We aim to build a multi-level biosensor that can be easily applied to the real world. The design of our project involves four parts:

Part 1: Cascade recording system

In order to construct a highly comprehensive cascade recording system that accurately records and reflects the sequence of events, three demands should be met:

• The system should exhibit high sensitivity in response to the selected signal.
• The recording operations should occur in a specific sequence, without interference.
• The recording scar should be as minimal as possible for a larger range of dimensions.

Here we demonstrate the design of the cascade recording system satisfying the demands, based on the principles of the cassette-recorder method, self-targeting CRISPR system, and self-recombination Lambda system.

The system consists of the signal input (stimulation part) and signal recording (cassette change). Initially, once stimulated, stgRNA1 in cassette 1 will be expressed leading to an in-situ knockout that introduces a double-strand break (DSB). The DSB, in conjunction with the Lambda Red recombination system, triggers intra-plasmid recombination^[1]. Consequently, cassette 1 is deleted, and cassette 2 is brought into proper expression position, right after the inducible promoter (Fig 1).

Despite the stimulation part, which will be discussed later, several technical details are incorporated to facilitate cassette change (Fig 2). In the self-targeting system, a nucleotide in the coding sequence of sgRNA has been altered to introduce a protospacer adjacent motif (PAM) immediately following the 20nts specificity-determining sequence (N20)^[2] (Fig 3, S. D. Perli et al., Science 2016). In the self-recombination system, the leading sequence of the subsequent cassette partially aligns with the promoter part, preparing it for intra-plasmid recombination^[3] (Fig 4, Zhao, D., Feng, X., Zhu, X. et al. Sci Rep 7, 16624 2017). As a result, the substitution of cassettes can be achieved as expected elegantly.

Part 2: EL222 directed evolution

The EL222 promoter, also known as the blue light promoter, is activated by blue light at approximately 465 nm. The system has two components: the EL222 factor and pel222.^[4] The EL222 factor is constitutively expressed in our cells. Upon exposure to blue light, El222 dimerizes and binds to pel222 sequence, triggering the expression of downstream genes (Fig5).

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In our design, we utilize EL222 to regulate gene editing at different levels. The experiment processes involving EL222 are bifurcated into two segments: test of EL222 and improvement of EL222.

To test the EL222 promoter and assess its functionality, we first synthesized the sequences of the EL222 factor along with pel222. Then we inserted them into the pET-28a-eGFP plasmid, positioned upstream of eGFP.

For the improvement phase, we substituted eGFP with blue fluorescent protein sBFP2 and red fluorescent protein mRFP1. As these proteins are not activated by blue light, this strategy circumvents the fluorescence quenching effect induced by blue light.

Through error-prone PCR (ep-PCR), different sequences of pel222 can be produced and replace the original pel222. After transformation, we measured leakiness and expression pre- and post-induction by imaging. Colonies exhibiting diminished fluorescence pre-induction and enhanced post-induction were selected. Ultimately, we validated fluorescent protein expression at the transcript level by qPCR and screened EL222 promoters with low leakage in response to different thresholds.

Part 3: sgRNA screening

To ensure optimal functionality of our system, we aim to generate high-efficiency sgRNA sequences. We developed an in-silico sgRNA generator based on the Escherichia coli genome database and existing experimental data (Fig 6). This approach leverages species genome information and cross-species efficiency data to generate a set of alternative sgRNA sequences with high on-target efficiency and low off-target rate.

We then tried evaluating their efficiency in wetlab using the Batch Random Experimentation Method (BREM). The wetlab results can be compared with theoretical predictions to refine our generator model.

We employed EGFP as the indicator of successful editing and constructed an N₂₀s screening plasmid (Fig 7). The plasmid contains a sgRNA targeting sequence inserted in the EGFP sequence and a sgRNA scaffold. The scaffold can load a specific N₂₀ sequence (20nts specificity-determining sequence) and conduct targeting fragment knockout, thereby driving EGFP expression. We randomly loaded a batch of different N₂₀ sequences onto the plasmid and identified efficient ones by sequencing green colonies. When the colony count is sufficiently high, our method will examine all N₂₀ sequences with near certainty. Moreover, we determined the threshold and confidence level for the experiment through probability calculations.

Part 4: Applications

The CRISPReporter, due to its remarkable recording capacity and potential to sense diverse signals, holds promise for a multitude of applications. We have explored its use in various domains such as diagnostics, environmental monitoring, and bioinformatics.

Diagnosis

Environmental monitering

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References

• Zhao D, Yuan S, Xiong B, et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact. 2016;15(1):205.
• Perli SD, Cui CH, Lu TK. Continuous genetic recording with self-targeting CRISPR-Cas in human cells. Science. 2016;353(6304):aag0511.
• Jayaraman P, Devarajan K, Chua TK, Zhang H, Gunawan E, Poh CL. Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic Acids Res. 2016;44(14):6994-7005.
• Zhao D, Feng X, Zhu X, Wu T, Zhang X, Bi C. CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency. Sci Rep. 2017;7(1):16624.
• Chuai G, Ma H, Yan J, et al. DeepCRISPR: optimized CRISPR guide RNA design by deep learning. Genome Biol. 2018;19(1):80. doi:10.1186/s13059-018-1459-4.