Coding

Part:BBa_K2200005

Designed by: Meredith Eating Xiang   Group: iGEM17_Shenzhen_SFLS   (2017-07-22)


hCas9

Expresses human codon optimized Cas9 nuclease for genome engineering


Contribution By Team 23 Zhejiang-United - BBa_K2200005 (cas9)

Contribution By Team 23 Zhejiang-United

BBa_K2200005 (cas9)

Group: Zhejiang-United iGEM 2023

Summary: Based on BBa_K2200005 (cas9), we mainly increase its application range. We verified the knockout effect of gntT and lacZ genes in E.coli Nissle 1917.

Documentation

a. Usage and Biology

Nissle 1917 is an E. coli strain with probiotic properties, but for certain applications, it is necessary to knock out specific genes[1]. Since CRISPR-Cas systems have high target DNA specificity and programmability, they can serve as gene editing tools[2]. The development and optimization of such tools can facilitate the construction of engineered cell lines for diverse production needs while accelerating the exploration of biological systems. However, unwanted escapers that do not undergo the intended editing frequently arise during CRISPR-Cas-mediated microbial genome editing, reducing editing efficiency[3].

b. Characterization/Measurement

We electroporated the Cas9 plasmids, gRNA plasmids, and homology arms (500 bp) into E. coli Nissle 1917. After 16 h growth, colony PCR identified transformants. Results showed p15A-Cas9 achieved 100% knockout of gntT and 80% for lacZ in E. coli Nissle 1917.

Figure 1: Editing efficiency results of p15A-Cas9 and p15A-Cas9-op in E. coli Nissle 1917.

Figure 1: Editing efficiency results of p15A-Cas9 in E. coli Nissle 1917.

Reference

  1. Buddenborg C., Daudel D., Liebrecht S., et al. Development of a tripartite vector system for live oral immunization using a gram-negative probiotic carrier [J]. Int J Med Microbiol, 2008, 298(1-2): 105-114.
  2. Vento J.M., Crook N., Beisel C.L. Barriers to genome editing with CRISPR in bacteria [J]. J Ind Microbiol Biotechnol, 2019, 46(9-10): 1327-1341.
  3. Li Q., Sun M., Lv L., et al. Improving the editing efficiency of CRISPR-Cas9 by reducing the generation of escapers based on the surviving mechanism [J]. ACS Synthetic Biology, 2023, 12(3): 672-680.

Improvement By Team iGEM23 Zhejiang_United - BBa_K2200005(Cas9)

Improvement By Team iGEM23 Zhejiang_United

New improved part BBa_K4876011(p15A-Cas9-λ-Red)

Group: Zhejiang-United iGEM 2023

Summary

Based on BBa_K2200005(Cas9), we constructed a new plasmid BBa_K4876011(p15A-Cas9-λ-Red) with replicon p15A and self-cleavage gRNA. The main purpose of p15A replicon is improved editing efficiency. Self-cutting gRNA mainly avoids the impact on the environment and protects the privacy of our products. Data supplement is carried out in the following two aspects:

  1. 1. Cas9 Protein Expression in E.coli BL21
  2. 2. Verification of Editing Efficiency at gntT and lacZ in Escherichia coli Nissle 1917

Usage and Biology

Cas9, originally named Csn1, is the large, multifunctional signature protein of type II CRISPR/Cas systems. It is well known even to general audiences because its RNA-guided endonuclease activity has made it a popular tool for custom editing of eukaryotic genomes[1].

Using CRISPR-Cas9, scientists can study the function of specific genes by selectively editing them. By observing the phenotypic changes in edited cells or organisms, it is possible to uncover the roles of genes in biological processes, enhancing our understanding of fundamental biological mechanisms[1].

Many cancers are caused by genetic mutations, and CRISPR-Cas9 technology can be used to study the mechanisms of cancer development and develop new therapeutic methods. It can be used to knock out, add, or correct mutated genes to simulate and study the roles of cancer-related genes[2-4].

CRISPR-Cas9 technology can be applied in the field of agriculture to help improve crops. Scientists can use this technology to enhance traits such as disease resistance, salt tolerance, yield, quality, and nutritional value in crops, thereby increasing food production efficiency and quality[5-8].

