scholarly journals Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

2019 ◽  
Author(s):  
Stephen K. Jones ◽  
John A. Hawkins ◽  
Nicole V. Johnson ◽  
Cheulhee Jung ◽  
Kuang Hu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Massively Parallel ◽  
Biophysical Model ◽  
Rna Sequences ◽  
Time Resolved ◽  
Guide Rna ◽  
Dna Library ◽  
Cleavage Specificity ◽  
Target Binding

AbstractEngineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeq— nuclease digestion and deep sequencing—a massively parallel platform that measures cleavage kinetics and captures the time-resolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a—reported as a more specific nuclease in cells—has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

scholarly journals Repressing Integrase attachment site operation with CRISPR-Cas9 in E. coli

2017 ◽  
Author(s):  
Andrey Shur ◽  
Richard M. Murray
Keyword(s):  
Dna Sequences ◽  
Dna Recombination ◽  
Attachment Site ◽  
Memory Systems ◽  
Rna Sequences ◽  
Guide Rna ◽  
Future Goals ◽  
Attachment Sites ◽  
Synthetic Cell ◽  
Specific Pair

AbstractSerine integrases are bacteriophage proteins responsible for integrating the phage genome into that of the host. Synthetic biologists have co-opted these proteins into useful tools for permanent DNA logic, utilizing their specific DNA recombination abilities to build synthetic cell differentiation and genetic memory systems. Each integrase has a specific pair of DNA sequences (attP/attB sites) that it recombines, but multiple identical sites can result in unpredictable recombination. We have developed a way to control integrase activity on identical attP/attB sites by using catalytically dead Cas9 (dCas9) as a programmable binding protein that can compete with integrase for binding to specific attachment sites. Utilizing a plasmid that contains two identical Bxb1 attP sites, integration can be repressed up to 8 fold at either one of the two attP sites when guide RNA and dCas9 are present. Guide RNA sequences that bind specifically to attB, or either of two attP sites, have been developed. Future goals are to utilize this technology to construct larger and more complex integrase logic circuits.

scholarly journals Genome reading by the NF-κB transcription factors

2019 ◽  
Vol 47 (19) ◽  
pp. 9967-9989 ◽  
Author(s):  
Maria Carmen Mulero ◽  
Vivien Ya-Fan Wang ◽  
Tom Huxford ◽  
Gourisankar Ghosh
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequences ◽  
Target Genes ◽  
Target Selection ◽  
Structural Features ◽  
Response Elements ◽  
Target Binding

Abstract The NF-κB family of dimeric transcription factors regulates transcription by selectively binding to DNA response elements present within promoters or enhancers of target genes. The DNA response elements, collectively known as κB sites or κB DNA, share the consensus 5′-GGGRNNNYCC-3′ (where R, Y and N are purine, pyrimidine and any nucleotide base, respectively). In addition, several DNA sequences that deviate significantly from the consensus have been shown to accommodate binding by NF-κB dimers. X-ray crystal structures of NF-κB in complex with diverse κB DNA have helped elucidate the chemical principles that underlie target selection in vitro. However, NF-κB dimers encounter additional impediments to selective DNA binding in vivo. Work carried out during the past decades has identified some of the barriers to sequence selective DNA target binding within the context of chromatin and suggests possible mechanisms by which NF-κB might overcome these obstacles. In this review, we first highlight structural features of NF-κB:DNA complexes and how distinctive features of NF-κB proteins and DNA sequences contribute to specific complex formation. We then discuss how native NF-κB dimers identify DNA binding targets in the nucleus with support from additional factors and how post-translational modifications enable NF-κB to selectively bind κB sites in vivo.

scholarly journals Purification of specific DNA species using the CRISPR system

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Toshitsugu Fujita ◽  
Hodaka Fujii
Keyword(s):  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
In Vitro Method ◽  
Guide Rna ◽  
Crispr System ◽  
Target Dna ◽  
Genomic Regions ◽  
Generation Sequencing ◽  
Subsequent Identification

AbstractIn 2013, we developed a new method of engineered DNA-binding molecule-mediated chromatin immunoprecipitation that incorporates the clustered regularly interspaced short palindromic repeats (CRISPR) system to purify specific DNA species. This CRISPR-mediated purification can be performed in-cell or in vitro; CRISPR complexes can be expressed to tag target DNA sequences in the cells to be analyzed, or a CRISPR ribonucleoprotein complex consisting of recombinant nuclease-dead Cas9 (dCas9) and synthetic guide RNA can be used to tag target DNA sequences in vitro. Both methods enable purification of specific DNA sequences in chromatin structures for subsequent identification of molecules (proteins, RNAs, and other genomic regions) associated with the target sequences. The in vitro method also enables enrichment of purified DNA sequences from a pool of heterogeneous sequences for next-generation sequencing or other applications. In this review, we outline the principle of CRISPR-mediated purification of specific DNA species and discuss recent advances in the technology.

scholarly journals In vitro transcription of cloned 5S RNA genes of the newt Notophthalmus.

