CRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering

Abstract Background Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR–Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR–Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR–Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions This study applied CRISPR–Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR–Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

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Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

Applied and Environmental Microbiology ◽

10.1128/aem.01453-16 ◽

2016 ◽

Vol 82 (17) ◽

pp. 5421-5427 ◽

Cited By ~ 102

Author(s):

Josef Altenbuchner

Keyword(s):

Bacillus Subtilis ◽

Genome Editing ◽

Genome Engineering ◽

Shuttle Vector ◽

Temperature Sensitive ◽

Target Sequence ◽

Target Specificity ◽

Content Type ◽

A Genome ◽

Single Plasmid

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system fromStreptococcus pyogeneshas recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing ofBacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication forEscherichia coli, the temperature-sensitive replication origin of plasmid pE194tsforB. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries thecas9gene under the control of theB. subtilismannose-inducible promoter PmanPand a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5′ end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in theB. subtilischromosome and by repair of thetrpC2mutation ofB. subtilis168.IMPORTANCEIn prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing ofB. subtilisovercomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

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Targeted editing and evolution of engineered ribosomes in vivo by filtered editing

Nature Communications ◽

10.1038/s41467-021-27836-x ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Felix Radford ◽

Shane D. Elliott ◽

Alanna Schepartz ◽

Farren J. Isaacs

Keyword(s):

Genome Editing ◽

Dna Sequences ◽

Genome Engineering ◽

23S Rrna ◽

Translational Efficiency ◽

Rna Molecules ◽

Genetic Elements ◽

A Genome ◽

Self Splicing

AbstractGenome editing technologies introduce targeted chromosomal modifications in organisms yet are constrained by the inability to selectively modify repetitive genetic elements. Here we describe filtered editing, a genome editing method that embeds group 1 self-splicing introns into repetitive genetic elements to construct unique genetic addresses that can be selectively modified. We introduce intron-containing ribosomes into the E. coli genome and perform targeted modifications of these ribosomes using CRISPR/Cas9 and multiplex automated genome engineering. Self-splicing of introns post-transcription yields scarless RNA molecules, generating a complex library of targeted combinatorial variants. We use filtered editing to co-evolve the 16S rRNA to tune the ribosome’s translational efficiency and the 23S rRNA to isolate antibiotic-resistant ribosome variants without interfering with native translation. This work sets the stage to engineer mutant ribosomes that polymerize abiological monomers with diverse chemistries and expands the scope of genome engineering for precise editing and evolution of repetitive DNA sequences.

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Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering

Microbial Cell Factories ◽

10.1186/s12934-019-1255-1 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 7

Author(s):

Ioannis Mougiakos ◽

Enrico Orsi ◽

Mohammad Rifqi Ghiffary ◽

Wilbert Post ◽

Alberto de Maria ◽

...

Keyword(s):

Metabolic Engineering ◽

Homologous Recombination ◽

Genome Editing ◽

Rhodobacter Sphaeroides ◽

Genome Engineering ◽

Start Codon ◽

Terpene Biosynthesis ◽

Knock Out ◽

A Genome ◽

Coa Reductase

Abstract Background Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides. Results Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected. Conclusions In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.

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Inducible Plasmid Self-Destruction (IPSD) assisted genome engineering in lactobacilli and bifidobacteria

10.1101/649921 ◽

2019 ◽

Author(s):

Fanglei Zuo ◽

Zhu Zeng ◽

Lennart Hammarström ◽

Harold Marcotte

Keyword(s):

Synthetic Biology ◽

Homologous Recombination ◽

Genetic Engineering ◽

Genome Editing ◽

Genome Engineering ◽

Bacterial Species ◽

Knock Out ◽

Recombination System ◽

A Genome ◽

Selection Of

ABSTRACTGenome engineering is essential for application of synthetic biology in probiotics including lactobacilli and bifidobacteria. Several homologous recombination system-based mutagenesis tools have been developed for these bacteria but still, have many limitations in different species or strains. Here we developed a genome engineering method based on an inducible self-destruction plasmid delivering homologous DNA into bacteria. Excision of the replicon by induced recombinase facilitates selection of homologous recombination events. This new genome editing tool called Inducible Plasmid Self-Destruction (IPSD) was successfully used to perform gene knock-out and knock-in in lactobacilli and bifidobacteria. Due to its simplicity and universality, the IPSD strategy may provide a general approach for genetic engineering of various bacterial species.

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In Vivo Genome Editing as a Therapeutic Approach

International Journal of Molecular Sciences ◽

10.3390/ijms19092721 ◽

2018 ◽

Vol 19 (9) ◽

pp. 2721 ◽

Cited By ~ 25

Author(s):

Beatrice Ho ◽

Sharon Loh ◽

Woon Chan ◽

Boon Soh

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Genetic Diseases ◽

Gene Mutations ◽

Genetic Mutation ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Therapeutic Benefits ◽

A Genome

Genome editing has been well established as a genome engineering tool that enables researchers to establish causal linkages between genetic mutation and biological phenotypes, providing further understanding of the genetic manifestation of many debilitating diseases. More recently, the paradigm of genome editing technologies has evolved to include the correction of mutations that cause diseases via the use of nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and more recently, Cas9 nuclease. With the aim of reversing disease phenotypes, which arise from somatic gene mutations, current research focuses on the clinical translatability of correcting human genetic diseases in vivo, to provide long-term therapeutic benefits and potentially circumvent the limitations of in vivo cell replacement therapy. In this review, in addition to providing an overview of the various genome editing techniques available, we have also summarized several in vivo genome engineering strategies that have successfully demonstrated disease correction via in vivo genome editing. The various benefits and challenges faced in applying in vivo genome editing in humans will also be discussed.

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Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395

International Journal of Molecular Sciences ◽

10.3390/ijms22020480 ◽

2021 ◽

Vol 22 (2) ◽

pp. 480

Author(s):

Jongrae Kim ◽

Kwang Suk Chang ◽

Sangmuk Lee ◽

EonSeon Jin

Keyword(s):

Genome Editing ◽

Chlorella Vulgaris ◽

Negative Selection ◽

Screening Strategy ◽

Feed Additive ◽

Cell Factory ◽

Dna Transformation ◽

Research Groups ◽

A Genome ◽

Food And Feed

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

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Recent advances in CRISPR/Cas9 and applications for wheat functional genomics and breeding

aBIOTECH ◽

10.1007/s42994-021-00042-5 ◽

2021 ◽

Author(s):

Jun Li ◽

Yan Li ◽

Ligeng Ma

Keyword(s):

Functional Genomics ◽

Genome Editing ◽

Functional Annotation ◽

Genome Engineering ◽

Agronomic Traits ◽

Wheat Breeding ◽

Breeding Programs ◽

Base Editing ◽

The World ◽

World Food Security

AbstractCommon wheat (Triticum aestivum L.) is one of the three major food crops in the world; thus, wheat breeding programs are important for world food security. Characterizing the genes that control important agronomic traits and finding new ways to alter them are necessary to improve wheat breeding. Functional genomics and breeding in polyploid wheat has been greatly accelerated by the advent of several powerful tools, especially CRISPR/Cas9 genome editing technology, which allows multiplex genome engineering. Here, we describe the development of CRISPR/Cas9, which has revolutionized the field of genome editing. In addition, we emphasize technological breakthroughs (e.g., base editing and prime editing) based on CRISPR/Cas9. We also summarize recent applications and advances in the functional annotation and breeding of wheat, and we introduce the production of CRISPR-edited DNA-free wheat. Combined with other achievements, CRISPR and CRISPR-based genome editing will speed progress in wheat biology and promote sustainable agriculture.

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