CRISPR-Cas9 has wide applications in biotechnology and synthetic biology. It can be used to create model organisms with specific gene modifications, enabling the study of the functionality of complex biological systems. Additionally, it can be used for strain improvement in the industrial production of microbes, such as the production of specific compounds or bioenergy[9-11].

Characterization

The original Cas9 encoding plasmid has a lower copy of pSC101 origin. We selected the higher copy p15A origin to increase Cas9 expression. This allows the retention of functional Cas9 during host editing to ensure normal CRISPR-Cas system operation, reducing escape rates and improving editing efficiency. In addition, the incorporation of the λ-Red recombination system can enhance editing efficiency, so we constructed the p15A-Cas9-λ-Red plasmid (Figure 1).

Figure 1: Engineering frame of the p15A-Cas9-λ-Red plasmid.

Figure 1: Engineering frame of the p15A-Cas9-λ-Red plasmid.

To construct p15A-Cas9-λ-Red, we amplified p15A and Cas9-λ-Red using primers with homology arms and recombined them using a cloning kit to generate p15A-Cas9-λ-Red. After transformation, single colonies grew on LB plates. Colony PCR and sequencing verified the correct construction of the p15A-Cas9-λ-Red plasmid (Figure 2).

Figure 2: The construction results of p15A-Cas9-λ-Red.

Figure 2: The construction results of p15A-Cas9-λ-Red.
(A) Colony PCR results. (B) Sequencing results.

Subsequently, we tested the induction of Cas9 expression by different concentrations of L-arabinose. The results showed that with higher concentrations of L-arabinose, the expression of Cas9 was higher, suggesting that we could control the level of Cas9 by regulating the addition of L-arabinose (Figure 3).

Figure 3: Results of Cas9 protein induction by L-arabinose.

Figure 3: Results of Cas9 protein induction by L-arabinose.
(A) Standard curve of BSA concentration. (B) The curve of Cas9 concentration.

To verify Cas9 expression in E. coli Nissle 1917, we transformed the constructed p15A-Cas9-λ-Red plasmid. After inducing expression, we lysed the cells by sonication and purified Cas9. The results showed that we successfully induced Cas9 protein expression (Figure 4).

Figure 4: The SDS-PAGE result of p15A-Cas9-λ-Red protein expression.

Figure 4: The SDS-PAGE result of p15A-Cas9-λ-Red protein expression.

Reference

  1. Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V. (2012). Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences, 109(39), E2579-E2586.
  2. Chen, B., Gilbert, L. A., Cimini, B. A., Schnitzbauer, J., Zhang, W., Li, G. W., … & Huang, J. (2013). Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell, 155(7), 1479-1491.
  3. Sanchez-Rivera, F. J., & Jacks, T. (2015). Applications of the CRISPR-Cas9 system in cancer biology. Nature Reviews Cancer, 15(7), 387-395.
  4. Platt, R. J., Chen, S., Zhou, Y., Yim, M. J., Swiech, L., Kempton, H. R., … & Gootenberg, J. S. (2014). CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell, 159(2), 440-455.
  5. Mahas, A., Neal Stewart Jr, C., & Mahfouz, M. (2019). Harnessing CRISPR/Cas systems for agricultural biotechnology. Biotechnology Advances, 37(6), 107448.
  6. Ricroch, A. E., Hénard-Damave, M. C., & Foueillassar, X. (2017). Barriers to the adoption of genetically modified (GM) crops in the European Union. Biotechnology Advances, 35(8), 189-197.
  7. Weeks, D. P., & Sparks, C. A. (2020). Precision genome engineering in crops: state of the art and future prospects. Plant Physiology, 183(2), 667-679.
  8. Zhu, J. K. (2016). Abiotic stress signaling and responses in plants. Cell, 167(2), 313-324.
  9. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., … & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339(6121), 819-823.
  10. Jao, L. E., Wente, S. R., & Chen, W. (2013). Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proceedings of the National Academy of Sciences, 110(34), 13904-13909.
  11. Port, F., & Bullock, S. L. (2016). Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs. Nature Methods, 13(10), 852-854.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3643
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2740
    Illegal NgoMIV site found at 3649
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 3768
    Illegal SapI.rc site found at 1159
    Illegal SapI.rc site found at 1401


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