Cpf1 Enzymes with Altered PAM Specificities

2016 ◽  
Author(s):  
Linyi Gao ◽  
David B.T. Cox ◽  
Winston X. Yan ◽  
John Manteiga ◽  
Martin Schneider ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Saturation Mutagenesis ◽  
Promising Tool ◽  
Genome Wide ◽  
Protospacer Adjacent Motif ◽  
Target Sites ◽  
High Level ◽  
Target Activity

SummaryThe RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1-5. Compared to other genome editing platforms, Cpf1 offers distinct advantages, such as the ability to easily target multiple genes simultaneously3, as well as low rates of off-target activity4, 5. However, the Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), which has been successfully harnessed for genome editing, can only robustly cleave target sites preceded by a TTTV protospacer adjacent motif (PAM), which may limit its practical utility. To address this limitation, we used a structure- guided saturation mutagenesis screen to increase the targeting range of Cpf1. We engineered two variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains. Together, these variants increase the targeting range of AsCpf1 to one cleavage site for every ~8.7 bp in non-repetitive regions of the human genome, providing a useful addition to the CRISPR/Cas genome engineering toolbox.

scholarly journals How p53 Molecules Solve the Target DNA Search Problem: A Review

2020 ◽  
Vol 21 (3) ◽  
pp. 1031 ◽  
Author(s):  
Kiyoto Kamagata ◽  
Yuji Itoh ◽  
Dwiky Rendra Graha Subekti
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Search Problem ◽  
Target Search ◽  
Cellular Processes ◽  
Target Dna ◽  
Target Sites ◽  
Dna Strands ◽  
Single Molecule Studies

Interactions between DNA and DNA-binding proteins play an important role in many essential cellular processes. A key function of the DNA-binding protein p53 is to search for and bind to target sites incorporated in genomic DNA, which triggers transcriptional regulation. How do p53 molecules achieve “rapid” and “accurate” target search in living cells? The search dynamics of p53 were expected to include 3D diffusion in solution, 1D diffusion along DNA, and intersegmental transfer between two different DNA strands. Single-molecule fluorescence microscopy enabled the tracking of p53 molecules on DNA and the characterization of these dynamics quantitatively. Recent intensive single-molecule studies of p53 succeeded in revealing each of these search dynamics. Here, we review these studies and discuss the target search mechanisms of p53.

DNA topology influences p53 sequence-specific DNA binding through structural transitions within the target sites

2008 ◽  
Vol 412 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Eva B. Jagelská ◽  
Václav Brázda ◽  
Petr Pečinka ◽  
Emil Paleček ◽  
Miroslav Fojta
Keyword(s):  
Dna Binding ◽  
Inverted Repeat ◽  
P53 Protein ◽  
Dna Supercoiling ◽  
Dna Topology ◽  
Relative Effect ◽  
Target Site ◽  
Additional Increase ◽  
Target Sites ◽  
Target Sequences

The tumour suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. P53 is a nuclear phosphoprotein and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild-type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences, differing in their internal symmetry. Binding of p53 to DNA increased with increasing negative superhelix density (−σ). At −σ≤0.03, the relative effect of DNA supercoiling on protein–DNA binding was similar for DNA containing both symmetrical and non-symmetrical target sites. On the other hand, at higher −σ, target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding as a result of increasing levels of negative DNA supercoiling. For −σ=0.07, an approx. 3-fold additional increase in binding was observed for a symmetrical target site compared with a non-symmetrical target site. The p53 target sequences possessing the inverted repeat symmetry were shown to form a cruciform structure in sufficiently negative supercoiled DNA. We show that formation of cruciforms in DNA topoisomers at −σ≥0.05 correlates with the extra enhancement of p53–DNA binding.

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