Difference between revisions of "Part:BBa K4630100"

Latest revision as of 23:32, 11 October 2023

stgRNA-barcode-cassette 1

This part is the barcode-cassette form of the self-targeting guide RNA 1 (stgRNA 1). It is the building block of the cascade recording system. It contains a Lac promoter, the upstream barcode, the stgRNA 1, a double terminator and the downstream barcode. It is used in the verification of the self-targeting and self-recombination system. The unique barcode is used to distinguish each cassette and provide unique homologous arm.

Background of the Cassette

Cassette recorder is the leading edge of DNA memory device. By changing cassettes, the entire recorder is capable to record many rounds of biological events. The method of DNA manipulation is the biological basis of the DNA-based recording. Perli et al. ’s work demonstrates that based on the CRISPR/Cas9 and self-targeting guide RNA (stgRNA), editing could occur in the stgRNA sequence¹ (fig B1a). Also, Zhao et al. ’s work enlightens us with the concept of intra-plasmid recombination² (fig B1b).

Figure B1. DNA manipulating methods from the literatures

Usage and Biology

We integrate the two methods into a single cassette, with unique barcode (fig B2). Once triggered, double strand break (DSB) is introduced into the working plasmid, enabling the intra-plasmid recombination. It is worth noting that the two homologous arms flank the stgRNA, so the entire cassette will be deleted after the recombination, and the next cassette is expected to be brought to the proper expressing place. The inducer triggers over and over, and recorder records it by keeping changing the cassette.

Figure B2. Working pattern of the change of cassette

Design

The key component, stgRNA 1, is generated by our guide RNA in-silico generator. As the first cassette, it contains Lac promoter to initiate the induced edition in the proof-of-concept. Also, the double terminator is designed to prevent it from expressing the next cassette. The barcode is also generated by the in-silico generator, to avoid recombination with the bacterial genome.

Figure D1. Design of stgRNA-barcode-cassette 1

Experiments

This part is the key element we use in the proof-of-concept of the recorder, and it help us better understand the recording event. Also, it is used to measure the basic parameter of the recording event. We cloned it onto pCDF-Duet1 plasmid and co-transformed it with pCas(BBa_K4630200) / pCasop(BBa_K4630201) into Escherichia coli DH5α.

The overall experiments we conducted on BBa_K4630100 include:

1. Verification of the self-targeting and self-recombination
2. Exploration of the concentration and induction time reliance of the inducer
3. Exclusion of the unexpected recombination

All experiments we conducted involving BBa_K4630100 include:

1. The construction of multi-cassette recorder with barcode
2. The verification of the multi-level recording

General Induction Protocol

1. Pick a colony from the verified plate, cultivate in 5ml liquid LB media for 12 hours.
2. Transfer 100µl bacteria solution to 3ml new liquid LB media. Cultivate to an OD600 of 0.3~0.5.
3. Add L-Arabinose and IPTG to certain final concentration and induce for a certain time (details are in each tests).
4. Spread the diluted solution to plates with appropriate antibiotics, and cultivate for 12 hours.
5. Pick the colonies and test the editing result.

Results

Strain Construction

We successfully co-transformed our working plasmid with pCas (BBa_K4630200), the vector for Cas9 and Lambda Red, into Escherichia coli DH5α (fig 1).

Pilot Experiment

We added a variable dosage of arabinose and IPTG close to the working concentration (tbl 1). Induction time for L-Arabinose is 22 hours and IPTG 5 hours. The sequencing results uncovered a successful editing (fig 2a). Large-scale screening revealed that condition 2 exhibited the highest efficiency, with efficiencies of 70%, 10%, and 38.1% for condition 2, 3, and 4, respectively (fig 2b). Non-induction controls substantiated that induction is the prerequisite for recording (fig 2c).

Concentration Matrix

First Concentration Matrix

To better measure the property of the cassette, we maintained the induction time at the previous preferred levels (Condition 2, L-Arabinose and IPTG inducing 22hrs and 5hrs respectively), we carried out a concentration matrix test (tbl 2).

It’s noted that the induction of arabinose inhibited the growth of bacteria strikingly (fig 3). Despite the missing data due to failure of sequencing, E6, E5, C2, B5 performed better (tbl 3, fig 4a, tbl 4), and the Lac promoter exhibited significant leakage expression (tbl 5, fig 4b). Furthermore, quality test based on electrophoresis provided parallel data for randomly picked groups, and the two data access showed a significant correlation, with paired t-test P = 0.7602, no significant difference (tbl 6, tbl 7, fig 5). Given the substantially larger amount of data from the electrophoresis test for E6 (N = 20) compared to sequencing (N = 4), we adjusted the knock-out ratio of E6 to 60%.

Second Concentration Matrix

To ensure the data reliability, we tried the concentration matrix again. Growth inhibition and leakage expression still occur (fig 6 a, b). However, the knock-out ratio of the second concontration matrix is generally lower than the first one.

Short Summary

To compare the two matrices, an integrated heatmap is plotted based on the mean value (fig 7a). The variation pattern of the two matrices showed some kind of correlation (fig 7b). Paired t test result of the two matrices showed a significant difference, with P = 0.0054(**) and mean of differences = -0.2071(fig 7c). Two-way ANOVA of the second matrix and the average matrix indicate that the IPTG concentration is the main variation factor (tbl 8).
The data demonstrate that there is a huge variation in the measurement, and more experimental data are needed. Moreover, the similarity of variation pattern indicates probability of logical regression. And the statistical analysis proves that IPTG concentration is the leading factor controlling the result. To sum up, the data can reflect the induction events to a certain extent, but accurate relationship relies on more data.

Time-Gradient Test

The test of knock-out showed a bell-type-like shape (tbl 9, fig 8a). The increasing trend ends on dot 4h and the extension of IPTG induction time does not help the editing afterwards.

Unexpected knock-out Exclusion

During the large-scale tests, we meet several cases of unexpected knock-out (tbl 10, fig 9). Theoretically, the larger the homologous arm is, the higher the recombination ratio. Interestingly, there are two sets of homologous arms intrinsically flanking the DSB target site. However, the preferred unexpected recombination occurred at the low ratio, 0.028, with a shorter homologous arm. The result demonstrates that the effect of distance is larger than the effect of homologous arm, and both of them are weak. We have more confidence that the change of cassette is sequential.

Reference

1. Perli SD, Cui CH, Lu TK. Continuous genetic recording with self-targeting CRISPR-Cas in human cells. Science. 2016;353(6304):aag0511. doi:10.1126/science.aag0511

2. 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. doi:10.1038/s41598-017-16998-8

Sequence and Features