Team-iGEM SJTU BioX 201

Safety-iGEM SJTU BioX 201

Overview

Our system incorporates two independently engineered microbes and one module to link those two gene circuits. These microbes are designed to fulfill a series of functions: (a) real-time detection of CRISPR off-target, (b) deferred detection of CRISPR off-target and (c) biostorage of information.

Fig.1 Gene circuit for real-time detection of CRISPR off-target

Fig.2 Gene circuit for transcription activation

Fig.3 Gene circuit for deferred detection of CRISPR off-target

Real-time off-target detection

1.How to construct off-target scenario in E. coli cell?

As the foundation of our work, we first built a model of off-target incidence. The key sequence is the part BBa_K2996505. We built the model in E. coli cell because it provides a quick and steady genetic operation platform. In our work to monitor off-target incidence, we built the system with “lure” (Fig.1), a sequence similar to the target, with the potential to recruit NdCas9-sgRNA complex. The lure sequence is placed upstream the DBD binding site, to which a fusion protein would bind. The space between the two sequences is determined by preliminary experiments. To ensure that our off-target model is effective, we made our choice out of several candidates of lure sequences that had been assessed by previously published literature[1]. Furthermore, we devised our algorithm and software to predict the of off-target incidence. We put the sgRNA construction module upstream the lure sequence in consideration of plasmid capability.

Fig.1 The key plasmid producing the off-target scenario

2.How to detect the off-target incidence in the constructed E. coli cell?

Two fusion proteins work as the detector to give the signal of off-target incident. One is N-luciferase linked to dCas9 (part BBa_K1689010, Fig.2),also called NdCas9, the other one is C-luciferase linked to zinc finger protein (part BBa_K2996503, Fig.3), also called Cluc-DBD. The Cluc-DBD fusion protein steadily binds to DBD binding site once it is translated. As for NdCas9, if the off-target incidence takes place, the dCas9 will bind to “lure” sequence in Fig.1 once combined with sgRNA. The N-terminal of this fusion protein, which is N-luciferase, swings in cytoplasmic matrix, and will soon C-luciferase combines it to make the whole enzyme (Fig.4). The two separated halves of luciferase can only be activated when physically pulled together. The whole system works when luciferin is added to the culture and excited by light of certain wavelength. In the detection system, the off-target signal is transferred to light signal.

Fig.2 The plasmid producing the N-dCas9 fusion protein

Fig.3 The key plasmid producing the Cluc-DBD fusion protein

Fig.4 The working pattern of the detection system

Deferred off-target detection: Transcription activation

1.Downstream response: CRISPRa

We flexibly linked RpoA, an RNAP α subunit which can activate transcription, to endonuclease-defective Cas9 (dCas9) as a bacterial transcription activator and innovated coding-sequence-targeting for RpoA-dCas9.

Compared to reported Cas9 bacterial transcription activator that fused dCas9 directly with RpoZ (RNA polymerase alpha subunit), dimerization of RpoA initiates RNAP assembly more effectively and RNAPα contains both determinants for promoter binding and determinants for RNAP assembly which will lower down off-target effect of the effector.

Using overlap PCR, RpoA is linked to dCas9 through a modified flexible Linker 4 (FL4), which has been proved to be effectively separate bifunction proteins.

Now we got the transcription activator part BBa_K2996701.

2.Triggered responses

For measurement, a reporter plasmid with the mRFP gene under the control of a constitutive promoter (BBa_J23117) that is preceded by a sequence rich in NGG PAM sequences on the NT strand. Thus, florescence intensity indicates the level of transcription activation.

To test the activation ability of our design, we first used gRNA that complements the NT strand from at least 20nt upstream from -55 point (With the transcription start denoted +1, RNAP complex covers from -55 to +20).

When dCas9 binds to lure and recruits RNA polymerase, promotor J23101 will be activated, thus increasing the expression of mRFP florescence.

As mentioned before, the recording of off-target incidence can be achieved through this transcription activation device, with luxI under control of this promotor. The increased expression of luxI gene leads to increase in AHL ambient concentration, activating the biostorage module.

CRISPR biostorage of off-target signal

1. How to record? The input

Overexpression of Cas1 and Cas2 nucleases leads to site-specific acquisition of a 33 bp protospacer at the leader end of the CRISPR locus in E. coli. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. The input signal should be able to initiate the expression of Cas1–Cas2 complex, which results in spacer acquisition into pRead-EGFP. Information can be transformed into binary format, with 1 corresponding the expression of Cas1–Cas2 complex.

