The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

http://stacks.cdc.gov/view/cdc/29957/cdc_29957_DS1.pdf

CRISPR-Cas systems for editing, regulating and targeting genomes

正しい方法がわかる臨床基本手技 : from the New England journal of medicine

EVidenceModeler (EVM) is presented as an automated eukaryotic gene structure annotation tool that reports eukaryotic gene structures as a weighted consensus of all available evidence. EVM, when combined with the Program to Assemble Spliced Alignments (PASA), yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms. Our experiments on both rice and human genome sequences demonstrate that EVM produces automated gene structure annotation approaching the quality of manual curation.

/pdf/automated-eukaryotic-gene-structure-annotation-using-3fo28wpira.pdf

Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments

In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation of pluripotent stem cells and genome engineering using IVT mRNA-encoded designer nucleases. This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.

/pdf/mrna-based-therapeutics-developing-a-new-class-of-drugs-3nmw5g8b9d.pdf

mRNA-based therapeutics — developing a new class of drugs

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.

/pdf/rs-1-enhances-crispr-cas9-and-talen-mediated-knock-in-3egird1n0h.pdf

RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency.

Rabbit models for the study of human atherosclerosis: from pathophysiological mechanisms to translational medicine.

Dear Editor,

Recently, zinc finger nuclease, transcription activator-like effector nuclease, and RNA-guided Cas9 endonuclease (Cas9) have emerged as powerful means for genome editing (Conklin, 2013; Gaj et al., 2013). These nucleases are efficient in generating double-strand breaks in the genome that can be repaired by error-prone nonhomologous end joining leading to a functional knockout (KO) of the targeted gene or used to integrate a DNA sequence at a specific locus through homologous recombination. Although the Cas9 system has been shown highly efficient in generating genetically engineered mice and rats (Li et al., 2013a, b; Wang et al., 2013), its feasibility in the rabbits still needs be determined. Here we report the use of Cas9 system to effectively generate targeted mutations in rabbit embryos and the production of KO rabbits.

The rabbit is a classic model animal species. It is useful for the study of many human diseases such as atherosclerosis, cystic fibrosis, and acquired immunodeficiency syndrome. However, production of gene targeted transgenic (GTT) rabbits has been an extreme challenge. This is mainly due to the lack of germline transmitting embryonic stem cells and the very low efficiency of somatic cell nuclear transfer in rabbits (Chesne et al., 2002).

In the present work, we used the Cas9 system to target the rabbit genome. Because embryo transfer work in large animal species is costly, we first established an in vitro system to test the efficacy of the RNA-guided Cas9 nucleases. Individual single guide RNAs (sgRNAs) were designed (Figure 1A and Supplementary Table S1) to target nine rabbit genes: apolipoprotein E (APOE), cluster of differentiation 36 (CD36), cystic fibrosis transmembrane conductance regulator, low-density lipoprotein receptor (LDLR), apolipoprotein CIII, scavenger receptor class B, member 1 (SCARB1), leptin, leptin receptor, and ryanodine receptor 2 (RyR2). For each gene, RNA mixture of Cas9 constructs (150 ng/μl Cas9 mRNA plus 6 ng/μl sgRNA) was microinjected into cytoplasm of pronuclear stage rabbit embryos (n = 290). We chose to use the concentration at 6 ng/μl for sgRNA because higher concentrations (12, 18, or 24 ng/μl) did not significantly improve the mutation rates (data not shown), and we speculate that higher quantity of sgRNA may increase the frequency of off-target events. Embryos were cultured in vitro, collected at blastocyst (BL) stage, and subjected to single embryo PCR and sequencing (n = 116) to identify mutations in the corresponding target locus. All nine sgRNAs generated mutations on their corresponding targeting loci with efficiencies ranging from 10% to nearly 100% (Figure 1D). High percentage (four out of nine) of the sgRNAs resulted in mutation rates higher than 50%; interestingly, bi-allelic mutations were also identified in these four, but not in those where mutation rates were 50% or lower (Figure 1D). 



Figure 1

Generation of KO rabbits with RNA-guided Cas9 nucleases. (A) Constructs of Cas9 RNA system used in this study. NLS, nuclear localization signal; bGH-pA, bovine growth hormone poly-A; PAM, protospacer adaptor motif. (B) Representative T7 endonuclease 1 ...



