scholarly journals Programming PAM antennae for efficient CRISPR-Cas9 DNA editing

2020 ◽  
Vol 6 (19) ◽  
pp. eaay9948
Author(s):  
Fei Wang ◽  
Yaya Hao ◽  
Qian Li ◽  
Jiang Li ◽  
Honglu Zhang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Super Resolution ◽  
Single Molecule Level ◽  
Guide Rna ◽  
Guide Rnas ◽  
Protospacer Adjacent Motif ◽  
Target Dna

Bacterial CRISPR-Cas9 nucleases have been repurposed as powerful genome editing tools. Whereas engineering guide RNAs or Cas nucleases have proven to improve the efficiency of CRISPR editing, modulation of protospacer-adjacent motif (PAM), indispensable for CRISPR, has been less explored. Here, we develop a DNA origami–based platform to program a PAM antenna microenvironment and address its performance at the single-molecule level with submolecular resolution. To mimic spatially controlled in vivo PAM distribution as may occur in chromatin, we investigate the effect of PAM antennae surrounding target DNA. We find that PAM antennae effectively sensitize the DNA cleavage by recruiting Cas9 molecules. Super-resolution tracking of single single-guide RNA/Cas9s reveals localized translocation of Cas9 among spatially proximal PAMs. We find that the introduction of the PAM antennae effectively modulates the microenvironment for enhanced target cleavage (up to ~50%). These results provide insight into factors that promote more efficient genome editing.

scholarly journals Quantification of the affinities of CRISPR–Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences

2020 ◽  
Vol 295 (19) ◽  
pp. 6509-6517 ◽  
Author(s):  
Vladimir Mekler ◽  
Konstantin Kuznedelov ◽  
Konstantin Severinov
Keyword(s):  
Genome Editing ◽  
Target Location ◽  
Dna Probes ◽  
Target Sequence ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9–gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with guide RNAs (gRNAs) (SpCas9–gRNA, SaCas9–gRNA, and FnCas9–gRNA, respectively) and of three engineered SpCas9–gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a “beacon” assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9–gRNA binding to fluorescently labeled target DNA derivatives called “Cas9 beacons.” We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9–gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

scholarly journals Engineering of the genome editing protein Cas9 to slide along DNA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Trishit Banerjee ◽  
Hiroto Takahashi ◽  
Dwiky Rendra Graha Subekti ◽  
Kiyoto Kamagata
Keyword(s):  
Fluorescence Microscopy ◽  
Genome Editing ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Sliding Friction ◽  
Single Molecule Fluorescence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Target Dna ◽  
Key Residues

AbstractThe genome editing protein Cas9 faces engineering challenges in improving off–target DNA cleavage and low editing efficiency. In this study, we aimed to engineer Cas9 to be able to slide along DNA, which might facilitate genome editing and reduce off-target cleavage. We used two approaches to achieve this: reducing the sliding friction along DNA by removing the interactions of Cas9 residues with DNA and facilitating sliding by introducing the sliding-promoting tail of Nhp6A. Seven engineered mutants of Cas9 were prepared, and their performance was tested using single-molecule fluorescence microscopy. Comparison of the mutations enabled the identification of key residues of Cas9 to enhance the sliding along DNA in the presence and absence of single guide RNA (sgRNA). The attachment of the tail to Cas9 mutants enhanced sliding along DNA, particularly in the presence of sgRNA. Together, using the proposed approaches, the sliding ability of Cas9 was improved up to eightfold in the presence of sgRNA. A sliding model of Cas9 and its engineering action are discussed herein.

scholarly journals Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient

2021 ◽  
Vol 22 (19) ◽  
pp. 10570
Author(s):  
Yuya Kato ◽  
Hirotaka Tabata ◽  
Kumiko Sato ◽  
Mariko Nakamura ◽  
Izumu Saito ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Genome Editing ◽  
Adenovirus Vector ◽  
Target Sequence ◽  
Guide Rna ◽  
Guide Rnas ◽  
B Virus ◽  
X Gene

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

scholarly journals Engineered dual selection for directed evolution of SpCas9 PAM specificity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gregory W. Goldberg ◽  
Jeffrey M. Spencer ◽  
David O. Giganti ◽  
Brendan R. Camellato ◽  
Neta Agmon ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Negative Selection ◽  
Selection Procedures ◽  
Guide Rna ◽  
Dna Targeting ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Selection For ◽  
Reduced Activity

AbstractThe widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.

scholarly journals Real-time observation of flexible domain movements in Cas9

2017 ◽  
Author(s):  
Saki Osuka ◽  
Kazushi Isomura ◽  
Shohei Kajimoto ◽  
Tomotaka Komori ◽  
Hiroshi Nishimasu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Potential Role ◽  
Domain Architecture ◽  
Guide Rna ◽  
Single Molecule Fret ◽  
Target Dna ◽  
Time Observation ◽  
Real Time Observation

ABSTRACTThe CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains, but whether these flexible movements occur in solution is unclear. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET. The flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain in Cas9 only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.

