High efficient multisites genome editing in allotetraploid cotton (Gossypium hirsutum) using CRISPR/Cas9 system

Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene‐specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site‐specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2‐edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7–100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode‐based high‐throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1‐edited T0 plants and it matched well with Sanger sequencing results. No off‐target editing was detected at the potential off‐target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing.     

Base editing with high efficiency in allotetraploid oilseed rape by A3A‐PBE system

CRISPR/Cas‐base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A‐PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base‐editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base‐edited plants of ALS conferred high herbicide resistance, while base‐edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T₀ to T₁ generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off‐target sites, large‐scale SNP variations were determined through whole‐genome sequencing between some base‐edited and wild‐type plants. These results revealed that A3A‐PBE base‐editing system could effectively convert C to T substitution with high‐editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.     

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA (gRNA) and LbCas12a‐CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium‐mediated transformation. On‐target mutation analysis showed that 90%–100% of the Cas9‐edited T0 plants carried indel mutations and 63%–77% of them were homozygous or biallelic mutants. In contrast, 0%–60% of Cas12a‐edited T0 plants had on‐target mutations. We then conducted CIRCLE‐seq analysis to identify genome‐wide potential off‐target sites for Cas9. A total of 18 and 67 potential off‐targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off‐target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.     

The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation

The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9‐induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9‐induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large‐scale off‐targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off‐target sites of a large number of T0 plants, low‐frequency mutations were found in only one off‐target site where the sequence had 1‐bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.     

Reduced fire blight susceptibility in apple cultivars using a high‐efficiency CRISPR/Cas9‐FLP/FRT‐based gene editing system

The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein MdDIPM4. In this work, MdDIPM4 knockout has been produced in two Malus × domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty‐seven transgenic lines were screened to identify CRISPR/Cas9‐induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss‐of‐function mutation were inoculated with the pathogen. Highly significant reduction in susceptibility was observed compared to control plants. Sequencing of five potential off‐target sites revealed no mutation event. Moreover, our construct contained a heat‐shock inducible FLP/FRT recombination system designed specifically to remove the T‐DNA harbouring the expression cassettes for CRISPR/Cas9, the marker gene and the FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat‐treated and screened by real‐time PCR to quantify the exogenous DNA elimination. The T‐DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9‐FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DNA.     

Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing

CRISPR/Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR/Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR/Cas9 complex. Due to the high frequency of gene silencing associated with co‐transferred plasmid backbone and low edit rate in wheat, a large T₀ transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium‐delivered CRISPR/Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR/Cas9 system, we have developed 68 edit mutants for four grain‐regulatory genes, TaCKX2‐1, TaGLW7, TaGW2, and TaGW8, in T₀, T₁, and T₂ generation plants at an average edit rate of 10% without detecting off‐target mutations in the most Cas9‐active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160‐bp deletion in TaCKX2‐D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium‐delivered CRISPR/Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR/Cas9 remains active for novel mutations through generations.     

毕赤酵母胞嘧啶碱基编辑器的设计与功能评价

【目的】在巴斯德毕赤酵母（Pichia pastoris）中建立一套分子靶向突变系统，为毕赤酵母的基因工程改造提供高效的编辑工具。【方法】基于规律成簇的间隔短回文重复序列/Cas9核酸酶（clustered regularly interspaced short palindromic repeats/Cas9 nuclease，CRISPR/Cas9）技术，设计并构建nCas9与胞苷脱氨酶融合表达的胞嘧啶碱基编辑器（cytosine base editor，CBE），并选择酵母基因组中富含碱基C的一段序列作为靶标以评价CBE的碱基编辑功能。电转化酵母后，利用高通量测序技术分析CBE的编辑效率及编辑模式，并进一步探究连接肽长度、融合蛋白相对位置和gRNA靶向序列（即spacer）长度等因素对CBE功能的影响。【结果】nCas9与PmCDA1融合组成的CBE能够实现毕赤酵母基因组碱基C的高效编辑。当连接肽长度为（GGGGS）₁₀时，CBE的编辑效率最高，编辑窗口位于前间隔序列邻近基序（protospacer adjacent motif，PAM）远端的C20–C14之间，其中C18的编辑效率可达85.1%。nCas9与PmCDA1相对位置的改变对CBE的编辑效率和编辑模式的影响不大。而gRNA靶向序列长度影响着CBE的编辑效率，且gRNA靶向序列长度不能低于17 nt，但19–23 nt之间均可引导CBE对基因组的高效编辑。【结论】本研究在巴斯德毕赤酵母中构建了一套具有高效碱基编辑活性的胞嘧啶碱基编辑器，为基于毕赤酵母的基础和应用研究提供了工具支持。     

One‐step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment

The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9‐expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off‐target effects were observed from off‐target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene‐disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild‐type CHO‐S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.     

