A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing

AbstractCRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.

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CRISPR-nRAGE, a Cas9 nickase-reverse transcriptase assisted versatile genetic engineering toolkit for E. coli

10.1101/2020.09.02.279141 ◽

2020 ◽

Author(s):

Yaojun Tong ◽

Tue S. Jørgensen ◽

Christopher M. Whitford ◽

Tilmann Weber ◽

Sang Yup Lee

Keyword(s):

Reverse Transcriptase ◽

Genome Engineering ◽

Genetic Manipulation ◽

Fragment Size ◽

Leukemia Virus ◽

Strand Break ◽

Engineering System ◽

E Coli ◽

Dna Engineering ◽

Nucleotide Resolution

AbstractIn most prokaryotes, missing and poorly active non-homologous end joining (NHEJ) DNA repair pathways heavily restrict the direct application of CRISPR-Cas for DNA double-strand break (DSB)-based genome engineering without providing editing templates. CRISPR base editors, on the other hand, can be directly used for genome engineering in a number of bacteria, including E. coli, showing advantages over CRISPR-Cas9, since they do not require DSBs. However, as the current CRISPR base editors can only engineer DNA by A to G or C to T/G/A substitutions, they are incapable of mediating deletions, insertions, and combinations of deletions, insertions and substitutions. To address these challenges, we developed a Cas9 nickase (Cas9n)-reverse transcriptase (Moloney Murine Leukemia Virus, M-MLV) mediated, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes, termed CRISPR-nRAGE (CRISPR-Cas9n Reverse transcriptase Assisted Genome Engineering) system. CRISPR-nRAGE can be used to introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli. Notably, small sized-deletion shows better editing efficiency compared to other kinds of DNA engineering. CRISPR-nRAGE has been used to delete and insert DNA fragments up to 97 bp and 33 bp, respectively. Efficiencies, however, drop sharply with the increase of the fragment size. It is not only a useful addition to the genome engineering arsenal for E. coli, but also may be the basis for the development of similar toolkits for other organisms.

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The efficacy of CRISPR-mediated cytosine base editing with the RPS5a promoter in Arabidopsis thaliana

Scientific Reports ◽

10.1038/s41598-021-87669-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Minkyung Choi ◽

Jae-Young Yun ◽

Jun-Hyuk Kim ◽

Jin-Soo Kim ◽

Sang-Tae Kim

Keyword(s):

Arabidopsis Thaliana ◽

Cytidine Deaminase ◽

Biological Research ◽

Loss Of Function ◽

Nucleotide Substitutions ◽

Viral Origin ◽

Nonsense Mutations ◽

Base Editing ◽

Low Efficiency ◽

Viable System

AbstractCRISPR/Cas9-mediated genome editing is an important and versatile technology in modern biological research. Recent advancements include base-editing CRISPR tools that enable targeted nucleotide substitutions using a fusion protein comprising a nickase variant of Cas9 and a base deaminase. Improvements in base editing efficiencies and inheritable of edited loci need to be made to make CRISPR a viable system in plants. Here, we report efficiency of cytosine base editors (CBEs) in Arabidopsis thaliana by applying the strong endogenous RPS5a promoter to drive the expression of nickase Cas9 and either rAPOBEC1 from rat (BE3) or the PmCDA1 activation-induced cytidine deaminase from sea lamprey (AIDv2). Compared with the strong heterologous CaMV35S promoter of viral origin, the RPS5a promoter improved CBE efficiency by 32% points with the number of T1 plants showing over 50% conversion ratio when the LFY gene was targeted. CBE induced nonsense mutations in LFY via C-to-T conversion, which resulted in loss-of-function lfy phenotypes; defects in LFY function were associated with the targeted base substitutions. Our data suggest that optimal promoter choice for CBE expression may affect base-editing efficiencies in plants. The results provide a strategy to optimize low-efficiency base editors and demonstrate their applicability for functional assays and trait development in crop research.

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Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA

mBio ◽

10.1128/mbio.00342-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Zhiwei Hu ◽

Yannan Wang ◽

Qian Liu ◽

Yan Qiu ◽

Zhiyu Zhong ◽

...

Keyword(s):

Genome Editing ◽

Dna Breaks ◽

Single Nucleotide ◽

Base Editing ◽

Guide Rna ◽

Guide Rnas ◽

Double Stranded Dna ◽

Target Sites ◽

Guide Sequence ◽

Sgrna Design

ABSTRACT Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing. IMPORTANCE Base editors are DSB-free genome editing tools and have been widely used in diverse living systems. However, it is reported that these tools can cause substantial off-target editings. To meet this challenge, we developed a new approach to improve the specificity of base editors by using hairpin sgRNAs with a bubble. Furthermore, our sgRNA design also dramatically reduced indels and unwanted base substitutions at on-target sites. We believe that the BH-sgRNA design is a significant improvement over existing sgRNAs of base editors, and our design promises to be adaptable to various base editors. We expect that it will make contributions to improving the safety of gene therapy.

