High-performance detection of adenosine triphosphate (ATP) is of great importance due to its irreplaceable roles in various physiological processes. In this work, a four-stage signal amplification was established for ATP detection based on an allosteric probe-conjugated strand displacement amplification (SDA) integrated with CRISPR/Cpf1 system (ASD-Cpf1). An allosteric probe (AH) with a hairpin structure was designed to reduce background interference from self-extension and non-specific amplification during the SDA reaction. ATP bound with the aptamer segment at 5′-terminal of AH probe and subsequently opened the AH probe to activate the SDA reaction. The amplification products were recognized by CRISPR/Cpf1 system and triggered the ssDNase activity of Cpf1, resulting in robust cleavage of surrounding DNA FQ-reporter (fluorophore-quencher labeled short single-stranded DNA sequence). With the four-step amplification, ATP was measured with high sensitivity and low background interference. Under the optimal conditions, a linear relationship was obtained between the fluorescence response and ATP concentrations ranging from 2 pM to 10 μM with a limit of detection (LOD) as low as 1.8 pM. Successful detection of ATP in diluted human serum samples further indicated that ASD-Cpf1 system provided a potential platform for detection of small molecules.
Yarrowia lipolytica, a nonconventional yeast, has become an industrial workhorse to synthesize valuable compounds, including lipids, oleochemicals, and nutraceuticals. While the synthetic biology toolkits to engineer the genome and endogenous metabolic pathways are not as developed as Baker’s yeast, it has emerged as a promising microbial host for industrial applications. In this study, we examined the multiplexed editing capability of the CRISPR-AsCpf1 coupled with gRNAs generated from either a yeast native promoter or an orthogonal T7 promoter, which yielded 73.3% editing efficiency for up to four target genes and 100% editing efficiency for two genes. We also attempted two strategies to enhance homology-directed recombination (HDR) efficiency; only minor improvements were observed. We further demonstrated that CRISPR-dAsCpf1 with T7-driven gRNA achieved significant gene knockdown compared to the CRISPR-RfxCas13d system. Its knockdown efficiency was comparable to that of an antisense T7 promoter system. Taken together, this work provides a facile toolkit that enables efficient and multiplexed genome editing and transcriptional knockdown of critical genes by combining CRISPR-Cpf1 with an orthogonal T7 transcription system in Y. lipolytica.
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