Background information for CRISPR-Cas system

CRISPR-Cas operons contain CRISPR arrays and CRISPR-associated (cas) genes. In the adaptation stage, short DNA fragments generated from phage DNA are captured by the adaptation complex. Following trimming of excess DNA, the adaptation complex integrates these fragments into the CRISPR array. During the expression and maturation stage, the CRISPR array is transcribed into long pre-CRISPR RNAs (pre-crRNAs). The pre-crRNAs are cleaved within the repeat regions to generate mature crRNAs. Each crRNA assembles with Cas proteins to form a surveillance complex. During the interference stage, the surveillance complex recognizes the target by complementary base-pairing with the crRNA sequence. Target binding triggers recruitment of a nuclease, which catalyzes degradation of the target nucleic acid.

The first nucleophilic attack occurs on the minus strand of the first repeat, distal to the leader, by the C 3'-OH end of the protospacer. After half-site intermediate formation, the second integration event occurs on the opposite strand at the leader-repeat border. The resulting single-stranded DNA gaps are repaired by yet uncharacterized mechanisms and the protospacer is fully integrated with the G as the first nucleotide at its 5' end. The asterisk denotes the duplication of the first repeat, as previously observed in vivo.

How to record response triggered by off-target? The idea is to clone Cas1–Cas2 complex downstream of an inducible promotor, pLuxR. With the addition of AHL, due to increased expression of LuxI enzyme from transcription activation device, Cas1–Cas2 complex can carry out the spacer adaptation.

In the preliminary testing stage, we used pRec and pTrig plasmid.

The pRec plasmid part BBa_K2996007 was generated by placing the E. coli cas1-cas2 cassette (amplified from NEB 10-beta) downstream of the PLTetO-1 promoter on a ColE1 plasmid containing chloramphenicol resistance marker and constitutively expressed TetR and LacI (LacI is required to repress the Lac promoter on pTrig).

The pTrig plasmid part BBa_K2996012 contains the mini-F origin and replication machinery, P1 lytic replication element RepL placed downstream of an IPTG-inducible Lac promoter, and kanamycin resistance marker. Upon induction, its copy number will sharply increase, we figure short DNA fragments generated from pTrig are captured and trimmed by the adaptation complex, in order to improve the effectiveness and accuracy of information input.

2.How to read? The output

We find a way to transform the expansion of CRISPR array into florescence signal, using a third reporter plasmid, pRead.

An original pRead contains a CRISPR array (We name it RSRL), which consists of 69 bp leader and one spacer flanked by two repeats. Integration cassette is put upstream of the complete coding sequence of an out-of-frame EGFP gene (EGFP +1).

Translation of transcripts generated from the tac promoter will stop at the leader, for those two stop codons. The wrong reading frame and existing stop codon together will prevent expression of the EGFP in the original situation.

Upon addition of inducer, spacer adaptation by Cas1-Cas2 will result in an addition of 61 base pairs (33bp new spacer and 28bp replicated repeat) into the RSRL array. This moves the stop codon out of frame and EGFP into the ORF. Expression of in-frame EGFP is further induced by IPTG under tac promoter, so that recorded information can be read whenever desired through addition of IPTG.

This way fluorescence produced by EGFP acts as output signal, observed by microplate fluorescence reader.

3.Expansion of storage: Hydrogel panel and Optical control

We have designed this CRISPR cell storage device like compact disc. To realize this, we choose microwell arrays filled with supramolecular hydrogel, for bacteria chassis can be fixed in certain places and inducers can penetrate the culture media.

Bacteria in early exponential stage are embedded into an enhanced hybrid hydrogel, sodium alginate aligned with 1,4-bi(phenylalanine-diglycol)-benzene (PDB), and seeded them into 96-well plate.

Each confined cell is a recording unit. Take it as a QR code, inducer is added to dots that are black. Information can be easily extract by scanning the plate by a microplate reader.

In the future, we will apply optical-controlled promotor to our input system. Light can initiate the expression of repL and Cas1-Cas2 complex, resulting in spacer acquisition. The biostorage disc record and read information are controlled by light.

Team:SJTU-BioX-Shanghai/Project/Design