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Microfluidic tool for rapid functional characterization of CRISPR complexes.

ABSTRACT: RNA guided nucleases are regarded as the future genome editing technologies. As such, they need to meet strong safety margins. Two major challenges in incorporating CRISPR technologies into the clinical world are off-target activity and editing efficiency. The common way to tackle such issues is to measure the binding and cleavage kinetics of the CRISPR enzyme. This can be challenging since, for example, DNA is not released from the CAS9 protein post cleavage. Here a promising new microfluidic approach to characterizing Enzymatic Interaction and Function of CRISPR complexes on a microfluidic platform (EnzyMIF) is presented. The method can rapidly detect the k_d, k_off, k_m and k_cat for various RNA guided nucleases. In this work, two single guide RNAs with significantly different in-cell cleavage efficiency, RAG2 and RAG1, are used as proof-of-concept. The EnzyMIF assay results provide biochemical characterization of these guide RNAs that can explain the difference in cleavage using both wild type (WT) CAS9 and HiFi CAS9. Notably, it is shown that EnzyMIF characterization correlates with cell culture genomic editing efficiency results. It is suggested that EnzyMIF can predict the quality of cleavage rapidly and quantitatively.

SUBMITTER: Peleg-Chen D

PROVIDER: S-EPMC8891023 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Microfluidic tool for rapid functional characterization of CRISPR complexes.

Peleg-Chen Dana D Shuvali Guy G Brio Lev L Ifrach Amit A Iancu Ortal O Barbiro-Michaely Efrat E Hendel Ayal A Gerber Doron D

New biotechnology 20220110

RNA guided nucleases are regarded as the future genome editing technologies. As such, they need to meet strong safety margins. Two major challenges in incorporating CRISPR technologies into the clinical world are off-target activity and editing efficiency. The common way to tackle such issues is to measure the binding and cleavage kinetics of the CRISPR enzyme. This can be challenging since, for example, DNA is not released from the CAS9 protein post cleavage. Here a promising new microfluidic a ...[more]

PMID: 35026470

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Project description:BackgroundCRISPR-Cas based diagnostic assays provide a portable solution which bridges the benefits of qRT-PCR and serological assays in terms of portability, specificity and ease of use. CRISPR-Cas assays are rapidly fieldable, specific and have been rigorously validated against a number of targets, including HIV and vector-borne pathogens. Recently, CRISPR-Cas12 and CRISPR-Cas13 diagnostic assays have been granted FDA approval for the detection of SARS-CoV-2. A critical step in utilizing this technology requires the design of highly-specific and efficient CRISPR RNAs (crRNAs) and isothermal primers. This process involves intensive manual curation and stringent parameters for design in order to minimize off-target detection while also preserving detection across divergent strains. As such, a single, streamlined bioinformatics platform for rapidly designing crRNAs for use with the CRISPR-Cas12 platform is needed. Here we offer PrimedSherlock, an automated, computer guided process for selecting highly-specific crRNAs and primers for targets of interest.ResultsUtilizing PrimedSherlock and publicly available databases, crRNAs were designed against a selection of Flavivirus genomes, including West Nile, Zika and all four serotypes of Dengue. Using outputs from PrimedSherlock in concert with both wildtype A.s Cas12a and Alt-R Cas12a Ultra nucleases, we demonstrated sensitive detection of nucleic acids of each respective arbovirus in in-vitro fluorescence assays. Moreover, primer and crRNA combinations facilitated the detection of their intended targets with minimal off-target background noise.ConclusionsPrimedSherlock is a novel crRNA design tool, specific for CRISPR-Cas12 diagnostic platforms. It allows for the rapid identification of highly conserved crRNA targets from user-provided primer pairs or PrimedRPA output files. Initial testing of crRNAs against arboviruses of medical importance demonstrated a robust ability to distinguish multiple strains by exploiting polymorphisms within otherwise highly conserved genomic regions. As a freely-accessible software package, PrimedSherlock could significantly increase the efficiency of CRISPR-Cas12 diagnostics. Conceptually, the portability of detection kits could also be enhanced when coupled with isothermal amplification technologies.

Dataset Information

Microfluidic tool for rapid functional characterization of CRISPR complexes.

Publications

Microfluidic tool for rapid functional characterization of CRISPR complexes.

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