Cas9 cleavage assay for pre-screening of sgRNAs using nicking triggered isothermal amplification

Abstract

A novel Cas9 cleavage assay was developed for quantitative evaluation of Cas9 cleavage efficiency and pre-screening of sgRNA to achieve highly specific and highly efficient CRISPR/Cas9 genome editing.

A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection

“…This unique conformational rearrangement may provide an ideal targeting site for various isothermal amplification techniques with enhanced robustness, specificity, and sensitivity due to the intrinsic properties of CRISPR effectors. For example, when combined with the exponential amplification reaction (EXPAR) and rolling cycle amplification (RCA), the CRISPR effector, Cas9, has been successfully applied in the efficient pre-screening of sgRNAs 27 and sensitive in situ genomic loci detection 28 , respectively. Thus, this CRISPR effectors-triggered strategy has great potentials to be applied in different situations.…”

Construction of an autonomously concatenated hybridization chain reaction for signal amplification and intracellular imaging

“…The isothermal amplified nucleic acid detection has attracted substantial research interests, 1 – 3 and numerous isothermal amplification means have been developed and applied in homogeneous diagnostics, forensics, medicine, and agriculture. 4 – 6 These include nucleic acid sequence-based amplification (NASBA), 7 , 8 signal-mediated amplification of RNA technology (SMART), 9 , 10 rolling circle amplification (RCA), 11 , 12 strand displacement amplification (SDA), 13 , 14 and loop-mediated isothermal amplification (LAMP). 15 , 16 Isothermal amplification provides a rapid and efficient tool at a constant temperature without thermocycling required in traditional polymerase chain reaction (PCR).…”

Microfluidic systems for rapid antibiotic susceptibility tests (ASTs) at the single-cell level

“…For example, some newly developed CRISPR systems (such as Cas12a and Cas13a) have now be used for genotypic and phenotypic analysis of bacterial pathogens. 47,[65][66][67][68][69][70] Specically, a CRISPR-Cas13a/C2c2-based SHERLOCK platform has been applied for ultrafast and sensitive RNA/DNA quantitation, which is suitable for rapid identication of bacterial pathogens by detection of specic bacterial genes. 66 Recently, an allosteric probe-initiated catalysis and CRISPR-Cas13a amplication reaction (termed 'APC-Cas') was developed for detecting very low numbers of a bacterial pathogens without isolation 71 and can selectively quantify bacterial cells with a concentration from 1 to 10 5 CFU mL À1 in various types of samples, and the cost of reagents can be reduced to below $1 per test.…”