Feel the burn: a collection of stories on hot’n’sharp DNA engineering

ABSTRACT Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5′-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5′-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95°C and retained at least half the activity after 9 min at 95°C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of k cat, Km , DNA, and Km , AdoMet. The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn2+ but not by Mg2+, Ca2+, or Mn2+. These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.

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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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Improving selectivity of DNA-RNA binding zinc finger using directed evolution

10.21203/rs.2.10756/v1 ◽

2019 ◽

Author(s):

Agata Agnieszka Sulej

Keyword(s):

Directed Evolution ◽

Zinc Finger ◽

Rna Binding ◽

Zinc Fingers ◽

Amino Acid Residues ◽

Double Stranded Dna ◽

Dna Engineering ◽

Materials Used ◽

Specific Sequences ◽

Selection Of

Abstract Objective Type C2H2 zinc fingers bind a variety of substrates among which, are specific sequences in the double-stranded DNA. Engineering efforts led to the discovery of a set of general rules that enable obtaining zinc fingers modules that bind to almost any given sequence. The objective of this work was to determine an analogical set of rules for the binding of specific sequences in DNA-RNA hybrid using directed evolution of ZfQQR zinc finger. The target regions for evolution included the amino acid residues that directly interact with the substrate and linkers between modules of the zinc finger. Results The directed evolution was performed using selection based on biopanning of phage-displayed libraries of randomized regions in the ZfQQR zinc finger. The applied strategy of randomization of the middle module of the zinc finger along with input library bias and materials used for biopanning hindered the selection of modules with altered specificity. However, the directed evolution of the linker sequence between modules enabled selection of variants with improved selectivity towards DNA-RNA hybrids in the presence of double-stranded DNA in comparison to the original ZfQQR. This confirms the necessity of linker optimization between modules in zinc finger domains.

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Engineering Protein Activity into Off-the-Shelf DNA Devices

10.1101/2022.01.03.474821 ◽

2022 ◽

Author(s):

Harsimranjit Sekhon ◽

Stewart N Loh

Keyword(s):

Biological Activity ◽

Protein Engineering ◽

Dna Sequence ◽

Cell Phone ◽

Biological Functions ◽

Protein Activity ◽

Rna Sequence ◽

Protein Switch ◽

Single Protein ◽

Dna Engineering

DNA-based devices are relatively straightforward to design by virtue of their predictable folding, but they lack biological activity. Conversely, protein-based devices offer a myriad of biological functions but are much more difficult to design due to their complex folding. This study bridges the fields of DNA engineering and protein engineering to generate a protein switch that is activated by a specific DNA sequence. A single protein switch, engineered from nanoluciferase using the alternate frame folding mechanism and herein called nLuc-AFF, is paired with different DNA technologies to create a biosensor for a DNA or RNA sequence of choice, sensors for serotonin and ATP, and a computational device that processes two DNA inputs. nLuc-AFF is a genetically-encoded, ratiometric, blue/green-luminescent biosensor whose output can be quantified by cell phone camera. nLuc-AFF is not falsely activated by decoy DNA and it retains full ratiometric readout in 100 % serum. The design approach can be applied to other proteins and enzymes to convert them into DNA-activated switches.

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Infoculture Methods

Proceedings ◽

10.3390/proceedings2020047063 ◽

2020 ◽

Vol 47 (1) ◽

pp. 63

Author(s):

Bo Gao

Keyword(s):

Artificial Intelligence ◽

Big Data ◽

Industry Data ◽

Dna Engineering ◽

Engineering Information

In big data, we know that it is important to have enough data to analyze, but where can one get more data? How does one get useful information? My novel idea of “infoculture” uses new BIONICS to resolve the problem of how to produce information for the artificial intelligence (AI) industry. Data can be cultivated by humans, just like how humans cultivate fish or shrimp. In the field of infoculture, the network is like an ocean, and the information machine is the fish and shrimp. However, key problems within this field are DATA DNA engineering, information disease engineering, infoculture engineering, and the ecology of information. The Google self-driving car is an example of a cultivated fish in infoculture theory.

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