The core experiments of ASFAST
ASFAST is a fast and portable device to detect the African swine fever virus (ASFV). The core experiments within ASFAST include the CRISPR-Cas12a system to specifically detect ASFV in a blood sample, and the PicoGreen fluorescent dye to indicate activation of the CRISPR-Cas12a system. The content below describes the steps and experiments in our device to provide a clear image of the experimental design for the development of ASFAST.
Introduction of CRISPR-Cas12a system
CRISPR-Cas12a system includes a LbCas12a protein with double stranded DNA (dsDNA) cleavage activity and a crRNA that has a guide protospacer adjacent motif (PAM) and a sequence complementary to the dsDNA target. Once the LbCas12a protein recognizes and binds with the target dsDNA through crRNA, LbCas12a protein executes cis cleavage activity to cut the target dsDNA. Furthermore, the cis cleavage activity of LbCas12a protein triggers the cleavage of non-specific single-stranded DNA (ssDNA), which is referred to as trans cleavage activity.
Steps to establish CRISPR-Cas12a system
Production and purification of crRNATo produce crRNAs for LbCas12a, we constructed a DNA template of crRNA with T7 promoter for in vitro transcription (IVT) assay. The DNA templates of crRNAs were flanked by prefix and suffix sequences, which can be easily amplified by PCR. Because the T7 terminator may add unexpected nucleotides to the 3’ terminus of crRNA in IVT assay, we treated DNA templates of crRNAs with KpnI to generate a 3’ protruding end and terminate the IVT directly. After crRNAs were generated by IVT assay, we further purified the crRNAs by phenol/chloroform extraction assay.
Confirmation of the cis-activity of Cas12a-crRNA complexIn our project, Cas12a-crRNA complex is designed to recognize the specific sequence of ASFV capsid gene p72, which shows the highest variation among ASFV strains. To improve the sensitivity of the Cas12a-crRNA complex, we constructed six different crRNAs targeting different parts of the p72 gene sequence. To confirm the cis-activity of Cas12a-crRNAs complex without using pathetic ASFV, we constructed a partial p72 gene sequence, which has no protein coding potential, into a yTA vector as a positive control. Similarly, vectors containing mutated partial p72 sequences serve as a negative control. In total, six pairs of positive and negative control vectors were generated for each crRNA. The co-incubation of Cas12a-crRNA complex with linearized positive control vectors should activate Cas12a-crRNA to cut the positive control vector into two fragments, whereas the linearized negative control vectors should remain as one fragment.
Confirmation of trans-activity of the Cas12a-crRNA complexThe activation of cis-activity of LbCas12a will in turn activate its trans-activity (collateral cleavage activity), which refers to the cleavage of non-specific ssDNA. Based on the principle of trans-activity of the Cas12a-crRNA complex, we added ssDNA to the activated (by positive control vector) or inactivated (by negative control vector) Cas12a-crRNA complex and confirmed the degradation of ssDNA (trans-activity) by gel electrophoresis.
1. Expression and purification of LbCas12a proteinThe Lachnospiraceae bacterium (Lb) Cas12a stands out among the LbCas12a orthologs for its trans nuclease activity. To express LbCas12a protein, we cultured E. coli BL21 (DE3) with LbCas12a expression vector (pHMT-Cas12a) at 37℃ until O.D. 600 approached 0.6 ~ 0.8, and then added IPTG to induce LbCas12a protein expression. The expressed LbCas12a protein is N-terminally fused with a His-tagged Maltose-Binding Protein (MBP) and Tobacco Etch Virus (TEV) Protease recognizing sequence, which increases the size of LbCas12a protein from 140kDa to 180kDa. Because the LbCas12a protein is huge, we induced LbCas12a protein expression at 16℃ for 14 hours to improve the correct protein folding.
After inducing LbCas12a protein expression, we extracted total protein from BL21 (DE3) by sonication. We then purified LbCas12a from the lysate using Nickel magnetic beads to pull down the His-tagged LbCas12a. The Nickel magnetic bead bound LbCas12a protein was further treated with TEV enzyme to cut the His-tagged MBP at the TEV recognition site to release LbCas12a protein from the beads. Finally, we used a dialysis membrane to filter LbCas12a into storage buffer, and stored it at –20 ℃.
Conjugation of ssDNA to magnetic bead
In ASFAST, the experiment (examination of ASFV) and detection of ASFV are connected by a magnetic bead transfer system. To conjugate ssDNA on magnetic beads for detection, we used EDC and NHS as the cross-linker to covalently conjugate amine-modified ssDNA to carboxylic magnetic beads by esterification.
Figure 1. The mechanism of the conjugation of ssDNA with magnetic beads.
The PicoGreen fluorescence dye
PicoGreen is an intercalating fluorochrome that has high sensitivity and affinity for dsDNA. In ASFAST, PicoGreen is used to optimize the detection of dsDNA after trans-activation of the Cas12a-crRNA complex. The quantitative measurement of the PicoGreen signal by fluorometer will indicate the presence or absence of ASFV in the sample.
Combination of Cas12a-crRNA complex, ssDNA-conjugated bead and PicoGreen system
After the feasibility of using the Cas12a-crRNA complex and ssDNA conjugated magnetic beads are confirmed, the final step is to put all the puzzle pieces together. First, the Cas12a-crRNA complex, ssDNA-conjugated magnetic beads, and positive control vector will be co-incubated. The positive control vector will activate the Cas12a-crRNA complex to cleave the ssDNA on magnetic beads in the trans reaction. The ssDNA-free magnetic beads will be transferred to a new tube, and PicoGreen added along with the complementary strand of magnetic beads bound ssDNA. The activation of the Cas12a-crRNA system by an ASFV specific sequence will decrease the strength of PicoGreen fluorescence.
Processing blood sample
To decrease the disruption of LbCas12a activity and PicoGreen fluorescence detection by hemoglobin in the blood, ASFAST will use pig serum as a starting material. The collection of blood samples and separation of serum in clot activator evacuated blood collection tubes will be performed by licensed experts, such as a veterinarian, in the future.