scholarly journals Kinetic basis for DNA target specificity of CRISPR-Cas12a

2018 ◽  
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
James R. Rybarski ◽  
Ilya J. Finkelstein ◽  
Rick Russell
Keyword(s):  
Dna Cleavage ◽  
Target Sequence ◽  
Seed Region ◽  
Target Specificity ◽  
Loop Formation ◽  
Guide Rna ◽  
Dna Targeting ◽  
Cas9 Nuclease ◽  
Protospacer Adjacent Motif ◽  
Late Transition

SUMMARYClass II CRISPR-Cas nucleases are programmable via a single guide RNA, enabling genome editing applications in nearly all organisms. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically-separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite the functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence, implying substantial reversibility during R-loop formation –a late transition state– and the absence of a ‘seed’ region. Our results provide a quantitative underpinning for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity of Cas12a than the Cas9 nuclease.

scholarly journals Mechanism of R-loop formation and conformational activation of Cas9

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Martin Jinek
Keyword(s):  
Dna Cleavage ◽  
Structural Basis ◽  
Seed Region ◽  
Loop Formation ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strand ◽  
Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

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 CrisPam: SNP-derived PAM analysis web tool and human pathogenic SNPs database for CRISPR allele-specific targeting

2019 ◽  
Author(s):  
Roy Rabinowitz ◽  
Roy Darnell ◽  
Daniel Offen
Keyword(s):  
Dna Cleavage ◽  
Variant Allele ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Guide Rna ◽  
Dna Targeting ◽  
Specific Targeting ◽  
Novel Technology ◽  
Protospacer Adjacent Motif ◽  
Allele Specific

Abstract Background CRISPR is a promising novel technology for treating genetic conditions. Therefore, it is essential to further develop and promote treatment’s safety and specificity. While the guide-RNA offers position-specific DNA targeting, it may tolerate small changes such as single-nucleotide polymorphisms (SNPs). To that end, an allele-specific targeting approach is in need for future treatments of heterozygous patients, suffering from genetic conditions caused by a SNP. The SNP-derived PAM approach allows highly allele-specific DNA cleavage by incorporating a protospacer adjacent motif (PAM) sequence only at the target allele. Description Here we present CrisPam, a tool that detects SNP-derived PAMs for allele-specific targeting by the CRISPR/Cas system. The algorithm scans the generation of each reported PAM for a given DNA sequence and its variations. A successful result is such that at least one PAM is generated by a SNP. Thus, the PAM shall be part of the variant allele only and the Cas protein will therefore be able to exclusively bind the variant allele for gene-editing, while the wildtype allele remains unchanged. Conclusion CrisPam is available online for researchers and also offers access to the CrisPamDB, a database that contains the CrisPam analysis for any reported pathogenic SNP in humans.

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

2019 ◽  
Vol 60 (10) ◽  
pp. 2255-2262 ◽  
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.

scholarly journals Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

2021 ◽  
Vol 7 (11) ◽  
pp. eabd6030
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
Bryan A. Gibson ◽  
Michael K. Rosen ◽  
Rick Russell ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Eukaryotic Cells ◽  
Loop Formation ◽  
Chromatin Compaction ◽  
Dna Targeting ◽  
Genomic Regions ◽  
Dna Bendability ◽  
Nucleosome Arrays

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding—PAM recognition and R-loop formation—are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

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

2020 ◽  
Vol 48 (15) ◽  
pp. 8601-8616 ◽  
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.

scholarly journals A cloning-free method for CRISPR/Cas9-mediated genome editing in fission yeast

2017 ◽  
Author(s):  
Xiao-Ran Zhang ◽  
Jia-Bei He ◽  
Yi-Zheng Wang ◽  
Li-Lin Du
Keyword(s):  
Fission Yeast ◽  
Genome Editing ◽  
Dna Cleavage ◽  
Genomic Sequence ◽  
Yeast Cells ◽  
Target Sequence ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Guide Rna ◽  
Sequence Deletion

ABSTRACTThe CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the ura4 or bsdMX marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.

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 DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a)

2018 ◽  
Vol 115 (21) ◽  
pp. 5444-5449 ◽  
Author(s):  
Digvijay Singh ◽  
John Mallon ◽  
Anustup Poddar ◽  
Yanbo Wang ◽  
Ramreddy Tippana ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Fluorescence Analysis ◽  
Dna Interaction ◽  
Target Sequence ◽  
Solution Ph ◽  
Guide Rna ◽  
Product Release ◽  
Time Observation

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5′ guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

scholarly journals Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

2020 ◽  
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
Bryan A. Gibson ◽  
Michael K. Rosen ◽  
Rick Russell ◽  
...  
Keyword(s):  
Dna Binding ◽  
Histone Modifications ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Eukaryotic Cells ◽  
Loop Formation ◽  
Core Particle ◽  
Dna Targeting ◽  
Dna Binding And Cleavage ◽  
Nucleosome Arrays

AbstractGenome engineering nucleases, including CRISPR-Cas12a, must access chromatinized DNA. Here, we investigate how Acidaminococcus sp. Cas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates DNA target accessibility to Cas12a. Nucleosome unwrapping determines the extent to which both steps of Cas12a binding–PAM recognition and R-loop formation–are inhibited by the nucleosome. Nucleosomes inhibit Cas12a binding even beyond the canonical core particle. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing a target-proximal nuclease-inactive Cas9 enhances DNA cleavage rates over 10-fold. Surprisingly, Cas12a readily cleaves DNA linking nucleosomes within chromatin-like phase separated nucleosome arrays—with DNA targeting reduced only ~4-fold. This work provides a mechanism for the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted regions of cells. We conclude that nucleosome wrapping restricts accessibility to CRISPR-Cas nucleases and anticipate that increasing nucleosome breathing dynamics will improve DNA binding and cleavage in eukaryotic cells.

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