scholarly journals CRISPR-Cas9 bends and twists DNA to read its sequence

2021 ◽  
Author(s):  
Joshua C. Cofsky ◽  
Katarzyna M. Soczek ◽  
Gavin J. Knott ◽  
Eva Nogales ◽  
Jennifer A. Doudna
Keyword(s):  
Genome Editing ◽  
Target Sequence ◽  
Base Flipping ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Capture ◽  
Dna Base ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Dna Base Pairs

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM)1. Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism2,3. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

scholarly journals CRISPR-Cas9 bends and twists DNA to read its sequence

2021 ◽  
Author(s):  
Jennifer Doudna ◽  
Joshua Cofsky ◽  
Katarzyna Soczek ◽  
Gavin Knott ◽  
Eva Nogales
Keyword(s):  
Genome Editing ◽  
Target Sequence ◽  
Base Flipping ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Capture ◽  
Dna Base ◽  
Protospacer Adjacent Motif ◽  
Target Dna ◽  
Dna Base Pairs

Abstract In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide-RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA at each PAM, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryo-electron-microscopy (EM) structures of Cas9:RNA:DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 “reads” snippets of DNA to locate target sites within a vast excess of non-target DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

scholarly journals Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9

2016 ◽  
Author(s):  
Digvijay Singh ◽  
Samuel H. Sternberg ◽  
Jingyi Fei ◽  
Jennifer A. Doudna ◽  
Taekjip Ha
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Dissociation Rate ◽  
Stable Complex ◽  
Guide Rna ◽  
Single Molecule Fret ◽  
Protospacer Adjacent Motif ◽  
Time Observation

Binding specificity of Cas9-guide RNA complexes to DNA is important for genome engineering applications, but how mismatches influence target recognition and rejection kinetics is not well understood. We used single-molecule FRET to probe real-time interactions between Cas9-RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence, but the dissociation rate greatly increases from < 0.006 s-1 to > 2 s-1 upon introduction of mismatches proximal to the protospacer adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 12 base pairs in length, which prevent DNA cleavage, still allow the formation of a stable complex (off-rate < 0.006 s-1), suggesting that extremely slow rejection could sequester Cas9-RNA, increasing the Cas9 expression level necessary for genome editing thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

scholarly journals In vivo genome editing using the Cpf1 ortholog derived from Eubacterium eligens

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Woo-Chan Ahn ◽  
Kwang-Hyun Park ◽  
In Seon Bak ◽  
Hyung-Nam Song ◽  
Yan An ◽  
...  
Keyword(s):  
Genome Editing ◽  
Knockout Mice ◽  
Dna Cleavage ◽  
Gene Editing ◽  
Essential Gene ◽  
Bacterial Species ◽  
A Genome ◽  
Target Dna ◽  
Metal Cofactor

Abstract Cpf1 is an RNA-guided endonuclease that can be programmed to cleave DNA targets. Specific features, such as containing a short crRNA, creating a staggered cleavage pattern and having a low off-target rate, render Cpf1 a promising gene-editing tool. Here, we present a new Cpf1 ortholog, EeCpf1, as a genome-editing tool; this ortholog is derived from the gut bacterial species Eubacterium eligens. EeCpf1 exhibits a higher cleavage activity with the Mn2+ metal cofactor and efficiently cuts the target DNA with an engineered, nucleotide extended crRNA at the 5′ target site. When mouse blastocysts were injected with multitargeting crRNAs against the IL2R-γ gene, an essential gene for immunodeficient mouse model production, EeCpf1 efficiently generated IL2R-γ knockout mice. For the first time, these results demonstrate that EeCpf1 can be used as an in vivo gene-editing tool for the production of knockout mice. The utilization of engineered crRNA with multiple target sites will help to explore the in vivo DNA cleavage activities of Cpf1 orthologs from other species that have not been demonstrated.

scholarly journals Expanding the range of editable targets in the wheat genome using the variants of the Cas12a and Cas9 nucleases

2020 ◽  
Author(s):  
Wei Wang ◽  
Bin Tian ◽  
Qianli Pan ◽  
Yueying Chen ◽  
Fei He ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Genome Editing ◽  
Agronomic Traits ◽  
Wheat Genome ◽  
Editing Efficiency ◽  
Francisella Novicida ◽  
Guide Rna ◽  
Guide Rnas ◽  
Protospacer Adjacent Motif ◽  
Endogenous Genes

AbstractThe development of CRISPR-based editors having different protospacer adjacent motif (PAM) recognition specificities, or guide RNA length/structure requirements broadens the range of possible genome editing applications. Here, we evaluated the natural and engineered variants of Cas12a (FnCas12a from Francisella novicida and LbCas12a from Lachnospiraceae bacterium) and Cas9 for wheat genome editing efficiency and ability to induce heritable mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a was able to induce mutations in the wheat genome in the current study, even though with a lower rate than that reported for SpCas9. The eight-fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guide RNAs flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides. A LbCas12a-MGE construct was successfully applied for generating heritable mutations in a gene controlling grain size and weight in wheat. We show that the range of editable loci in the wheat genome could be expanded by using the engineered variants of Cas12a (LbCas12a-RVR) and Cas9 (Cas9-NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9-NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports the set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to Cas9-based modification.

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