CRISPR-Cas9 bends and twists DNA to read its sequence

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.

Download Full-text

CRISPR-Cas9 bends and twists DNA to read its sequence

10.1101/2021.09.06.459219 ◽

2021 ◽

Cited By ~ 2

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.

Download Full-text

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012239 ◽

2020 ◽

Vol 295 (19) ◽

pp. 6509-6517 ◽

Cited By ~ 1

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.

Download Full-text

Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

Nucleic Acids Research ◽

10.1093/nar/gkaa605 ◽

2020 ◽

Vol 48 (15) ◽

pp. 8601-8616 ◽

Cited By ~ 1

Author(s):

Hanseop Kim ◽

Wi-jae Lee ◽

Yeounsun Oh ◽

Seung-Hun Kang ◽

Junho K Hur ◽

...

Keyword(s):

Dna Sequences ◽

Genome Engineering ◽

Target Genes ◽

Target Sequence ◽

Energy Potential ◽

Guide Rna ◽

Sequence Recognition ◽

Protospacer Adjacent Motif ◽

Effective Manner ◽

Target Dna

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

Download Full-text

Developing Heritable Mutations in Arabidopsis thaliana Using a Modified CRISPR/Cas9 Toolkit Comprising PAM-Altered Cas9 Variants and gRNAs

Plant and Cell Physiology ◽

10.1093/pcp/pcz118 ◽

2019 ◽

Vol 60 (10) ◽

pp. 2255-2262 ◽

Cited By ~ 5

Author(s):

Akihiro Yamamoto ◽

Takashi Ishida ◽

Mika Yoshimura ◽

Yuri Kimura ◽

Shinichiro Sawa

Keyword(s):

Arabidopsis Thaliana ◽

Genome Editing ◽

Target Genes ◽

Molecular Genetic ◽

Science Research ◽

Target Sequence ◽

Guide Rna ◽

Cas9 Nuclease ◽

Protospacer Adjacent Motif ◽

Model Plant Arabidopsis Thaliana

Abstract Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), comprising an RNA-guided DNA endonuclease and a programmable guide RNA (gRNA), is currently recognized to be a powerful genome-editing tool and is widely used in biological science. Despite the usefulness of the system, a protospacer-adjacent motif (PAM) immediately downstream of the target sequence needs to be taken into account in the design of the gRNA, a requirement which limits the flexibility of the CRISPR-based genome-editing system. To overcome this limitation, a Cas9 isolated from Streptococcus pyogenes, namely SpCas9, engineered to develop several variants of Cas9 nuclease, has been generated. SpCas9 recognizes the NGG sequence as the PAM, whereas its variants are capable of interacting with different PAMs. Despite the potential advantage of the Cas9 variants, their functionalities have not previously been tested in the widely used model plant, Arabidopsis thaliana. Here, we developed a plant-specific vector series harboring SpCas9-VQR (NGAN or NGNG) or SpCas9-EQR (NGAG) and evaluated their functionalities. These modified Cas9 nucleases efficiently introduced mutations into the CLV3 and AS1 target genes using gRNAs that were compatible with atypical PAMs. Furthermore, the generated mutations were passed on to their offspring. This study illustrated the usefulness of the SpCas9 variants because the ability to generate heritable mutations will be of great benefit in molecular genetic analyses. A greater number of potential SpCas9-variant-recognition sites in these genes are predicted, compared with those of conventional SpCas9. These results demonstrated the usefulness of the SpCas9 variants for genome editing in the field of plant science research.

Download Full-text

Programming PAM antennae for efficient CRISPR-Cas9 DNA editing

Science Advances ◽

10.1126/sciadv.aay9948 ◽

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.

Download Full-text

New 2-Oxopyridine/2-Thiopyridine Derivatives Tethered to a Benzotriazole with Cytotoxicity on MCF7 Cell Lines and with Antiviral Activities

Letters in Drug Design & Discovery ◽

10.2174/1570180816666190220123547 ◽

2020 ◽

Vol 17 (2) ◽

pp. 124-137 ◽

Cited By ~ 2

Author(s):

Adel Mahmoud Attia ◽

Ahmed Ibrahin Khodair ◽

Eman Abdelnasser Gendy ◽

Mohammed Abu El-Magd ◽

Yaseen Ali Mosa Mohamed Elshaier

Keyword(s):

