In 2020, Prof. Emmanuelle Charpentier and Prof. Jennifer Doudna were awarded the Nobel Prize in Chemistry for developing the CRISPR-based gene editing method[1]. Since then, the CRISPR-based gene editing method has received more and more public attention, due to its advantages of facile synthesis, easy use, low cost, and high specificity. It is also versatile as it can be applied in many fields such as genome engineering, inherited disease therapy, high throughput functional genomic screens, genetic disease diagnostics and etc[2].

However, CRISPR-based genome engineering strategy is not that perfect. Off-target cleaves, variable gRNA efficiency, protospacer-adjacent motif (PAM) specificity and other factors contribute to its limited development[3-5]. These problems make it difficult to obtain strains that are accurately edited, forcing us to increase the number of colonies tested to select the strains that we wanted. Especially in the process of high-throughput gene editing, these problems not only prolong the time of strain construction and screening, but also cost more manpower and materials.

According to previous researches, double stranded DNA breaks (DSBs) are known to induce the SOS response[6], which inspired us that the SOS system can act as a switch to cause downstream responses. Escherichia coli expressing the levansucrase encoded by the sacB gene cannot grow on LB plates containing 5% sucrose, therefore this gene has been used as a counter-selection marker in many studies[7], which inspired us that we can link the SOS system with the sacB-based negative selection system.

Herein, we developed a CRISPR-based purification system "strainer" in E. coli, which can remove the unsuccessfully edited cells, and then improve the overall editing efficiency. The "strainer" method utilized the double stranded DNA breaks (DSBs) as a signal to start the transcription of gRNA targeting on the plasmid harboring inducible toxic gene(sacB). Thus, the strain with successful recombineering can survive in the media with sucrose(without sacB gene). The strain without DSBs, still has sacB gene plasmid, cannot survive in the media with sucrose. This is why we can increase the overall editing efficiency (Fig.1).

Fig. 1. The schematic diagram of "strainer"

In addition, the dry-lab group optimized the design of gRNA to help mitigate the off-target problem of CRISPR/Cas system, which further helped to improve the editing efficiency using our "strainer" for the strain or library construction. We then applied "strainer" for the construction of isopropanol producing strains. In the process, we have done single gene editing, large DNA fragment integration, and large scale gene editing in a shorter time.

In conclusion, our "strainer" can remove the cells unedited and thereby improving the overall editing efficiency during the high-throughput genome engineering process, single gene editing and large fragment gene editing process, which seemed that our strainer system can be potentially used as a universal method for cell factory construction.

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[2] Wang S, Du Y, Wang D, Ma J, Tang A, Kong D. Signal amplification and output of CRISPR/Cas-based biosensing systems: A review. Anal Chim Acta. 2021, 1185: 338882.

[3] Tan Y, Chu AHY, Bao S, Hoang DA, Kebede FT, Xiong W, Ji M, Shi J, Zheng Z. Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity. Proc Natl Acad Sci U S A. 2019, 116 (42): 20969-20976.

[4] Liu R, Liang L, Freed EF, Gill RT. Directed evolution of CRISPR/Cas systems for precise gene editing. Trends Biotechnol. 2021, 39 (3): 262-273.

[5] Xiang X, Corsi GI, Anthon C, Qu K, Pan X, Liang X, Han P, Dong Z, Liu L, Zhong J, Ma T, Wang J, Zhang X, Jiang H, Xu F, Liu X, Xu X, Wang J, Yang H, Bolund L, Church GM, Lin L, Gorodkin J, Luo Y. Enhancing CRISPR-Cas9 gRNA efficiency prediction by data integration and deep learning. Nat Commun. 2021, 12 (1): 3238.

[6] Moreb EA, Hoover B, Yaseen A, Valyasevi N, Roecker Z, Menacho-Melgar R, Lynch MD. Managing the SOS response for enhanced CRISPR-Cas-based recombineering in E. coli through transient inhibition of host RecA activity. ACS Synth Biol. 2017, 6 (12): 2209-2218.

[7] Gao S, Yao S, Hart DJ, An Y. Signal peptide-dependent protein translocation pathway is crucial for the sucrose sensitivity of SacB-expressing Escherichia coli. Biochem Eng J. 2017, 122: 71-74.