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The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato

10.1101/628107 ◽

2019 ◽

Author(s):

Florian Veillet ◽

Laura Chauvin ◽

Marie-Paule Kermarrec ◽

François Sevestre ◽

Mathilde Merrer ◽

...

Keyword(s):

Solanum Tuberosum ◽

Genome Editing ◽

Genome Engineering ◽

Basic Research ◽

Loss Of Function ◽

Dna Breaks ◽

Nucleotide Substitutions ◽

Discrete Variation ◽

Tetraploid Potato ◽

Base Editing

AbstractGenome editing has recently become a method of choice for basic research and functional genomics, and holds great potential for molecular plant breeding applications. The powerful CRISPR-Cas9 system that typically produces double-strand DNA breaks is mainly used to generate knockout mutants. Recently, the development of base editors has broadened the scope of genome editing, allowing precise and efficient nucleotide substitutions. In this study, we produced mutants in two cultivated elite cultivars of the tetraploid potato (Solanum tuberosum) using stable or transient expression of the CRISPR-Cas9 components to knockout the amylose-producing StGBSSI gene. We set up a rapid, highly sensitive and cost-effective screening strategy based on high-resolution melting analysis followed by direct Sanger sequencing and trace chromatogram analysis. Most mutations consisted of small indels, but unwanted insertions of plasmid DNA were also observed. We successfully created tetra-allelic mutants with impaired amylose biosynthesis, confirming the loss-of-function of the StGBSSI protein. The second main objective of this work was to demonstrate the proof of concept of CRISPR-Cas9 base editing in the tetraploid potato by targeting two loci encoding catalytic motifs of the StGBSSI enzyme. Using a cytidine base editor (CBE), we efficiently and precisely induced DNA substitutions in the KTGGL-encoding locus, leading to discrete variation in the amino acid sequence and generating a loss-of-function allele. The successful application of base editing in the tetraploid potato opens up new avenues for genome engineering in this species.Key MessageThe StGBSSI gene was successfully and precisely edited in the tetraploid potato using gene and base editing strategies, leading to plants with impaired amylose biosynthesis.

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Base editing in Streptomyces with Cas9-deaminase fusions

10.1101/630137 ◽

2019 ◽

Cited By ~ 5

Author(s):

Zhiyu Zhong ◽

Junhong Guo ◽

Liang Deng ◽

Li Chen ◽

Jian Wang ◽

...

Keyword(s):

Genome Editing ◽

Target Genes ◽

Genetic Manipulation ◽

Strand Break ◽

High Fidelity ◽

Nucleotide Substitutions ◽

Base Editing ◽

Dna Double Strand Break ◽

Repair Template ◽

Adenine Base

AbstractConventional CRISPR/Cas genetic manipulation has been profitably applied to the genus Streptomyces, the most prolific bacterial producers of antibiotics. However, its reliance on DNA double-strand break (DSB) formation leads to unacceptably low yields of desired recombinants. We have adapted for Streptomyces recently-introduced cytidine base editors (CBEs) and adenine base editors (ABEs) which enable targeted C-to-T or A-to-G nucleotide substitutions, respectively, bypassing DSB and the need for a repair template. We report successful genome editing in Streptomyces at frequencies of around 50% using defective Cas9-guided base editors and up to 100% by using nicked Cas9-guided base editors. Furthermore, we demonstrate the multiplex genome editing potential of the nicked Cas9-guided base editor BE3 by programmed mutation of nine target genes simultaneously. Use of the high-fidelity version of BE3 (HF-BE3) essentially improved editing specificity. Collectively, this work provides a powerful new tool for genome editing in Streptomyces.

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Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice

Science ◽

10.1126/science.aaw7166 ◽

2019 ◽

pp. eaaw7166 ◽

Cited By ~ 77

Author(s):

Shuai Jin ◽

Yuan Zong ◽

Qiang Gao ◽

Zixu Zhu ◽

Yanpeng Wang ◽

...

Keyword(s):

High Fidelity ◽

Single Nucleotide Variants ◽

Disease Treatment ◽

Single Nucleotide ◽

Genetic Changes ◽

Base Editing ◽

Guide Rna ◽

Genome Wide ◽

Adenine Base

Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole genome sequencing (WGS) analysis of rice plants treated with BE3, high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C→T type of single nucleotide variants (SNVs) and appear to be enriched in genic regions. Notably, treatment of rice with BE3 or HF1-BE3 in the absence of single-guide RNA also results in the rise of genome-wide SNVs. Thus, the base editing unit of BE3 or HF1-BE3 needs to be optimized in order to attain high fidelity.

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Phage Peptides Mediate Precision Base Editing with Focused Targeting Window

10.1101/2020.11.02.364430 ◽

2020 ◽

Author(s):

Kun Jia ◽

Yan-ru Cui ◽

Shisheng Huang ◽

Peihong Yu ◽

Zhengxing Lian ◽

...

