scholarly journals Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

2017 ◽  
Vol 114 (21) ◽  
pp. 5443-5448 ◽  
Author(s):  
Vladimir Mekler ◽  
Leonid Minakhin ◽  
Konstantin Severinov
Keyword(s):  
Dna Sequences ◽  
Rna Binding ◽  
Proximal Region ◽  
Target Sequence ◽  
Duplex Dna ◽  
Rna Molecules ◽  
Guide Rna ◽  
Double Stranded Dna ◽  
Protospacer Adjacent Motif ◽  
Dna Complex

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA–DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA–DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

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 Concurrent binding to DNA and RNA facilitates the pluripotency reprogramming activity of Sox2

2020 ◽  
Vol 48 (7) ◽  
pp. 3869-3887 ◽  
Author(s):  
Linlin Hou ◽  
Yuanjie Wei ◽  
Yingying Lin ◽  
Xiwei Wang ◽  
Yiwei Lai ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Target Genes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
High Mobility ◽  
Binding Motif ◽  
Dna And Rna ◽  
Somatic Cell Reprogramming ◽  
Double Stranded Dna

Abstract Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.

scholarly journals Heterogeneous nuclear ribonucleoprotein D0B is a sequence-specificDNA-binding protein

1999 ◽  
Vol 338 (2) ◽  
pp. 417-425 ◽  
Author(s):  
Mate TOLNAY ◽  
Lyudmila A. VERESHCHAGINA ◽  
George C. TSOKOS
Keyword(s):  
Dna Sequences ◽  
Rna Binding ◽  
Binding Protein ◽  
Double Helix ◽  
Physiological Role ◽  
Single Stranded Dna ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Double Stranded Dna ◽  
Nuclear Ribonucleoprotein

Complement receptor 2 (CR2) is important in the regulation of the B lymphocyte response; the regulation of its expression is therefore of central importance. We recently reported that a 42 kDa heterogeneous nuclear ribonucleoprotein (hnRNP) is involved in the transcriptional regulation of the human CR2 gene [Tolnay, Lambris and Tsokos (1997) J. Immunol. 159, 5492–5501]. We cloned the cDNA encoding this protein and found it to be identical with hnRNP D0B, a sequence-specific RNA-binding protein. By using a set of mutated oligonucleotides, we demonstrated that the recombinant hnRNP D0B displays sequence specificity for double-stranded oligonucleotide defined by the CR2 promoter. We conducted electrophoretic mobility-shift assays to estimate the apparent Kd of hnRNP D0B for the double-stranded DNA motif and found it to be 59 nM. Interestingly, hnRNP D0B displayed affinities of 28 and 18 nM for the sense and anti-sense strands of the CR2 promoter-defined oligonucleotide respectively. The significantly greater binding affinity of hnRNP D0B for single-stranded DNA than for double-stranded DNA suggests that the protein might melt the double helix. The intranuclear concentration of sequence-specific protein was estimated to be 250–400 nM, indicating that the protein binds to the CR2 promoter in vivo. Co-precipitation of a complex formed in vivo between hnRNP D0B and the TATA-binding protein demonstrates that hnRNP D0B interacts with the basal transcription apparatus. Our results suggest a new physiological role for hnRNP D0B that involves binding to double- and single-stranded DNA sequences in a specific manner and functioning as a transcription factor.

scholarly journals Enhancement of Target Specificity of CRISPR-Cas12a by Using a Chimeric DNA-RNA Guide

2020 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Seung-Hun Kang ◽  
Junho K. Hur ◽  
Hyomin Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Genetic Mutation ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Effective Manner ◽  
Target Dna

AbstractThe CRISPR-Cas9 system is widely used for target-specific genome engineering. Cpf1 is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cpf1 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 Cpf1 activity is improved for therapeutic purposes. In our study, we investigated off-target cleavage by Cpf1 and modified the Cpf1 (cr)RNA to address the off-target cleavage issue. We developed a CRISPR-Cpf1 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-Cpf1 and SpCas9 nickase effectively work in the intracellular genome is suggested. In our results, CRISPR-Cpf1 induces less off-target mutations at the cell level, when chimeric DNA-RNA guide was used for genome editing. This study has a potential for therapeutic applications in incurable diseases caused by genetic mutation.

scholarly journals A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

2016 ◽  
Vol 2 (8) ◽  
pp. e1600699 ◽  
Author(s):  
Hiroshi Arakawa
Keyword(s):  
Dna Sequences ◽  
Genetic Screening ◽  
Restriction Enzymes ◽  
Mrna Sequence ◽  
Complementary Sequence ◽  
Complementary Dna ◽  
Random Primer ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Target Dna

The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally making gRNA. I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species.

