An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip

CRISPR‐based platform for rapid, sensitive and field‐deployable detection of scale drop disease virus in Asian sea bass (Lates calcarifer)

“…In contrast, samples with fewer copies of target produced a strong control line and a barely visible test line, identical to the pattern observed in the notemplate control. To reduce the colour of the test line in no-target control (NTC) even further, we tried increasing the concentrations of FB reporter from 125 nM to 500, 750 and 1000 nM, but a faint test line could still be observed, in agreement with previous studies (Figure S6) (Gong et al, 2021;Patchsung et al, 2020).…”

“…For instance, it has been challenging to combine RPA and Cas12a reagents into one reaction due to the possibility of activated Cas12a trans-cleaving RPA primers (Wang et al, 2019). A recent finding that RPA primers with phosphorothioate bonds are refractory to Cas12a-mediated trans-cleavage may aid the development of one-pot workflows that afford even shorter test times and lower risks for cross-contamination (Gong et al, 2021). Another study also reported that the incorporation of DNA into guide RNAs enhanced the sensitivity of Cas12a detection by at least 10-fold, providing a simpler strategy for enhancing the sensitivity of RPA-Cas12a than extensive screening of RPA primers and optimization of reaction parameters (Ooi et al, 2021).…”

Rational Programming of Cas12a for Early-Stage Detection of COVID-19 by Lateral Flow Assay and Portable Real-Time Fluorescence Readout Facilities

“…For instance, two different groups [ 27 , 28 ] reported an LOD of 10 copies/µL using CRISPR/Cas12a, LAMP, and commercial Milenia HybriDetect dipsticks for colorimetric detection and a laboratory-centered fluorophotometer. Similarly, by substituting LAMP with RPA, Talwar et al [ 2 ] and Gong et al [ 31 ] also achieved LODs of 10 and 5 copies/reaction, respectively. In the HybriDetect dipstick, a test line is immobilized with an anti-goat antibody that captures AuNPs with goat anti-FITC antibody, whereas the control line is coated with a streptavidin that captures the FITC-biotinylated reporter.…”

“…This switching mechanism of the HybriDetect dipstick has disadvantages in that it can lead to intuitive misinterpretation due to the competitive binding of the reporter to the control and test lines. More recently, Gong et al [ 31 ] demonstrated a novel reporter in a method named TESTOR (Targeting DNA by Cas12a-based Eye Sight Testing in a Onepot Reaction) to solve the unspecificity of the HybriDetect dipstick. The concept of TESTOR is similar to that of the HybriDetect dipstick; however, the difference lies in TESTOR’s novel reporter, which was designed with a phosphorothioate in the middle of the digoxin and biotin labels.…”

Ionizable lipid-assisted efficient hepatic delivery of gene editing elements for oncotherapy

“…It has been applied to treat cancer [ 1 ], blister skin disease [ 2 ], Huntington's disease [ 3 ], sickle cell disease (SCD) [ 4 ], HIV-1 infection [ 5 ], and to establish specific animal model carrying a modified genome [ 6 ], etc. It can also be used to detect certain nucleic acid molecules, facilitating disease diagnosis, pathogenic microorganism analysis and environmental monitoring [ 7 , 8 ]. To some extent, the emergence of CRISPR/Cas reflects the significant advances of human gene editing technology, and provides an effective and powerful platform to further understand gene functions, underlying biological or pathological mechanisms of various disease, and ultimately development of novel treatment regimens.…”