BE3型胞嘧啶碱基编辑器在谷氨酸棒杆菌中的开发及应用

碱基编辑技术结合了CRISPR/Cas系统的靶向特异性与碱基脱氨酶的催化活性,因其不产生双链DNA断裂、不需要外源DNA模板、不依赖同源重组修复,自开发以来,便受到研究者的追捧,在哺乳动物细胞、植物、微生物等领域相继得到开发与应用。为了进一步丰富碱基编辑系统在谷氨酸棒杆菌中的应用,将鼠源胞嘧啶脱氨酶(rAPOBEC1)与nCas9蛋白融合,实现了在谷氨酸棒杆菌中C到T的编辑,编辑比例较低(0-20%);在上述融合蛋白C端添加UGI蛋白,构建BE3型胞嘧啶碱基编辑器,抑制体内的DNA碱基切除修复机制,显著的提高了碱基编辑效率,使得C到T的碱基编辑效率高达90%;为了简化操作,将双质粒碱基编辑系统优化为单质粒碱基编辑系统,并显著提高转化效率;最后通过单质粒碱基编辑系统对基因组中其他位点的编辑测试,进一步证明了BE3型碱基编辑器在谷氨酸棒杆菌中的高效性,同时发现该碱基编辑器具有较宽的编辑窗口(PAM上游-11到-19位),有助于覆盖更多的基因组靶标位点,为谷氨酸棒杆菌的基因组改造提供了更多的工具选择。     

Assessment of Cas12a‐mediated gene editing efficiency in plants

The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA‐guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB‐assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target‐dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on‐target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a‐free segregating T2 lines to assess possible off‐target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of ?10 to ?2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off‐target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.     

Multiplex Gene Disruption by Targeted Base Editing of Yarrowia lipolytica Genome Using Cytidine Deaminase Combined with the CRISPR/Cas9 System

The oleaginous yeast Yarrowia lipolytica has a tendency to use the non‐homologous end joining repair (NHEJ) over the homology directed recombination as double‐strand breaks (DSB) repair system, making it difficult to edit the genome using homologous recombination. A recently developed Target‐AID (activation‐induced cytidine deaminase) base editor, designed to recruit cytidine deaminase (CDA) to the target DNA locus via the CRISPR/Cas9 system, can directly induce C to T mutation without DSB and donor DNA. In this study, this system is adopted in Y. lipolytica for multiplex gene disruption. Target‐specific gRNA(s) and a fusion protein consisting of a nickase Cas9, pmCDA1, and uracil DNA glycosylase inhibitor are expressed from a single plasmid to disrupt target genes by introducing a stop codon via C to T mutation within the mutational window. Deletion of the KU70 gene involved in the NHEJ prevents the generation of indels by base excision repair following cytidine deamination, increasing the accuracy of genome editing. Using this Target‐AID system with optimized expression levels of the base editor, single gene disruption and simultaneous double gene disruption are achieved with the efficiencies up to 94% and 31%, respectively, demonstrating this base editing system as a convenient genome editing tool in Y. lipolytica.     

Patterns of CRISPR/Cas9 activity in plants,animals and microbes

The CRISPR/Cas9 system and related RNA‐guided endonucleases can introduce double‐strand breaks (DSBs) at specific sites in the genome, allowing the generation of targeted mutations in one or more genes as well as more complex genomic rearrangements. Modifications of the canonical CRISPR/Cas9 system from Streptococcus pyogenes and the introduction of related systems from other bacteria have increased the diversity of genomic sites that can be targeted, providing greater control over the resolution of DSBs, the targeting efficiency (frequency of on‐target mutations), the targeting accuracy (likelihood of off‐target mutations) and the type of mutations that are induced. Although much is now known about the principles of CRISPR/Cas9 genome editing, the likelihood of different outcomes is species‐dependent and there have been few comparative studies looking at the basis of such diversity. Here we critically analyse the activity of CRISPR/Cas9 and related systems in different plant species and compare the outcomes in animals and microbes to draw broad conclusions about the design principles required for effective genome editing in different organisms. These principles will be important for the commercial development of crops, farm animals, animal disease models and novel microbial strains using CRISPR/Cas9 and other genome‐editing tools.     

Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants

The CRISPR/Cas9 system has been extensively applied for crop improvement. However, our understanding of Cas9 specificity is very limited in Cas9‐edited plants. To identify on‐ and off‐target mutation in an edited crop, we described whole genome sequencing (WGS) of 14 Cas9‐edited cotton plants targeted to three genes, and three negative (Ne) control and three wild‐type (WT) plants. In total, 4188–6404 unique single‐nucleotide polymorphisms (SNPs) and 312–745 insertions/deletions (indels) were detected in 14 Cas9‐edited plants compared to WT, negative and cotton reference genome sequences. Since the majority of these variations lack a protospacer‐adjacent motif (PAM), we demonstrated that the most variations following Cas9‐edited are due either to somaclonal variation or/and pre‐existing/inherent variation from maternal plants, but not off‐target effects. Of a total of 4413 potential off‐target sites (allowing ≤5 mismatches within the 20‐bp sgRNA and 3‐bp PAM sequences), the WGS data revealed that only four are bona fide off‐target indel mutations, validated by Sanger sequencing. Moreover, inherent genetic variation of WT can generate novel off‐target sites and destroy PAMs, which suggested great care should be taken to design sgRNA for the minimizing of off‐target effect. These findings suggested that CRISPR/Cas9 system is highly specific for cotton plants.     

Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy,a web‐based target finding tool

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus genome engineering of CHO cells for improved product quality and yield is of great interest. Here, we demonstrate for the first time the efficacy of the CRISPR Cas9 technology in CHO cells by generating site‐specific gene disruptions in COSMC and FUT8, both of which encode proteins involved in glycosylation. The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome‐editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. Finally, we have developed a user‐friendly bioinformatics tool, named “CRISPy” for rapid identification of sgRNA target sequences in the CHO‐K1 genome. The CRISPy tool identified 1,970,449 CRISPR targets divided into 27,553 genes and lists the number of off‐target sites in the genome. In conclusion, the proven functionality of Cas9 to edit CHO genomes combined with our CRISPy database have the potential to accelerate genome editing and synthetic biology efforts in CHO cells. Biotechnol. Bioeng. 2014; 111: 1604–1616. © 2014 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

CRISPR/Cas9 engineering of albino cystinuria Type A mice

Cystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co‐injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off‐target editing. No off‐target indels were observed for the top 10 predicted off‐targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.     

Multiplex genome editing using a dCas9-cytidine deaminase fusion in Streptomyces

CRISPR/Cas-mediated genome editing has greatly facilitated the study of gene function in Streptomyces. However, it could not be efficiently employed in streptomycetes with low homologous recombination(HR) ability. Here, a deaminase-assisted base editor d Cas9-CDA-UL_(str) was developed in Streptomyces, which comprises the nuclease-deficient Cas9(dCas9), the cytidine deaminase from Petromyzon marinus(PmCDA1), the uracil DNA glycosylase inhibitor(UGI) and the protein degradation tag(LVA tag). Using d Cas9-CDA-UL_(str) , we achieved single-, double-and triple-point mutations(cytosine-to-thymine substitutions)at target sites in Streptomyces coelicolor with efficiency up to 100%, 60% and 20%, respectively. This base editor was also demonstrated to be highly efficient for base editing in the industrial strain, Streptomyces rapamycinicus, which produces the immunosuppressive agent rapamycin. Compared with base editors derived from the cytidine deaminase rAPOBEC1, the PmCDA1-assisted base editor dCas9-CDA-UL_(str) could edit cytosines preceded by guanosines with high efficiency, which is a great advantage for editing Streptomyces genomes(with high GC content). Collectively, the base editor dCas9-CDA-UL_(str) could be employed for efficient multiplex genome editing in Streptomyces. Since the d Cas9-CDA-UL_(str) -based genome editing is independent of HR-mediated DNA repair, we believe this technology will greatly facilitate functional genome research and metabolic engineering in Streptomyces strains with weak HR ability.     

Increasing the efficiency of CRISPR‐Cas9‐VQR precise genome editing in rice

Clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR‐Cas9) is a revolutionary technology that enables efficient genomic modification in many organisms. Currently, the wide use of Streptococcus pyogenes Cas9 (SpCas9) primarily recognizes sites harbouring a canonical NGG protospacer adjacent motif (PAM). The newly developed VQR (D1135V/R1335Q/T1337R) variant of Cas9 has been shown to cleave sites containing NGA PAM in rice, which greatly expanded the range of genome editing. However, the low editing efficiency of the VQR variant remains, which limits its wide application in genome editing. In this study, by modifying the single guide RNA (sgRNA) structure and strong endogenous promoters, we significantly increased the editing efficiency of the VQR variant. The modified CRISPR‐Cas9‐VQR system provides a robust toolbox for multiplex genome editing at sites containing noncanonical NGA PAM.     

Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

The fast growth, ease of metabolic labelling and potential for feedstock and biofuels production make duckweeds not only an attractive model system for understanding plant biology, but also a potential future crop. However, current duckweed research is constrained by the lack of efficient genetic manipulation tools. Here, we report a case study on genome editing in a duckweed species, Lemna aequinoctialis, using a fast and efficient transformation and CRISPR/Cas9 tool. By optimizing currently available transformation protocols, we reduced the duration time of Agrobacterium‐mediated transformation to 5–6 weeks with a success rate of over 94%. Based on the optimized transformation protocol, we generated 15 (14.3% success rate) biallelic LaPDS mutants that showed albino phenotype using a CRISPR/Cas9 system. Investigations on CRISPR/Cas9‐mediated mutation spectrum among mutated L. aequinoctialis showed that most of mutations were short insertions and deletions. This study presents the first example of CRISPR/Cas9‐mediated genome editing in duckweeds, which will open new research avenues in using duckweeds for both basic and applied research.     

Front Cover Image,Volume 116, Number 11, November 2019

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome ( www.ibiodesign.net/gBIG ).