An RNA-aptamer-based two-color CRISPR labeling system

Wang, Siyuan; Su, Jessica; Zhang, Feng; Zhuang, Xiaowei

doi:10.1038/srep26857

Cited by 81 publications

(86 citation statements)

References 24 publications

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“…Shorter and simpler guide-RNA 9 for Cpf1 could potentially be deleterious for its engineering or extension, as is done for Cas9's guide-RNA 39 . For e.g., an extra 5' guanine in the guide-RNA was extremely deleterious for LbCpf1 (Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

Singh

Mallon

Poddar

et al. 2017

Preprint

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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 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 have used single-molecule fluorescence imaging and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologues. Like Cas9, Cpf1 initially binds DNA in search of PAM (protospacer-adjacent motif) sequences, verifies the target sequence unidirectionally from the PAM-proximal end and rapidly rejects any targets that lack a PAM or that are poorly matched with the guide-RNA. Cpf1 requires ~ 17 bp sequence match for both stable binding and cleavage, contrasting it with Cas9 which requires 9 bp for stable binding and ~16 bp for cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAMdistal cleavage product, but not the PAM-proximal product. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

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Section: Discussionmentioning

confidence: 99%

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

Singh

Mallon

Poddar

et al. 2017

Preprint

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“…Since its introduction as a biological tool, the CRISPR/Cas9 system has been harnessed to create model organism genetic knockouts (KO), label chromatin, and even modulate gene activity (Dahlman et al, 2015; Konermann et al, 2015; Platt et al, 2014; Sung et al, 2014; Wang et al, 2016). Advantages of the CRISPR/Cas9 system include its ease of use and versatility, making it the tool of choice for genetic manipulation.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9 system-mediated impairment of synaptobrevin/VAMP function in postmitotic hippocampal neurons

Horvath

Kavalali

Monteggia³

2017

Journal of Neuroscience Methods

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Background The use of the CRISPR/Cas9 system is becoming widespread, however current studies have predominantly focused on dividing cells. It is currently unknown if CRISPR/Cas9 can be used in a postmitotic setting to examine non-cell autonomous/presynaptic phenotypes in the resulting genetically heterogeneous cell population. New method A single CRISPR/Cas9 lentivirus was used to transfect a high percentage of primary cultured neurons and target synaptobrevin 2 (Syb2, also called VAMP2). Results Primary hippocampal cultures infected with the Syb2 targeting virus displayed dramatic reductions in Syb2 protein and immunocytochemical staining. In many boutons Syb2 was completely undetected. These cultures recapitulated the known functional phenotypes of Syb2 knockout neurons, which are non-cell autonomous and presynaptic in origin, indicating that Syb2 was knocked out in a large fraction of neurons. Comparison with existing method(s) Previous methods used multiple viruses or sparse transfection methods and only examined cell autonomous or postsynaptic phenotypes. The current method demonstrates that the CRISPR/Cas9 system can be used to alter network dynamics by removing or lowering the target gene from a majority of cells in the culture. Conclusions A combination of CRISPR/Cas9 system and single high efficiency lentivirus infection can be used to examine non-cell autonomous and presynaptic phenotypes in postmitotic neurons.

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“…Furthermore, the RNA‐binding viral proteins MCP and PCP can be labelled with different colours via fluorescent proteins so that two distinct targets can be visualized simultaneously. As in Yi Fu's study, Siyuan Wang conducted analogous research and found that extending the tetraloop and stem loop 2 of the sgRNA with MS2 or PP7 aptamers can increase the signal‐to‐background ratio of chromatin imaging. In the meantime, Hanhui Ma et al proposed an approach similar to but more advanced than the above method.…”

Section: Applications To Detecting Specific Sequences Of Dna or Rnamentioning

confidence: 82%

The applications of CRISPR/Cas system in molecular detection

Zhou

Peng

Zhang

2018

J Cellular Molecular Medi

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The Streptococcus pyogenes CRISPR/Cas system has found widespread applications as a gene‐editing and regulatory tool for the simultaneous delivery of the Cas9 protein and guide RNAs into the cell, thus making the recognition of specific DNA sequences possible. The recent study that shows that Cas9 can also bind to and cleave RNA in an RNA‐programmable manner is suggestive of potential utility of this system as a universal nucleic‐acid recognition tool. To increase the signal intensity of the CRISPR/Cas system, a signal amplification technique has to be exploited appropriately; this requirement is also a challenge for the detection of DNA or RNA. Furthermore, the CRISPR/Cas system may be used to detect point mutations or single‐nucleotide variants because of the specificity of the recognition between the target sequence and the CRISPR/Cas system. These lines of evidence make this technique capable of detecting pathogens during infection via analysis of their DNA or RNA. Thus, here we summarize applications of the CRISPR/Cas system to the recognition and detection of DNA and RNA molecules as well as the signal amplification. We also describe its potential ability to detect mutations and single‐nucleotide variants. Finally, we sum up its applications to testing for pathogens and potential barriers for its implementation.

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An RNA-aptamer-based two-color CRISPR labeling system

Cited by 81 publications

References 24 publications

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1

CRISPR/Cas9 system-mediated impairment of synaptobrevin/VAMP function in postmitotic hippocampal neurons

The applications of CRISPR/Cas system in molecular detection

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