1981 ◽  
Vol 90 (2) ◽  
pp. 323-331 ◽  
Author(s):  
B K Kay ◽  
O Schmidt ◽  
J G Gall
Keyword(s):  
Dna Sequences ◽  
Gene Segment ◽  
In Vitro Transcription ◽  
Xenopus Laevis Oocyte ◽  
Coding Region ◽  
5S Rna ◽  
Rna Sequences ◽  
Repeat Units ◽  
5S Gene

Recombinant plasmids that carried genes coding for 5S ribosomal RNA of the newt, Notophthalmus viridescens, were transcribed in vitro with extracts of Xenopus laevis oocyte nuclei. Plasmids containing multiple repeats of the 5S gene and spacer directed accurate transcription of 5S RNA (120 bases). Individual repeat units were recloned by inserting Sau 3A restriction fragments into the Bam HI site of plasmid pBR322. Because each repeat was cut by the enzyme within the coding region, the inserts had incomplete coding regions at their ends and spacer sequences in the middle. The DNA of these subclones directed synthesis of a 5S-size RNA that contained both plasmid and 5S RNA sequences. Transcription initiated in the vector, proceeded through the gene segment coding for nucleotides 41-120, and terminated at the end of the gene. The initiation of in vitro transcription required neither the original 5' flanking sequences of the spacer nor the first third of the gene. We conclude that intragenic DNA sequences control the initiation of transcription. Other subclones that include pseudogenes gave rise to some transcripts 156 nucleotides long. These long transcripts represented continuation of transcription through the 36-base-pair pseudogene that is located immediately downstream from the 5S gene. However, most transcripts of these subclones terminated at the end of the normal gene before the beginning of the pseudogene. It is probable that a run of four or more Ts serves as part of the termination signal.

scholarly journals Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

2020 ◽  
Author(s):  
Ida Höijer ◽  
Josefin Johansson ◽  
Sanna Gudmundsson ◽  
Chen-Shan Chin ◽  
Ignas Bunikis ◽  
...  
Keyword(s):  
Binding Sites ◽  
De Novo ◽  
Target Prediction ◽  
Human Cell Line ◽  
Guide Rna ◽  
Target Sites ◽  
Long Read ◽  
Target Binding

AbstractA much-debated concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target sites and activity is challenging. Here we present SMRT-OTS and Nano-OTS, two amplification-free long-read sequencing protocols for detection of gRNA driven digestion of genomic DNA by Cas9. The methods were assessed using the human cell line HEK293, which was first re-sequenced at 18x coverage using highly accurate (HiFi) SMRT reads to get a detailed view of all on- and off-target binding regions. We then applied SMRT-OTS and Nano-OTS to investigate the specificity of three different gRNAs, resulting in a set of 55 high-confidence gRNA binding sites identified by both methods. Twenty-five (45%) of these sites were not reported by off-target prediction software, either because they contained four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. We further discovered that a heterozygous SNP can cause allele-specific gRNA binding. Finally, by performing a de novo genome assembly of the HiFi reads, we were able to re-discover 98.7% of the gRNA binding sites without any prior information about the human reference genome. This suggests that CRISPR-Cas9 off-target sites can be efficiently mapped also in organisms where the genome sequence is unknown. In conclusion, the amplification-free sequencing protocols revealed many gRNA binding sites in vitro that would be difficult to predict based on gRNA sequence alignment to a reference. Nevertheless, it is still unknown whether in vivo off-target editing would occur at these sites.

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.

In vitro Assay Revealed Mismatches between Guide RNA and Target DNA can Enhance Cas9 Nuclease Activity

2020 ◽  
Vol 1 (1) ◽  
pp. 69-72
Author(s):  
Ji Luan ◽  
Zhen Li ◽  
Hailong Wang ◽  
Jun Fu ◽  
Youming Zhang
Keyword(s):  
Dna Sequences ◽  
High Efficiency ◽  
Nuclease Activity ◽  
Ease Of Use ◽  
Unexpected Finding ◽  
Vitro Assay ◽  
Guide Rna ◽  
Cleavage Activity ◽  
Target Dna