Keyword(s):

Genome Engineering ◽

Single Point ◽

Disease Model ◽

Point Mutations ◽

Gene Correction ◽

Nucleotide Substitutions ◽

Base Editing ◽

A Cell ◽

Single Point Mutations ◽

Adenine Base

AbstractCytidine base editors (CBE) are novel genome engineering tools that can generate C-to-T nucleotide substitutions without introducing double-stranded breaks (DSBs). Instead of generating single-point mutations, CBEs induce nucleotide substitutions at wobble positions within the 20-nucleotide target site. A variety of strategies have been developed to improve the targeting scope and window of CBEs. Among these strategies, molecular switches that can temporally control CBE activities represent compelling options. In this study, we investigated the feasibility of using a bacteriophage-derived peptide, referred to as G8PPD, as the off-switch of CBEs. We showed that G8PPD could be employed to control the activities of and improve the targeting window of A3A and BE3 CBEs and adenine base editor 7.10 (ABE7.10). Notably, in a cell-based disease model of Marfan syndrome, G8PPD facilitated A3A CBE-based gene correction with a more focused targeting window and improved the percentage of perfectly edited gene alleles from less than 4% to more than 38% of the whole population. Our study presents the first peptide off-switch that can improve the targeting scope of CBEs, thus highlighting the importance of the temporal control of BE activity for precision base editing.

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Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system

10.1101/169441 ◽

2017 ◽

Cited By ~ 4

Author(s):

Ryan Marshall ◽

Colin S. Maxwell ◽

Scott P. Collins ◽

Michelle L. Luo ◽

Thomas Jacobsen ◽

...

Keyword(s):

Protein Purification ◽

Dna Cleavage ◽

Genome Engineering ◽

Gene Repression ◽

Live Cells ◽

Translation System ◽

Guide Rna ◽

E Coli ◽

Translation Systems

ABSTRACTCRISPR-Cas systems have offered versatile technologies for genome engineering, yet their implementation has been outpaced by the ongoing discovery of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.

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Highly Efficient CRISPR-Mediated Base Editing in Sinorhizobium meliloti

Frontiers in Microbiology ◽

10.3389/fmicb.2021.686008 ◽

2021 ◽

Vol 12 ◽

Author(s):

Longxiang Wang ◽

Yuan Xiao ◽

Xiaowei Wei ◽

Jimin Pan ◽

Deqiang Duanmu

Keyword(s):

Nitrogen Fixation ◽

Sinorhizobium Meliloti ◽

Cytidine Deaminase ◽

Base Editing ◽

Guide Rna ◽

Genetic Manipulations ◽

Highly Efficient ◽

Adenine Deaminase ◽

Nucleotide Resolution ◽

Biological Nitrogen

Rhizobia are widespread gram-negative soil bacteria and indispensable symbiotic partners of leguminous plants that facilitate the most highly efficient biological nitrogen fixation in nature. Although genetic studies in Sinorhizobium meliloti have advanced our understanding of symbiotic nitrogen fixation (SNF), the current methods used for genetic manipulations in Sinorhizobium meliloti are time-consuming and labor-intensive. In this study, we report the development of a few precise gene modification tools that utilize the CRISPR/Cas9 system and various deaminases. By fusing the Cas9 nickase to an adenine deaminase, we developed an adenine base editor (ABE) system that facilitated adenine-to-guanine transitions at one-nucleotide resolution without forming double-strand breaks (DSB). We also engineered a cytidine base editor (CBE) and a guanine base editor (GBE) that catalyze cytidine-to-thymine substitutions and cytidine-to-guanine transversions, respectively, by replacing adenine deaminase with cytidine deaminase and other auxiliary enzymes. All of these base editors are amenable to the assembly of multiple synthetic guide RNA (sgRNA) cassettes using Golden Gate Assembly to simultaneously achieve multigene mutations or disruptions. These CRISPR-mediated base editing tools will accelerate the functional genomics study and genome manipulation of rhizobia.

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Precise and programmable C:G to G:C base editing in genomic DNA

10.1101/2020.07.21.213827 ◽

2020 ◽

Author(s):

Liwei Chen ◽

Jung Eun Park ◽

Peter Paa ◽

Priscilla D. Rajakumar ◽

Yi Ting Chew ◽

...

Keyword(s):

Therapeutic Potential ◽

Excision Repair ◽

Genetic Diseases ◽

Cytosine Deaminase ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Base Editing ◽

Guide Rnas ◽

Base Excision ◽

Nucleotide Resolution

AbstractMany genetic diseases are caused by single-nucleotide polymorphisms (SNPs). Base editors can correct SNPs at single-nucleotide resolution, but until recently, only allowed for C:G to T:A and A:T to G:C transition edits, addressing four out of twelve possible DNA base substitutions. Here we developed a novel class of C:G to G:C Base Editors (CGBEs) to create single-base genomic transversions in human cells. Our CGBEs consist of a nickase CRISPR-Cas9 (nCas9) fused to a cytosine deaminase and base excision repair (BER) proteins. Characterization of >30 CGBE candidates and 27 guide RNAs (gRNAs) revealed that CGBEs predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean C:G to G:C edits at 15% and up to 37%). CGBEs target cytosine in WCW, ACC or GCT sequence contexts and within a precise two-nucleotide window of the target protospacer. We further targeted genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of CGBE in interrogating and correcting human genetic diseases.

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