Probing CRISPR-Cas12a Nuclease Activity Using Double-Stranded DNA-Templated Fluorescent Substrates

Smith, Christopher W.; Nandu, Nidhi; Kachwala, Mahera J; Chen, Yu-Sheng; Uyar, Taha Bilal; Yigit, Mehmet V.

doi:10.1021/acs.biochem.0c00140

Cited by 31 publications

(20 citation statements)

References 31 publications

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“…Moreover, the Cas12a nuclease is smaller in size than Sp Cas9 and uses a shorter CRISPR RNA (crRNA) for activity [ 14 ]. Notably, target site-activated Cas12a exhibits nonspecific trans activity against bystander nucleic acids [ 15 , 16 ], an attribute that has been harnessed in a burgeoning range of biosensors [ 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

Ittiprasert

Chatupheeraphat

Mann

et al. 2022

IJMS

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The efficiency of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. was compared with SpCas9 from Streptococcus pyogenes, for functional genomics in Schistosoma mansoni. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key schistosome enzyme termed omega-1. Programmed cleavages catalyzed by Cas12a and Cas9 resulted in staggered- and blunt-ended strand breaks, respectively. AsCas12a was more efficient than SpCas9 for gene knockout, as determined by TIDE analysis. CRISPResso2 analysis confirmed that most mutations were deletions. Knockout efficiency of both nucleases markedly increased in the presence of single-stranded oligodeoxynucleotide (ssODN) template. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands were tested, resulting in KO efficiencies of 15.67, 28.71, and 21.43% in the SpCas9 plus ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans-cleavage against the ssODNs by activated AsCas12a was not apparent in vitro. SpCas9 catalyzed more precise transgene insertion, with knock-in efficiencies of 17.07% for the KI_Cas9 group, 14.58% for KI_Cas12a-NT-ssODN, and 12.37% for KI_Cas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases.

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Section: Introductionmentioning

confidence: 99%

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

Ittiprasert

Chatupheeraphat

Mann

et al. 2022

IJMS

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“…The breakpoints for interpreting the results of in vitro susceptibility testing do not consider the pharmacokinetic factors. 28 Therefore, rapid lab detection and genotyping of carbapenem-resistant strains is very important for the antimicrobial treatment and outcomes of CRE infections.…”

Section: Discussionmentioning

confidence: 99%

A New Method Based on LAMP-CRISPR–Cas12a-Lateral Flow Immunochromatographic Strip for Detection

Xu¹,

Tang²,

Li³

et al. 2022

IDR

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Introduction Carbapenemase-mediated antimicrobial resistance is currently a hot spot of global concern. Carbapenem-resistant organisms are highly prevalent in hospitals associated with difficult-to-treat infections, resulting in poor clinical outcome due to limited treatment options. It is urgently needed to have a rapid, efficient, and convenient molecular assay for identifying such resistant strains. Methods For this end, we developed a new laboratory assay targeting Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM) based on loop-mediated isothermal amplification, CRISPR–Cas12a, and lateral flow immunochromatographic strip (CRISPR–Cas-LAMP-lateral flow strip). The method was designed to use a guide RNA (gRNA) to recognize the target DNA and guide Cas12a to cleave the target DNA, and simultaneously cleave any single-stranded DNA within the cleavage reaction system. Results The cleavage products are visible to the naked eye on the lateral flow strip. This method is highly sensitive in direct detection of bacteria in samples containing at least 3×10 5 CFU/mL without the need for bacterial culture. Discussion It provides shorter turnaround time and higher specificity than the conventional bacterial culture and susceptibility testing method. This new assay is applicable for extensive use in hospital infection control, as well as identification and treatment of resistant strains due to simple operation and inexpensive apparatuses.

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“…2) [122,148]. Moreover, in 2020 Christopher W. Smith and co-authors [149] research proved that this CRISPR-Cas12a trans-cleavage is not limited to ssDNA substrates, and Cas12a-based diagnostics can be extended to ssDNA/dsDNA hybrid substrates. Several variables of NaCl (50-150 mM) concentration and fluorescently silent ssDNA/dsDNA (0-12 bp nicked) hybrid substrates were applied in the bioassays to monitor CRISPR-Cas12a cis-(target) and trans-(nontarget) activities.…”

Section: Potentials and Limitation Of Crispr-cas12amentioning

confidence: 99%

Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review)

2022

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Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as ‘genetic scissors’, that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5–7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR–Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction. Graphical Abstract

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Probing CRISPR-Cas12a Nuclease Activity Using Double-Stranded DNA-Templated Fluorescent Substrates

Cited by 31 publications

References 31 publications

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

RNA-Guided AsCas12a- and SpCas9-Catalyzed Knockout and Homology Directed Repair of the Omega-1 Locus of the Human Blood Fluke, Schistosoma mansoni

A New Method Based on LAMP-CRISPR–Cas12a-Lateral Flow Immunochromatographic Strip for Detection

Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review)

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