Saturation Mutagenesis Genome Engineering of Infective ΦX174 Bacteriophage via Unamplified Oligo Pools and Golden Gate Assembly

Here we present a novel protocol for the construction of saturation single-site—and massive multi-site—mutant libraries of a bacteriophage. We segmented the ΦX174 genome into 14 non-toxic and non-replicative fragments compatible with golden gate assembly. We next used nicking mutagenesis with oligonucleotides prepared from unamplified oligo pools with individual segments as templates to prepare near-comprehensive single-site mutagenesis libraries of genes encoding the F capsid protein (421 amino acids scanned) and G spike protein (172 amino acids scanned). Libraries possessed greater than 99% of all 11,860 programmed mutations. Golden Gate cloning was then used to assemble the complete ΦX174 mutant genome and generate libraries of infective viruses. This protocol will enable reverse genetics experiments for studying viral evolution and, with some modifications, can be applied for engineering of therapeutically relevant bacteriophages with larger genomes.

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Highly Multiplexed Genome Engineering Using CRISPR/Cas9 gRNA Arrays

10.1101/331371 ◽

2018 ◽

Author(s):

Morito Kurata ◽

Natalie K. Wolf ◽

Walker S. Lahr ◽

Madison T. Weg ◽

Samantha Lee ◽

...

Keyword(s):

Single Cell ◽

Adaptive Immunity ◽

Genome Engineering ◽

Biological Processes ◽

Golden Gate ◽

Pol Ii ◽

Guide Rna ◽

Multiple Loci ◽

Model Complex ◽

Pol Iii

ABSTRACTThe CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limiting due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

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Golden Mutagenesis: An efficient multi-site-saturation mutagenesis approach by Golden Gate cloning with automated primer design

Scientific Reports ◽

10.1038/s41598-019-47376-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 15

Author(s):

Pascal Püllmann ◽

Chris Ulpinnis ◽

Sylvestre Marillonnet ◽

Ramona Gruetzner ◽

Steffen Neumann ◽

...

Keyword(s):

Primer Design ◽

Saturation Mutagenesis ◽

Golden Gate ◽

Site Saturation ◽

Golden Gate Cloning

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Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia coli

mSystems ◽

10.1128/msystems.00232-20 ◽

2020 ◽

Vol 5 (2) ◽

Author(s):

Cong Chen ◽

Alaksh Choudhury ◽

Shuanghong Zhang ◽

Andrew D. Garst ◽

Xin Song ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Genome Engineering ◽

Transcriptomic Analysis ◽

Saturation Mutagenesis ◽

Receptor Protein ◽

Novel Mutations ◽

Content Type ◽

Global Regulators ◽

E Coli

ABSTRACT It is important to expedite our understanding of antibiotic resistance to address the increasing numbers of fatalities and environmental pollution due to the emergence of antibiotic resistance and multidrug-resistant strains. Here, we combined the CRISPR-enabled trackable genome engineering (CREATE) technology and transcriptomic analysis to investigate antibiotic tolerance in Escherichia coli. We developed rationally designed site saturation mutagenesis libraries targeting 23 global regulators to identify fitness-conferring mutations in response to diverse antibiotic stresses. We identified seven novel mutations that confer resistance to the ribosome-targeting antibiotics doxycycline, thiamphenicol, and gentamicin in E. coli. To the best of our knowledge, these mutations that we identified have not been reported previously during treatment with the indicated antibiotics. Transcriptome sequencing-based transcriptome analysis was further employed to evaluate the genome-wide changes in gene expression in E. coli for SoxR G121P and cAMP receptor protein (CRP) V140W reconstructions, and improved fitness in response to doxycycline and gentamicin was seen. In the case of doxycycline, we speculated that SoxR G121P significantly increased the expression of genes involved in carbohydrate metabolism and energy metabolism to promote cell growth for improved adaptation. In the CRP V140W mutant with improved gentamicin tolerance, the expression of several amino acid biosynthesis genes and fatty acid degradation genes was significantly changed, and these changes probably altered the cellular energy state to improve adaptation. These findings have important significance for understanding such nonspecific mechanisms of antibiotic resistance and developing new antibacterial drugs. IMPORTANCE The growing threat of antimicrobial resistance poses a serious threat to public health care and motivates efforts to understand the means by which resistance acquisition occurs and how this can be combatted. To address these challenges, we expedited the identification of novel mutations that enable complex phenotypic changes that result in improved tolerance to antibiotics by integrating CREATE and transcriptomic analysis of global regulators. The results give us a better understanding of the mechanisms of resistance to tetracycline antibiotics and aminoglycoside antibiotics and also indicate that the method may be used for quickly identifying resistance-related mutations.

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Engineered Cpf1 Enzymes with Altered PAM Specificities

10.1101/091611 ◽

2016 ◽

Cited By ~ 6

Author(s):

Linyi Gao ◽

David B.T. Cox ◽

Winston X. Yan ◽

John Manteiga ◽

Martin Schneider ◽

...

Keyword(s):

Genome Editing ◽

Genome Engineering ◽

Saturation Mutagenesis ◽

Promising Tool ◽

Genome Wide ◽

Protospacer Adjacent Motif ◽

Target Sites ◽

High Level ◽

Target Activity

SummaryThe RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1-5. Compared to other genome editing platforms, Cpf1 offers distinct advantages, such as the ability to easily target multiple genes simultaneously3, as well as low rates of off-target activity4, 5. However, the Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), which has been successfully harnessed for genome editing, can only robustly cleave target sites preceded by a TTTV protospacer adjacent motif (PAM), which may limit its practical utility. To address this limitation, we used a structure- guided saturation mutagenesis screen to increase the targeting range of Cpf1. We engineered two variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains. Together, these variants increase the targeting range of AsCpf1 to one cleavage site for every ~8.7 bp in non-repetitive regions of the human genome, providing a useful addition to the CRISPR/Cas genome engineering toolbox.

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