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NAR Top Articles - Synthetic Biology and Chemistry

Synthetic Biology and Chemistry

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May 2015

Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
DiCarlo, JE; Norville, JE; Mali, P; Rios, X; Aach, J; Church, GM
Nucleic Acids Res. 2013, 41, 4336-4343
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast.

CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences
Lin, YN; Cradick, TJ; Brown, MT; Deshmukh, H; Ranjan, P; Sarode, N; Wile, BM; Vertino, PM; Stewart, FJ; Bao, G
Nucleic Acids Res. 2014, 42, 7473-7485
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CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain insertions ('DNA bulge') or deletions ('RNA bulge') compared to the RNA guide strand, and Cas9 nickases used for paired nicking can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand...

Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice
Prakash, TP; Graham, MJ; Yu, JH; Carty, R; Low, A; Chappell, A; Schmidt, K; Zhao, CG; Aghajan, M; Murray, HF; Riney, S; Booten, SL; Murray, SF; Gaus, H; Crosby, J; Lima, WF; Guo, SL; Monia, BP; Swayze, EE; Seth, PP
Nucleic Acids Res. 2014, 42, 8796-8807
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Triantennary N-acetyl galactosamine (GalNAc, GN3), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6-10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2'-O-Et-2',4'-bridged nucleic acid) gapmer ASOs, similar to 60-fold enhancement in potency relative to the parent MOE (2'-O-methoxyethyl RNA) ASO was observed. GN3-conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3-ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3-ASO conjugate was detected in plasma suggesting that GN3 acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver...

Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice
Zhou, HB; Liu, B; Weeks, DP; Spalding, MH; Yang, B
Nucleic Acids Res. 2014, 42, 10903-10914
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The Cas9/sgRNA of the CRISPR/Cas system has emerged as a robust technology for targeted gene editing in various organisms, including plants, where Cas9/sgRNA-mediated small deletions/insertions at single cleavage sites have been reported in transient and stable transformations, although genetic transmission of edits has been reported only in Arabidopsis and rice. Large chromosomal excision between two remote nuclease-targeted loci has been reported only in a few non-plant species. Here we report in rice Cas9/sgRNA-induced large chromosomal segment deletions, the inheritance of genome edits in multiple generations and construction of a set of facile vectors for high-efficiency, multiplex gene targeting. Four sugar efflux transporter genes were modified in rice at high efficiency; the most efficient system yielding 87-100% editing in T0 transgenic plants, all with di-allelic edits. Furthermore, genetic crosses segregating Cas9/sgRNA transgenes away from edited genes yielded several genome-edited but transgene-free rice plants...

CRISPR/Cas9 systems targeting {beta}-globin and CCR5 genes have substantial off-target activity
Cradick, TJ; Fine, EJ; Antico, CJ; Bao, G
Nucleic Acids Res. 2013, 41, 9584-9592
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The ability to precisely modify endogenous genes can significantly facilitate biological studies and disease treatment, and the clustered regularly interspaced short palindromic repeats (CRISPR) systems have the potential to be powerful tools for genome engineering. However, the target specificity of CRISPR systems is largely unknown. Here we demonstrate that CRISPR/Cas9 systems targeting the human hemoglobin beta and C-C chemokine receptor type 5 genes have substantial off-target cleavage, especially within the hemoglobin delta and C-C chemokine receptor type 2 genes, respectively, causing gross chromosomal deletions. The guide strands of the CRISPR/Cas9 systems were designed to have a range of mismatches with the sequences of potential off-target sites. Off-target analysis was performed using the T7 endonuclease I mutation detection assay and Sanger sequencing. We found that the repair of the on-and off-target cleavage resulted in a wide variety of insertions, deletions and point mutations. Therefore, CRISPR/Cas9 systems need to be carefully designed to avoid potential off-target cleavage sites...

A platform for rapid prototyping of synthetic gene networks in mammalian cells
Duportet, X; Wroblewska, L; Guye, P; Li, YQ; Eyquem, J; Rieders, J; Rimchala, T; Batt, G; Weiss, R
Nucleic Acids Res. 2014, 42, 13440-13451
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Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi) gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries...

Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay
Lee, ME; Aswani, A; Han, AS; Tomlin, CJ; Dueber, JE
Nucleic Acids Res. 2013, 41, 10668-10678
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Engineered metabolic pathways often suffer from flux imbalances that can overburden the cell and accumulate intermediate metabolites, resulting in reduced product titers. One way to alleviate such imbalances is to adjust the expression levels of the constituent enzymes using a combinatorial expression library. Typically, this approach requires high-throughput assays, which are unfortunately unavailable for the vast majority of desirable target compounds. To address this, we applied regression modeling to enable expression optimization using only a small number of measurements. We characterized a set of constitutive promoters in Saccharomyces cerevisiae that spanned a wide range of expression and maintained their relative strengths irrespective of the coding sequence. We used a standardized assembly strategy to construct a combinatorial library and express for the first time in yeast the five-enzyme violacein biosynthetic pathway. We trained a regression model on a random sample comprising 3% of the total library, and then used that model to predict genotypes that would preferentially produce each of the products in this highly branched pathway...

CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages
Martel, B; Moineau, S
Nucleic Acids Res. 2014, 42, 9504-9513
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Bacteriophages are now widely recognized as major players in a wide variety of ecosystems. Novel genes are often identified in newly isolated phages as well as in environmental metavirome studies. Most of these novel viral genes have unknown functions but appear to be coding for small, non-structural proteins. To understand their biological role, very efficient genetic tools are required to modify them, especially in the genome of virulent phages. We first show that specific point mutations and large deletions can be engineered in the genome of the virulent phage 2972 using the Streptococcus thermophilus CRISPR-Cas Type II-A system as a selective pressure to increase recombination efficiencies. Of significance, all the plaques tested contained recombinant phages with the desired mutation. Furthermore, we show that the CRISPR-Cas engineering system can be used to efficiently introduce a functional methyltransferase gene into a virulent phage genome. Finally, synthetic CRISPR bacteriophage insensitive mutants were constructed by cloning a spacer-repeat unit in a low-copy vector illustrating the possibility to target multiple regions of the phage genome...

On the biophysics and kinetics of toehold-mediated DNA strand displacement
Srinivas, N; Ouldridge, TE; Sulc, P; Schaeffer, JM; Yurke, B; Louis, AA; Doye, JPK; Winfree, E
Nucleic Acids Res. 2013, 41, 10641-10658
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Dynamic DNA nanotechnology often uses toehold-mediated strand displacement for controlling reaction kinetics. Although the dependence of strand displacement kinetics on toehold length has been experimentally characterized and phenomenologically modeled, detailed biophysical understanding has remained elusive. Here, we study strand displacement at multiple levels of detail, using an intuitive model of a random walk on a 1D energy landscape, a secondary structure kinetics model with single base-pair steps and a coarse-grained molecular model that incorporates 3D geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Two factors explain the dependence of strand displacement kinetics on toehold length: (i) the physical process by which a single step of branch migration occurs is significantly slower than the fraying of a single base pair and (ii) initiating branch migration incurs a thermodynamic penalty, not captured by state-of-the-art nearest neighbor models of DNA, due to the additional overhang it engenders at the junction...

A split intein T7 RNA polymerase for transcriptional AND-logic
Schaerli, Y; Gili, M; Isalan, M
Nucleic Acids Res. 2014, 42, 12322-12328
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Synthetic biology has developed numerous parts for building synthetic gene circuits. However, few parts have been described for prokaryotes to integrate two signals at a promoter in an AND fashion, i.e. the promoter is only activated in the presence of both signals. Here we present a new part for this function: a split intein T7 RNA polymerase. We divide T7 RNA polymerase into two expression domains and fuse each to a split intein. Only when both domains are expressed does the split intein mediate protein trans-splicing, yielding a full-length T7 RNA polymerase that can transcribe genes via a T7 promoter. We demonstrate an AND gate with the new part: the signal-to-background ratio is very high, resulting in an almost digital signal. This has utility for more complex circuits and so we construct a band-pass filter in Escherichia coli. The split intein approach should be widely applicable for engineering artificial gene circuit parts.

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