CRISPR-dCas9 Mediated Cytosine Deaminase Base Editing in Bacillus subtilis

Abstract

Base editing technology based on Clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) is a recent addition to the family of CRISPR technologies. Compared with the traditional CRISPR/Cas9 technology, it does not rely on DNA double strand break and homologous recombination, and can realize gene inactivation and point mutation more quickly and simply. Herein, we first developed a base editing method for genome editing in Bacillus subtilis utilizing CRISPR/dCas9 (a fully nuclease-deficient mutant of Cas9 from S. pyogenes) and activation-induced cytidine deaminase (AID). This method achieved three and four loci simultaneous editing with editing efficiency up to 100% and 50%, respectively. Our base editing system in B. subtilis has a 5 nt editing window, which is similar to previous reported base editing in other microorganisms. We demonstrated that the plasmid curing rate is almost 100%, which is advantageous for multiple rounds of genome engineering in B. subtilis. Finally, we applied multiplex genome editing to generate a B. subtilis 168 mutant strain with eight inactive extracellular proteases genes in just two rounds of base editing and plasmid curing, suggesting that it is a powerful tool for gene manipulation in B. subtilis and industrial applications in the future.

Development of a base editor for protein evolution via in situ mutation in vivo

“…Due to the diverse genome environments in diverse cells and host-dependent factors of some tools, one tool is usually applied for one kind of cell. In addition, the tools also displayed significantly different conversion efficiency even in one kind of cell because of the different expression version and construction strategy ( 31 , 32 ). No matter how to use these tools, a BE with high stability and high conversion efficiency is expected.…”

Cytosine Base Editor-Mediated Multiplex Genome Editing to Accelerate Discovery of Novel Antibiotics in Bacillus subtilis and Paenibacillus polymyxa

“…Since the development of CBE system in eukaryotes (Komor et al, 2016), base editors have been gradually applied to prokaryotes such as E. coli (Banno et al, 2018), P. aeruginosa (Chen et al, 2018), S. aureus (Gu et al, 2018), Streptomyces (Tong et al, 2019), Corynebacterium glutamicum (Wang et al, 2018), and B. subtilis (Yu et al, 2020). While the eukaryotic CBE system contains nCas9, we used dCas9 as previously reported (Banno et al, 2018;Yu et al, 2020) because we found that nCas9 affects transformation efficiency in B. subtilis (Supplementary Figure S1A). For base editing, cytidine deaminases, rAPOBEC1, and PmCDA1 have been widely used as the component of CBEs.…”

“…Recently, the use of PmCDA1-mediated base editing has been reported in B. subtilis (Yu et al, 2020). The system showed high editing efficiency at position 18 from PAM alone.…”

“…The CBE is highly valuable for multiplex genome editing due to its simplicity, high efficiency, high specificity, and low genome damage (Komor et al, 2016;Nishida et al, 2016); further, it does not require recombination, foreign DNA templates, or DSBs, unlike previous genome editing tools (Vento et al, 2019). Recently, a CBE system for Bacillus subtilis has been reported (Yu et al, 2020). However, the system had a narrow optimal editing window and the editing efficiency was significantly decreased when more than three targets were edited at the same time, indicating that the base editor for multiplex editing needs to be improved.…”

Engineering of Multiple Modules to Improve Amorphadiene Production in Bacillus subtilis Using CRISPR-Cas9

“…This system can avoid the dependence on HDR and reduce the fatality rate caused by DSBs, but it requires the participation of cytosine deaminase. 38 After checking the nucleotide sequences of T399 and H448, we found that it is not feasible to introduce the desired mutations by base editing events ( Figure 3 B). Therefore, to introduce our desired mutations into GFPADS, it is necessary to create the DSBs by SpCas9 near T399 and H448 and introduce editing templates containing inconsistent sequences with N20 nucleotides.…”