Precise DNA Cloning via PAMless CRISPR-SpRYgests

While restriction enzymes (REs) remain the gold-standard for manipulating DNA in vitro, they have notable drawbacks including a dependence on short binding motifs that constrain their ability to cleave DNA substrates. Here we overcome limitations of REs by developing an optimized molecular workflow that leverages the PAMless nature of a CRISPR-Cas enzyme named SpRY to cleave DNA at practically any sequence. Using SpRY for DNA digests (SpRYgests), we establish a method that permits the efficient cleavage of DNA substrates at any base pair. We demonstrate the effectiveness of SpRYgests using more than 130 gRNAs, illustrating the versatility of this approach to improve the precision of and simplify several cloning workflows, including those not possible with REs. We also optimize a rapid and simple one-pot gRNA synthesis protocol, which reduces cost and makes the overall SpRYgest workflow comparable to that of RE digests. Together, SpRYgests are straightforward to implement and can be utilized to improve a variety of DNA engineering applications.

Engineering Protein Activity into Off-the-Shelf DNA Devices

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.

Design and Assembly of DNA Molecules Using Multi-Objective Optimisation

Rapid engineering of biological systems is currently hindered by limited integration of manufacturing constraints into the design process, ultimately reducing the yield of many synthetic biology workflows. Here we tackle DNA engineering as a multi-objective optimization problem aiming at finding the best tradeoff between design requirements and manufacturing constraints. We developed a new open-source algorithm for DNA engineering, called Multi-Objective Optimisation algorithm for DNA Design and Assembly (MOODA), available as a Python and Anaconda package, as well as a Docker image. Experimental results show that our method provides near optimal constructs and scales linearly with design complexity, effectively paving the way to rational engineering of DNA molecules from genes to genomes.

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

In 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.

Infoculture Methods

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.

Design and assembly of DNA molecules using multi-objective optimisation

Rapid engineering of biological systems is currently hindered by limited integration of manufacturing constraints into the design process, ultimately limiting the yield of many synthetic biology workflows.Here we tackle DNA engineering as a multi-objective optimization problem aiming at finding the best tradeoff between design requirements and manufacturing constraints. We developed a new open-source algorithm for DNA engineering, called Multi-Objective Optimisation algorithm for DNA Design and Assembly (MOODA), available as a Python package and web application at http://mooda.stracquadaniolab.org.Experimental results show that our method provides near optimal constructs and scales linearly with design complexity, effectively paving the way to rational engineering of DNA molecules from genes to genomes.

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

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.

Stable mutants of restriction-deficient/modification-proficient Bacillus subtilis 168: hub strains for giant DNA engineering

Bacillus subtilis 168 has been explored as a platform for the synthesis and transmission of large DNA. Two inherent DNA incorporation systems, natural transformation and pLS20-based conjugation transfer, enable rapid handling of target DNA. Both systems are affected by the Bsu restriction–modification system that recognizes and cleaves unmethylated XhoI sites, limiting the choice of target DNA. We constructed B. subtilis 168 with stable mutation for restriction-deficient and modification-proficient (r−m+). It was demonstrated that the r−m+ strains can incorporate and transfer synthesized DNA with multiple XhoI sites. These should be of value as hub strains to integrate and disseminate giant DNA between B. subtilis 168 derivatives.