Dna Damage ◽

Anticancer Agents ◽

Active Compound ◽

Docking Study ◽

Base Pairs ◽

Dna Base ◽

Dna Base Pairs ◽

Antiviral Activities ◽

Active Methylene ◽

Damage Study

Background:Perturbation of nucleic acids structures and confirmation by small molecules through intercalation binding is an intriguing application in anticancer therapy. The planar aromatic moiety of anticancer agents was inserted between DNA base pairs leading to change in the DNA structure and subsequent functional arrest.Objective:The final scaffold of the target compounds was annulated and linked to a benzotriazole ring. These new pharmacophoric features were examined as antiviral and anticancer agents against MCF7 and their effect on DNA damage was also assessed.Methods:A new series of fully substituted 2-oxopyridine/2-thioxopyridine derivatives tethered to a benzotriazole moiety (4a-h) was synthesized through Michael cyclization of synthesized α,β- unsaturated compounds (3a-e) with appropriate active methylene derivatives. The DNA damage study was assessed by comet assay. In silico DNA molecular docking was performed using Open Eye software to corroborate the experimental results and to understand molecule interaction at the atomic level.Results:The highest DNA damage was observed in Doxorubicin, followed by 4h, then, 4b, 4g, 4f, 4e, and 4d. The docking study showed that compound 4h formed Hydrogen Bonds (HBs) as a standard ligand with GSK-3. Compound 4h was the most active compound against rotavirus Wa, HAVHM175, and HSV strains with a reduction of 30%, 40%, and 70%, respectively.Conclusion:Compound 4h was the most active compound and could act as a prospective lead molecule for anticancer agent.

Download Full-text

On the change of the order od stability of DNA base pairs as a result of the methylation of guanine

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19881943 ◽

1988 ◽

Vol 53 (9) ◽

pp. 1943-1945

Author(s):

Pavel Hobza ◽

Camille Sandorfy

Keyword(s):

Ab Initio ◽

Base Pairs ◽

Dispersion Energy ◽

Dna Base ◽

Dna Base Pairs

The interaction of the 6-O methylguanine cation with cytosine and thymine was studied using the ab initio SCF method in combination with a London type expression for dispersion energy. The structure of the complex formed with cytosine differs from that found previously with guanine itself.

Download Full-text

Efficient implementation of the single-reference algebraic diagrammatic construction theory for charged excitations: Applications to the TEMPO radical and DNA base pairs

The Journal of Chemical Physics ◽

10.1063/5.0040317 ◽

2021 ◽

Vol 154 (7) ◽

pp. 074105

Author(s):

Samragni Banerjee ◽

Alexander Yu. Sokolov

Keyword(s):

Efficient Implementation ◽

Base Pairs ◽

Dna Base ◽

Dna Base Pairs ◽

Tempo Radical

Download Full-text

RNA Delivery via DNA-Inspired Janus Base Nanotubes for Extracellular Matrix Penetration

MRS Advances ◽

10.1557/adv.2020.47 ◽

2020 ◽

Vol 5 (16) ◽

pp. 815-823

Author(s):

Ian Sands ◽

Jinhyung Lee ◽

Wuxia Zhang ◽

Yupeng Chen

Keyword(s):

Extracellular Matrix ◽

Small Rnas ◽

Base Pairs ◽

Major Barrier ◽

Dna Base ◽

Dna Base Pairs ◽

Negative Surface ◽

Negative Surface Charge ◽

Delivery Vehicles ◽

Low Cell Density

AbstractRNA delivery into deep tissues with dense extracellular matrix (ECM) has been challenging. For example, cartilage is a major barrier for RNA and drug delivery due to its avascular structure, low cell density and strong negative surface charge. Cartilage ECM is comprised of collagens, proteoglycans, and various other noncollagneous proteins with a spacing of 20nm. Conventional nanoparticles are usually spherical with a diameter larger than 50-60nm (after cargo loading). Therefore, they presented limited success for RNA delivery into cartilage. Here, we developed Janus base nanotubes (JBNTs, self-assembled nanotubes inspired from DNA base pairs) to assemble with small RNAs to form nano-rod delivery vehicles (termed as “Nanopieces”). Nanopieces have a diameter of ∼20nm (smallest delivery vehicles after cargo loading) and a length of ∼100nm. They present a novel breakthrough in ECM penetration due to the reduced size and adjustable characteristics to encourage ECM and intracellular penetration.

Download Full-text