Bo Zhou,Ye Chen,Lifang Li,Jiashuo Liu,Yuyan Wang,Zheying Huang,Zheng Hu,Rui Tian,Zhen Li
Summary Here, we present a protocol for generating LtCas12a protein recognizing distinct TTNA (N represented A, T, C, G) protospacer adjacent motif sequence. We describe steps for transforming and harvesting bacterial cells and protein purification including nickel affinity chromatography and dialysis. We then detail procedures for verification of LtCas12a with cis- and trans-cleavage activities. For complete details on the use and execution of this protocol, please refer to Chen et al. (2023). 1
Songkuan Zhuang,Xiaojun Huang,Muyan Diao,Ruiling Dong,Yueyu Dong,Jun Liu,Yizhen Liu
Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.
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