PMID- 36625577 OWN - NLM STAT- MEDLINE DCOM- 20230302 LR - 20230314 IS - 2150-7511 (Electronic) VI - 14 IP - 1 DP - 2023 Feb 28 TI - Improved Dual Base Editor Systems (iACBEs) for Simultaneous Conversion of Adenine and Cytosine in the Bacterium Escherichia coli. PG - e0229622 LID - 10.1128/mbio.02296-22 [doi] LID - e02296-22 AB - Genome-editing (GE) techniques like base editing are ideal for introducing novel gain-of-function mutations and in situ protein evolution. Features of base editors (BEs) such as higher efficacy, relaxed protospacer adjacent motif (PAM), and a broader editing window enables diversification of user-defined targeted locus. Cytosine (CBE) or adenine (ABE) BEs alone can only alter C-to-T or A-to-G in target sites. In contrast, dual BEs (ACBEs) can concurrently generate C-to-T and A-to-G modifications. Although BE tools have recently been applied in microbes, there is no report of ACBE for microbial GE. In this study, we engineered four improved ACBEs (iACBEs) tethering highly active CBE and ABE variants that can introduce synchronized C-to-T and A-to-G mutations in targeted loci. iACBE4 generated by evoCDA1-ABE9e fusion demonstrated a broader editing window (positions -6 to 15) and is also compatible with the multiplex editing approach in Escherichia coli. We further show that the iACBE4-NG containing PAM-relaxed nCas9-NG expands the targeting scope beyond NGG (N-A/G/C/T) PAM. As a proof-of-concept, iACBE was effectively utilized to identify previously unknown mutations in the rpoB gene, conferring gain-of-function, i.e., rifampicin resistance. The iACBE tool would expand the CRISPR-GE toolkit for microbial genome engineering and synthetic biology. IMPORTANCE Dual base editors are DSB-free CRISPR tools applied in eukaryotes but not yet in bacteria. We developed an improved ACBE toolset for bacteria, combining highly processive deaminases. We believe that the bacterial optimized iACBE toolset is a significant advancement in CRISPR-based E. coli genome editing and adaptable to other microbes. FAU - Shelake, Rahul Mahadev AU - Shelake RM AUID- ORCID: 0000-0003-0691-560X AD - Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South Korea. FAU - Pramanik, Dibyajyoti AU - Pramanik D AUID- ORCID: 0000-0002-7996-1124 AD - Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South Korea. FAU - Kim, Jae-Yean AU - Kim JY AUID- ORCID: 0000-0002-1180-6232 AD - Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South Korea. AD - Division of Life Science, Gyeongsang National University, Jinju, South Korea. AD - Nulla Bio Inc., Jinju, South Korea. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20230110 PL - United States TA - mBio JT - mBio JID - 101519231 RN - EC 3.1.- (CRISPR-Associated Protein 9) RN - JAC85A2161 (Adenine) RN - 8J337D1HZY (Cytosine) RN - 10191-18-1 (BES) SB - IM MH - *CRISPR-Associated Protein 9/genetics MH - *CRISPR-Cas Systems MH - Escherichia coli/genetics/metabolism MH - Adenine MH - Cytosine MH - Gene Editing/methods PMC - PMC9973308 OTO - NOTNLM OT - CRISPR OT - base editing OT - dual base editor OT - genome editing OT - microbial engineering COIS- The authors declare no conflict of interest. EDAT- 2023/01/11 06:00 MHDA- 2023/03/03 06:00 PMCR- 2023/01/10 CRDT- 2023/01/10 09:03 PHST- 2023/01/11 06:00 [pubmed] PHST- 2023/03/03 06:00 [medline] PHST- 2023/01/10 09:03 [entrez] PHST- 2023/01/10 00:00 [pmc-release] AID - 02296-22 [pii] AID - mbio.02296-22 [pii] AID - 10.1128/mbio.02296-22 [doi] PST - ppublish SO - mBio. 2023 Feb 28;14(1):e0229622. doi: 10.1128/mbio.02296-22. Epub 2023 Jan 10.