Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes.1–7. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a novel transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TAL) effector proteins8, 9. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effector proteins can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

Digital information is accumulating at an astounding rate, straining our ability to store and archive it. DNA is among the most dense and stable information media known. The development of new technologies in both DNA synthesis and sequencing make DNA an increasingly feasible digital storage medium. We developed a strategy to encode arbitrary digital information in DNA, wrote a 5.27-megabit book using DNA microchips, and read the book by using next-generation DNA sequencing.

The ability to direct functional domains to specific DNA sequences is a long sought-after goal for studying and engineering biological processes. Transcription activator like effectors (TALEs) from Xanthomonas sp. present a promising platform for designing sequence-specific DNA binding proteins. Here we describe a robust and rapid method for overcoming the difficulty of constructing TALE repeat domains. We synthesized 17 designer TALEs (dTALEs) that are customized to recognize specific DNA binding sites, and demonstrate that dTALEs can specifically modulate transcription of endogenous genes (Sox2 and Klf4) from the native genome in human cells. dTALEs provide a designable DNA targeting platform for the interrogation and engineering of biological systems.

Most amino acids are encoded by multiple codons, and codon choice has strong effects on protein expression. Rare codons are enriched at the N terminus of genes in most organisms, although the causes and effects of this bias are unclear. Here, we measure expression from >14,000 synthetic reporters in Escherichia coli and show that using N-terminal rare codons instead of common ones increases expression by ~14-fold (median 4-fold). We quantify how individual N-terminal codons affect expression and show that these effects shape the sequence of natural genes. Finally, we demonstrate that reduced RNA structure and not codon rarity itself is responsible for expression increases. Our observations resolve controversies over the roles of N-terminal codon bias and suggest a straightforward method for optimizing heterologous gene expression in bacteria.

For over 60 years, the synthetic production of new DNA sequences has helped researchers understand and engineer biology. Here we summarize methods and caveats for the de novo synthesis of DNA, with particular emphasis on recent technologies that allow for large-scale and low-cost production. In addition, we discuss emerging applications enabled by large-scale de novo DNA constructs, as well as the challenges and opportunities that lie ahead.

The inability to predict heterologous gene expression levels precisely hinders our ability to engineer biological systems. Using well-characterized regulatory elements offers a potential solution only if such elements behave predictably when combined. We synthesized 12,563 combinations of common promoters and ribosome binding sites and simultaneously measured DNA, RNA, and protein levels from the entire library. Using a simple model, we found that RNA and protein expression were within twofold of expected levels 80% and 64% of the time, respectively. The large dataset allowed quantitation of global effects, such as translation rate on mRNA stability and mRNA secondary structure on translation rate. However, the worst 5% of constructs deviated from prediction by 13-fold on average, which could hinder large-scale genetic engineering projects. The ease and scale this of approach indicates that rather than relying on prediction or standardization, we can screen synthetic libraries for desired behavior.next-generation sequencing | synthetic biology | systems biology O rganisms can be engineered to produce chemical, material, fuel, and medical products that are often superior to nonbiological alternatives (1). Biotechnologists have sought to discover, improve, and industrialize such products through the use of recombinant DNA technologies (2, 3). In recent years, these efforts have increased in complexity from expressing a few genes at once to optimizing multicomponent circuits and pathways (4-7). To attain desired systems-level function reliably, careful and time-consuming optimization of individual components is required (8-11).To mitigate this slow trial-and-error optimization, two dominant approaches have taken hold. The first approach seeks to predict expression levels by elucidating the biophysical relationships between sequence and function. For example, several groups have modified promoters (12, 13) and ribosome binding sites (RBSs) (14-16) to see how small sequence changes affect transcription or translation. Such studies are fundamentally challenging due to the vastness of sequence space. In addition, because these approaches mostly look at either transcription or translation individually, they are rarely able to investigate interactions between these processes.The second approach uses combinations of individually characterized elements to attain desired expression without directly considering their DNA sequences (17-25). Current efforts have focused on approaches to limit the number of time-consuming steps required to characterize potential interactions and on identifying existing or engineered elements that act predictably when used in combination (26-28). However, these studies still suggest there are enough idiosyncratic interactions and context effects that it will be necessary to construct and measure many variants of a circuit to achieve desired function (29). For larger circuits, such approaches are necessarily limited in scope due to the difficulty in measuring large numbers of combinations (26, 27...

Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol or sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.

Natural biological systems are selected by evolution to continue to exist and evolve. Evolution likely gives rise to complicated systems that are difficult to understand and manipulate. Here, we redesign the genome of a natural biological system, bacteriophage T7, in order to specify an engineered surrogate that, if viable, would be easier to study and extend. Our initial design goals were to physically separate and enable unique manipulation of primary genetic elements. Implicit in our design are the hypotheses that overlapping genetic elements are, in aggregate, nonessential for T7 viability and that our models for the functions encoded by elements are sufficient. To test our initial design, we replaced the left 11 515 base pairs (bp) of the 39 937 bp wild-type genome with 12179 bp of engineered DNA. The resulting chimeric genome encodes a viable bacteriophage that appears to maintain key features of the original while being simpler to model and easier to manipulate. The viability of our initial design suggests that the genomes encoding natural biological systems can be systematically redesigned and built anew in service of scientific understanding or human intention.