Efficient sequence-specific isolation of DNA fragments and chromatin by<i>in vitro</i>enChIP technology using recombinant CRISPR ribonucleoproteins

Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

doi:10.1101/033241

Cited by 5 publications

(5 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3a ). We utilized S. pyogenes dCas9 and gRNA consisting of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) sequences that are functional at 37 °C 12 . As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations

2021

Self Cite

View full text Add to dashboard Cite

Isothermal DNA amplification, such as recombinase polymerase amplification (RPA), is well suited for point-of-care testing (POCT) as it does not require lengthy thermal cycling. By exploiting DNA amplification at low temperatures that do not denature heat-sensitive molecules such as proteins, we have developed a blocking RPA method to detect gene mutations and examine the epigenetic status of DNA. We found that both nucleic acid blockers and nuclease-dead clustered regularly interspaced short palindromic repeats (CRISPR) ribonucleoproteins suppress RPA reactions by blocking elongation by DNA polymerases in a sequence-specific manner. By examining these suppression events, we are able to discriminate single-nucleotide mutations in cancer cells and evaluate genome-editing events. Methyl-CpG binding proteins similarly inhibit elongation by DNA polymerases on CpG-methylated template DNA in our RPA reactions, allowing for the detection of methylated CpG islands. Thus, the use of heat-sensitive molecules such as proteins and ribonucleoprotein complexes as blockers in low-temperature isothermal DNA amplification reactions markedly expands the utility and application of these methods.

show abstract

“…3a ). We utilized S. pyogenes dCas9 and gRNA consisting of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) sequences that are functional at 37 °C 12 . As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…3xFLAG-tagged S. pyogenes dCas9 protein (r3xFLAG-Sp-dCas9-D) was produced in our previous study 12 . To anneal crRNA and tracrRNA, 1 µl of crRNA (10 µM) and 1 µl of tracrRNA (10 µM) were annealed in 2 µl of nuclease-free water (total 4 µl) at 98 °C for 2 min and then cooled at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations

2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…Locus-tagging can be achieved by expression of engineered DNA-binding molecules in the cells to be analyzed (in-cell enChIP) (1). Alternatively, it can be achieved in vitro using recombinant or synthetic engineered DNA-binding molecules (in vitro enChIP) (7). After tagging with an engineered DNA-binding molecule, the locus is isolated by a nity puri cation and its associated proteins are identi ed by mass spectrometry (MS) (1,8).…”

Section: Introductionmentioning

confidence: 99%

MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis

Yuno¹,

Fujita

Fujii

2021

Preprint

Self Cite

View full text Add to dashboard Cite

Engineered DNA-binding molecule–mediated chromatin immunoprecipitation (enChIP) is a technology for purifying specific genomic regions to facilitate identification of their associated molecules, including proteins, RNAs, and other genomic regions. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, e.g., a variant of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). In this study, to increase the flexibility of enChIP and expand the range of target cells, we generated murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9. We constructed MSCV-based retroviral plasmids expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and various drug resistance genes. We showed that by using these plasmids, it is feasible to purify target genomic regions with yields comparable to those reported using other systems. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells.

show abstract

“…In a recent study by using recombinant CRISPR ribonucleoproteins, recovery yields of 40−60% of target fragments from the human genome with associated contamination of nonspecific fragments of ∼4−5% were reported. 35 Because of the incompleteness of the site-specific scission by pcPNAs/S1 nuclease and purification steps, the recovery yield of this method becomes <10%. To isolate simple restriction fragments, our previous method using biotinylated pcPNA yielded >40% of the target fragments.…”

mentioning

confidence: 99%

One-Pot Isolation of a Desired Human Genome Fragment by Using a Biotinylated pcPNA/S1 Nuclease Combination

et al. 2018

View full text Add to dashboard Cite

Scission of the human genome at predetermined sites and isolation of a particular fragment are of great interest for the analysis of lesion/modification sites, in proteomics, and for gene therapy. However, methods for human genome scission and specific fragment isolation are limited. Here, we report a novel one-pot method for the site-specific scission of DNA by using a biotinylated pcPNA/S1 nuclease combination and isolation of a desired fragment by streptavidin-coated magnetic beads. The proof of concept was initially demonstrated for the clipping of plasmid DNA and isolation of the required fragment. Our method was then successfully applied for the isolation of a fragment from the cell-derived human genome.

show abstract

Efficient sequence-specific isolation of DNA fragments and chromatin byin vitroenChIP technology using recombinant CRISPR ribonucleoproteins

Cited by 5 publications

References 30 publications

Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations

Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations

MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis

One-Pot Isolation of a Desired Human Genome Fragment by Using a Biotinylated pcPNA/S1 Nuclease Combination

Contact Info

Product

Resources

About

Efficient sequence-specific isolation of DNA fragments and chromatin byin vitroenChIP technology using recombinant CRISPR ribonucleoproteins

Cited by 5 publications

References 30 publications

Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations

Protein or ribonucleoprotein-mediated blocking of recombinase polymerase amplification enables the discrimination of nucleotide and epigenetic differences between cell populations

MSCV-based retroviral plasmids expressing&nbsp;3xFLAG-Sp-dCas9&nbsp;for enChIP analysis

One-Pot Isolation of a Desired Human Genome Fragment by Using a Biotinylated pcPNA/S1 Nuclease Combination

Contact Info

Product

Resources

About

MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis