A method for the quantitative detection of Cas12a ribonucleoproteins.

Cas12a ribonucleoprotein (RNP) is an RNA-guided CRISPR-associated nuclease used widely for genome editing and molecular diagnostics. Conventional detection methods rely on adopting antibody-based reagents that are expensive and lack scalability, and, moreover, only detect Cas12 enzyme rather than RNP, which is the true effector. Here, we describe a method for the rapid and quantitative detection of the effective Cas12a RNPs by the combined use of anti-CRISPR protein AcrVA1 and stem-loop RT-qPCR, achieving a limit of detection (LOD) of 1 fM in reaction buffer and 0.1 pM under biologically representative conditions.

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

The CRISPR-Cas12a nuclease shreds short single-stranded DNA (ssDNA) substrates indiscriminately through trans-cleavage upon activation with a specific target DNA. This shredding activity offered the potential for development of ssDNA-templated probes with fluorescent dye (F) and quencher (Q) labels. However, the formulations of double-stranded DNA (dsDNA)-templated fluorescent probes have not been reported possibly due to unknown (or limited) activity of Cas12a against short dsDNAs. The ssDNA probes have been shown to be powerful for diagnostic applications; however, limiting the probe selections to short ssDNAs could be restrictive from an application and probe diversification standpoint. Here, we report a dsDNA substrate (probe-full) for probing Cas12a trans-cleavage activity upon target detection. A diverse set of Cas12a substrates with alternating dsDNA character were designed and studied using fluorescence spectroscopy. We have observed that probe-full without any nick displayed trans-cleavage performance that was better than that of the form that contains a nick. Different experimental conditions of salt concentration, target concentration, and mismatch tolerance were examined to evaluate the probe performance. The activity of Cas12a was programmed for a dsDNA frame copied from a tobacco curly shoot virus (TCSV) or hepatitis B virus (HepBV) genome by using crRNA against TCSV or HepBV, respectively. While on-target activity offered detection of as little as 10 pM dsDNA target, off-target activity was not observed even at 1 nM control DNAs. This study demonstrates that trans-cleavage of Cas12a is not limited to ssDNA substrates, and Cas12a-based diagnostics can be extended to dsDNA substrates.

Sortase-mediated fluorescent labeling of CRISPR complexes.

Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. The sortase tag consists of just a few amino acids that are specifically recognized at either the N- or the C-terminus, making this strategy advantageous when the protein is part of a larger complex. Sortase is active at high ionic strength, 4°C, and with a broad range of organic fluorophores. We discuss the design, optimization, and single-molecule fluorescent imaging of CRISPR-Cas complexes on DNA curtains. Sortase-mediated transpeptidation is a versatile addition to the protein labeling toolkit.

Imaging genomic elements in living cells using CRISPR/Cas9.

In addition to their applications in genome editing and gene expression regulation, programmable DNA recognition systems, including both CRISPR and TALE, have been recently engineered for the visualization of endogenous genomic elements in living cells. This capability greatly helps the study of genome function regulation by its physical organization and interaction with other nuclear structures. This chapter first discusses the general considerations in designing and implementing the imaging system. The subsequent sections provide detailed protocols to use the CRISPR/Cas9 system to label and image specific genomic loci, including the establishment of expression systems for dCas9-GFP and sgRNA, the procedure to label repetitive sequences of telomeres and protein-coding genes, the simultaneous expression of many sgRNAs to label a nonrepetitive locus, and the verification of signal specificity by FISH.