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 Random-PE: an efficient integration of random sequences into mammalian genome by prime editing

2021 ◽  
Vol 2 (1) ◽  
Author(s):  
Yaoge Jiao ◽  
Lifang Zhou ◽  
Rui Tao ◽  
Yanhong Wang ◽  
Yun Hu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Mammalian Cells ◽  
Fine Tuning ◽  
Random Sequences ◽  
Strand Breaks ◽  
Guide Rna ◽  
Simple Strategy ◽  
Protospacer Adjacent Motif ◽  
Efficient Integration

AbstractPrime editing (PE) enables efficiently targeted introduction of multiple types of small-sized genetic change without requiring double-strand breaks or donor templates. Here we designed a simple strategy to introduce random DNA sequences into targeted genomic loci by prime editing, which we named random prime editing (Random-PE). In our strategy, the prime editing guide RNA (pegRNA) was engineered to harbor random sequences between the primer binding sequence (PBS) and homologous arm (HA) of the reverse transcriptase templates. With these pegRNAs, we achieved efficient targeted insertion or substitution of random sequences with different lengths, ranging from 5 to 10, in mammalian cells. Importantly, the diversity of inserted sequences is well preserved. By fine-tuning the design of random sequences, we were able to make simultaneously insertions or substitutions of random sequences in multiple sites, allowing in situ evolution of multiple positions in a given protein. Therefore, these results provide a framework for targeted integration of random sequences into genomes, which can be redirected for manifold applications, such as in situ protospacer adjacent motif (PAM) library construction, enhancer screening, and DNA barcoding.

scholarly journals Cas12a trans-cleavage can be modulated in vitro and is active on ssDNA, dsDNA, and RNA

2019 ◽  
Author(s):  
Ryan T. Fuchs ◽  
Jennifer Curcuru ◽  
Megumu Mabuchi ◽  
Paul Yourik ◽  
G. Brett Robb
Keyword(s):  
Dna Sequences ◽  
Nuclease Activity ◽  
Target Sequence ◽  
Sequence Composition ◽  
Guide Rna ◽  
Dna Targeting ◽  
Single Turnover ◽  
Target Dna ◽  
Buffer Composition

ABSTRACTCRISPR-Cas12a (Cpf1) are RNA-guided nuclease effectors of acquired immune response that act in their native organisms by cleaving targeted DNA sequences. Like CRISPR-Cas9 RNA-guided DNA targeting enzymes, Cas12a orthologs have been repurposed for genome editing in non-native organisms and for DNA manipulationin vitro. Recent studies have shown that activation of Cas12a via guide RNA-target DNA pairing causes multiple turnover, non-specific ssDNA degradation intrans, after single turnover on-target cleavage incis. We find that the non-specifictransnuclease activity affects RNA and dsDNA in addition to ssDNA, an activity made more evident by adjustment of reaction buffer composition. The magnitude of thetransnuclease activity varies depending on features of the guide RNA being used, specifically target sequence composition and length. We also find that the magnitude oftransnuclease activity varies between the three most well-studied Cas12a orthologs and that the Cas12a fromLachnospiraceaebacterium ND2006 appears to be the most active.

scholarly journals Functional Identification of the Xanthomonas oryzae pv. oryzae Type I-C CRISPR-Cas System and Its Potential in Gene Editing Application

2021 ◽  
Vol 12 ◽  
Author(s):  
Qibing Liu ◽  
Siwei Wang ◽  
Juying Long ◽  
Zhuoyue Chen ◽  
Bing Yang ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Gene Editing ◽  
Xanthomonas Oryzae ◽  
Functional Study ◽  
Target Sequence ◽  
Type I ◽  
Functional Identification ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
New Vision

The type I clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is one of five adaptive immune systems and exists widely in bacteria and archaea. In this study, we showed that Xanthomonas oryzae pv. oryzae (Xoo) possesses a functional CRISPR system by engineering constructs mimicking its CRISPR cassette. CRISPR array analysis showed that the TTC at the 5′-end of the target sequence is a functional protospacer-adjacent motif (PAM) of CRISPR. Guide RNA (gRNA) deletion analysis identified a minimum of 27-bp spacer that was required to ensure successful self-target killing in PXO99A strain. Mutants with deletion of individual Cas genes were constructed to analyze the effects of Cas proteins on mature CRISPR RNA (crRNA), processing intermediates and DNA interference. Results showed that depleting each of the three genes, cas5d, csd1, and csd2 inactivated the pre-crRNA processing, whereas inactivation of cas3 impaired in processing pre-crRNA. Furthermore, the Xoo CRISPR/Cas system was functional in Pseudomonas syringae pv. tomato. Collectively, our results would contribute to the functional study of CRISPR/Cas system of Xoo, and also provide a new vision on the use of bacterial endogenous systems as a convenient tool for gene editing.

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.

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