Detection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique

Zhang, Beibei; Wang, Qiao; Xu, Xinhui; Xia, Qiang; Long, Feifei; Li, Weiwei; Shui, Ying-chun; Xia, Xinyi; Wang, Jinke

doi:10.1007/s00216-018-0873-5

Cited by 58 publications

(47 citation statements)

References 40 publications

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“…Due to extensive presence of PAM sequences in human genome, sgRNA design and selection are less of an issue for CATE. For preparing csgRNA simply and rapidly by in vitro transcription, a new sgRNA transcription template preparation method recently developed by our lab was used (56). The sgRNA transcription template can be rapidly prepared by a three-round PCR protocol.…”

Section: Discussionmentioning

confidence: 99%

CRISPR-assisted targeted enrichment-sequencing (CATE-seq)

Xia

Zhang

et al. 2019

Preprint

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The current targeted sequencing of genome is mainly dependent on various hybridization-based methods. However, the hybridization-based methods are still limited by the intrinsic shortcomings of nucleic acid hybridization. This study developed a new CRISPR-based targeted sequencing technique, CRISPR-assisted targeted enrichment-sequencing (CATE-seq). In this technique, the input genomic DNA (gDNA) was firstly bound by a complex of dCas9 and capture sgRNA (csgRNA). The DNA-dCas9-csgRNA complex was then captured on magnetic beads through an easy room-temperature annealing between a short universal capture sequence (24 bp) at the 3′ end of csgRNA and capture oligonucleotide coupled on magnetic beads. The enriched DNAs were finally analyzed by next generation sequencing. Using this technique, three different scales of targeted enrichments were successfully performed, including enriching 35 target exons of 6 genes from 6 gDNA samples with 54 csgRNAs, 339 target exons of 186 genes from 9 gDNA samples with 367 csgRNAs, and 2031 target exons of 451 genes from 2 gDNA samples with 2302 csgRNAs. This technique has several significant advantages over the current hybridization-based methods, including high simplicity, specificity, sensitivity, throughput, and scalability.

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Section: Discussionmentioning

confidence: 99%

CRISPR-assisted targeted enrichment-sequencing (CATE-seq)

Xia

Zhang

et al. 2019

Preprint

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“…For accurate detection and genotyping of HPV, which is critical for identifying those at risk of HPV‐related cancers, three versions of CRISPR‐typing PCR (ctPCR) methods were recently established (Figure 5a). In the first two versions, the target DNA was first identified and cut by a pair of Cas9–sgRNA complexes, then the cleaved DNA was ligated into a format that can be amplified and detected through PCR or qPCR (Q. Wang et al, 2018; B. Zhang et al, 2018). Specifically, the cleaved DNA was tailed by an adenine (A) and ligated by a T adaptor in ctPCR version 1.0, enabling amplification by a pair of general‐specific primers (Q. Wang et al, 2018); ctPCR version 2.0 ligated the cleaved product into the intermolecular concatenated linear or intramolecular circular DNA and amplified it with a pair of reverse primers, which could not amplify the target DNA without cleavage and ligation because of the reverse orientation of the two primers (B. Zhang et al, 2018).…”

Section: Detection Of Pathogenic Virusmentioning

confidence: 99%

Novel nucleic acid detection strategies based on CRISPR‐Cas systems: From construction to application

Zhu

Liu

Xie

et al. 2020

Biotech & Bioengineering

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Beyond their widespread application as genome‐editing and regulatory tools, clustered regularly interspaced short palindromic repeats (CRISPR)‐CRISPR‐associated (Cas) systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity. Recently developed Cas family effectors have opened the door to the development of new strategies for detecting different types of nucleic acids for a variety of purposes. Precise and efficient nucleic acid detection using CRISPR‐Cas systems has the potential to advance both basic and applied biological research. In this review, we summarize the CRISPR‐Cas systems used for the recognition and detection of specific nucleic acids for different purposes, including the detection of genomic DNA, nongenomic DNA, RNA, and pathogenic microbe genomes. Current challenges and further applications of CRISPR‐based detection methods will be discussed according to the most recent developments.

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“…Each DNA target had a pair of sgRNA, named sgRNAa and sgRNAb, respectively. According to the designed sgRNA, primers (Table S2) were synthesized to amplify sgRNA template by PCR using our previous protocol (42). The PCR-amplified sgRNA template has a T7 promoter sequence.…”

Section: Preparation Of Sgrnamentioning

confidence: 99%

“…The PCR-amplified sgRNA template has a T7 promoter sequence. SgRNA was then prepared by in vitro transcription using sgRNA template as previously described (42). The prepared sgRNA had a 5′-end 20 bp target DNA-specific sequence and a 3′-end capture sequence.…”

Section: Preparation Of Sgrnamentioning

confidence: 99%

“…For example, the CRISPR/Cas9 system has been early used to detect Zika viruses and can type Zika viruses (13). Recently, several DNA detection methods were developed by using its typical double-stranded DNA (dsDNA) cleavage activity of Cas9, such as CRISPR-typing PCR (ctPCR) (14)(15)(16), CUT-LAMP (17), FLASH (18), and . Several DNA detection methods were developed by using the Cas9 nickase activity, such as Cas9nAR (Cas9 nickase-based amplification reaction) (19), and CRISDA (CRISPR-Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification) (20).…”

Section: Introductionmentioning

confidence: 99%

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CRISPR-Assisted DNA Detection, a novel dCas9-based DNA detection technique

Luo

Gao

et al. 2020

Preprint

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Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present COVID-19 pandemic. The simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, the current nucleic acid detection techniques are still limited the traditional amplification and hybridization. To overcome the limitation, we here develop a CRISPR/Cas9-assisted DNA detection (CADD). In this detection, DNA sample is incubated with a pair of capture sgRNAs (sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa and the signal readout-related probe is captured by the capture sequence of sgRNAb.Finally the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cell and virus. Especially, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples were successfully detected by the method. All detections can be finished in 30 minutes at room temperature. This detection holds promise for rapid on-the-spot detection or point-of-care testing (POCT).

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Detection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique

Cited by 58 publications

References 40 publications

CRISPR-assisted targeted enrichment-sequencing (CATE-seq)

CRISPR-assisted targeted enrichment-sequencing (CATE-seq)

Novel nucleic acid detection strategies based on CRISPR‐Cas systems: From construction to application

CRISPR-Assisted DNA Detection, a novel dCas9-based DNA detection technique

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