Background: CRISPR-Cas9 is a powerful technology that allows us to modify DNA sequences in a specific manner across a variety of organisms. Due to its high efficiency and specificity, and ease of use, it becomes a commonly used method for gene editing. Although many structural and biochemical studies have been carried out to understand the fundamental mechanism of CRISPR/Cas9, our understanding of CRISPR/Cas9 caused off-target effects is still lacking. Methods: The enhanced in vitro cleavage activity of Cas9 protein from Streptococcus pyogenes (SpCas9) was evaluated by both synthetic crRNA-tracrRNA duplexes and in vitro transcribed single guide RNAs. Results: Here, we report an unexpected finding that mismatches between the guide RNA and target DNA significantly enhanced the in vitro cleavage activity of SpCas9 by more than 2 folds. Conclusion: Our observation that mismatches between the guide RNA and target DNA can dramatically increase the in vitro cleavage of Cas9 suggests the potential sequence preference for the CRSIPR/Cas9 system.

scholarly journals Viral Hormones: Expanding Dimensions in Endocrinology

Endocrinology ◽  
2019 ◽  
Vol 160 (9) ◽  
pp. 2165-2179 ◽  
Author(s):  
Qian Huang ◽  
C Ronald Kahn ◽  
Emrah Altindis
Keyword(s):  
Dna Sequences ◽  
Sequence Similarity ◽  
Endocrine System ◽  
Peptide Hormones ◽  
Rna Sequences ◽  
High Sequence Similarity ◽  
In Vivo Models ◽  
Viral Mimicry

AbstractViruses have developed different mechanisms to manipulate their hosts, including the process of viral mimicry in which viruses express important host proteins. Until recently, examples of viral mimicry were limited to mimics of growth factors and immunomodulatory proteins. Using a comprehensive bioinformatics approach, we have shown that viruses possess the DNA/RNA with potential to encode 16 different peptides with high sequence similarity to human peptide hormones and metabolically important regulatory proteins. We have characterized one of these families, the viral insulin/IGF-1–like peptides (VILPs), which we identified in four members of the Iridoviridae family. VILPs can bind to human insulin and IGF-1 receptors and stimulate classic postreceptor signaling pathways. Moreover, VILPs can stimulate glucose uptake in vitro and in vivo and stimulate DNA synthesis. DNA sequences of some VILP-carrying viruses have been identified in the human enteric virome. In addition to VILPs, sequences with homology to 15 other peptide hormones or cytokines can be identified in viral DNA/RNA sequences, some with a very high identity to hormones. Recent data by others has identified a peptide that resembles and mimics α-melanocyte-stimulating hormone’s anti-inflammatory effects in in vitro and in vivo models. Taken together, these studies reveal novel mechanisms of viral and bacterial pathogenesis in which the microbe can directly target or mimic the host endocrine system. These findings also introduce the concept of a system of microbial hormones that provides new insights into the evolution of peptide hormones, as well as potential new roles of microbial hormones in health and disease.

scholarly journals Sequence-specific inhibition of reverse transcription by recombinant CRISPR/dCas13a ribonucleoprotein complexes in vitro

2021 ◽  
Author(s):  
Toshitsugu Fujita ◽  
Shoko Nagata ◽  
Miyuki Yuno ◽  
Hodaka Fujii
Keyword(s):  
Dna Sequences ◽  
Reverse Transcription ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Binding Activity ◽  
Guide Rna ◽  
Ribonucleoprotein Complexes ◽  
Specific Manner ◽  
Rnp Complex

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for genome editing because of its ability to cleave specific DNA sequences. Recently, RNA-specific CRISPR systems have been reported. CRISPR systems, consisting of a guide RNA (gRNA) and a nuclease-dead form of Cas13a (dCas13a), can be used for RNA editing and visualization of target RNA. In this study, we examined whether a recombinant CRISPR/dCas13a ribonucleoprotein (RNP) complex could be used to inhibit reverse transcription (RT) in a sequence-specific manner in vitro. Recombinant Leptotrichia wadei dCas13a was expressed using the silkworm-baculovirus expression system and affinity-purified. We found that the CRISPR/dCas13a RNP complex, combined with a chemically-synthesized gRNA sequence, could specifically inhibit RT of EGFR and NEAT1, but not non-specific RNA. Thus, the CRISPR/dCas13a RNP complex can inhibit RT reactions in a sequence-specific manner. RT inhibition by the CRISPR/dCas13a system may be useful to assess target binding activity, to discriminate RNA species retaining target sequences of gRNA, or to suppress RT from undesirable RNA species.

scholarly journals Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement

2021 ◽  
Vol 7 (8) ◽  
pp. eabe5496
Author(s):  
Evan A. Boyle ◽  
Winston R. Becker ◽  
Hua B. Bai ◽  
Janice S. Chen ◽  
Jennifer A. Doudna ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Dna Cleavage ◽  
High Specificity ◽  
Biophysical Model ◽  
Target Sequence ◽  
Sequence Context ◽  
Guide Rnas ◽  
Target Dna ◽  
Target Binding ◽  
Model Sequence

The RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data have hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage and then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target modulates Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to “proofreading” capability. Lastly, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.

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