After validating the in vitro gene targeting capacity of these Cas9 constructs, we continued to use four of them (APOE, CD36, LDLR, and RYR2) to produce KO rabbits. CD36, LDLR, and APOE KO rabbits are useful to study lipid metabolisms and atherosclerosis. The fourth line (RyR2 KO) will be used as a model to study heart arrhythmia. This is in recognition of the increasing demand for non-murine models for cardiovascular diseases in the research community. Cardiac physiology of rabbit better mimics that of human in a number of key areas than mouse does (Fan and Watanabe, 2003). For example, cholesteryl ester transfer protein, which plays a central role in the atherosclerotic process, is abundant in both human and rabbit plasma but absent in the mouse. Like humans, rabbits are very susceptible to diet-induced atherosclerosis, whereas wild-type (WT) mice do not develop atherosclerosis naturally.

A total of 301 embryos were injected with one of the four Cas9 constructs and transferred to 10 pseudo-pregnant recipient rabbits (20–35 embryos per recipient). After 1 month gestation, nine (90%) recipients gave birth to 68 live kits (7.6/L), out of which 38 were identified as positive KO after initial T7 endonuclease assay and final confirmation by PCR sequencing (Figure 1B, C, and E). The term rate calculated as total term kits/total embryos is 22.6% (68/301). The KO rate calculated as total KO kits/total term is 55.9% (38/68). Consistent with the prediction based on the in vitro results, three (i.e. APOE, CD36, and RyR2) out of the four Cas9 constructs resulted in higher than 50% mutation rates and bi-allelic mutations. It remains to be tested whether mutations in these founder animals will faithfully transmit to the next generation. In agreement with previous reports, Δ15 mutant alleles were repeatedly discovered in six out of nine LDLR KO founder rabbits (Figure 1C, left lower panel), likely caused by microhomology-mediated end joining (Wang et al., 2013).

One main concern with the Cas9 system for gene targeting is the off-target effects (Fu et al., 2013; Hsu et al., 2013). Recently, Hsu et al. (2013) examined Cas9-induced off-target mutation events in human 293T and 293FT cells. They found that sgRNA can tolerate as much as 4 nt changes in the seed sequence, and that the change of the protospacer adaptor motif (PAM) sequence from NGG to NAG does not totally abolish the targeting capacity. Their work suggested that there may be up to hundreds of potential off-target loci in a mammalian genome for one particular Cas9 seed sequence. In the present work, we examined off-target effects in all the KO founders (LDLR, RYR2, CD36, and APOE), following similar strategies described by Wang et al. (2013) in their mouse work. We used BLASTn to identify exact match to the 15 nt sequence (12 nt seed region and 3 nt NGG). A total of 160 potential off-target loci were identified, of which 9 were within an exon region (Supplementary Table S2). Considering the fact that mutations in an exon region are more likely to cause gene mutation and phenotype changes in the animals, we looked at these nine loci (Supplementary Table S3). None of the 38 founders contained mutations in these exon regions.

This finding substantially alleviated the concerns of using Cas9-based gene targeting in rabbits. Notably, we used the most stringent way (i.e. exact match of 12 nt seed sequence plus the 3 nt NGG in the PAM), yet failed to identify any off-target events. Several factors may have contributed to the low frequency of off-target events. First, we used very low concentration of the sgRNA (6 ng/μl). In contrast, 12.5–50 ng/μl were used in similar mouse and rat studies (Li et al., 2013a; Wang et al., 2013). Secondly, we used RNA, whereas Hsu et al. (2013) used plasmid DNA. The much shorter half-life of RNAs may also reduce the off-target frequencies. Thirdly, we worked in an in vivo system of a different species, quite different from the in vitro system using immortalized human cell lines (e.g. 293T and 293FT) (Hsu et al., 2013). Consistent with our findings, recent reports of GTT mice and rats generated via Cas9 system also had very few detectable off-target mutations (Li et al., 2013b; Wang et al., 2013). Nevertheless, we admit that additional work may be necessary to examine all potential off-target loci. It is also noteworthy that the current assembly of rabbit genome is still incomplete. On the other hand, in the context of transgenic animal production, we argue that the initial focus could be narrowed to those that fall in an exon region, therefore making off-target examination feasible and affordable. Researchers who use these novel rabbit models in their future studies should however bear in mind that there may be mutations, although remotely, in other regions of the animal genome.

To our knowledge, our work represents the first report of successful generation of GTT rabbits using RNA-guided Cas9 nucleases. We used 10 donor rabbits to harvest embryos, transferred 301 embryos to 10 recipient rabbits, and generated 38 KO founders. On average, for a given targeted gene, five rabbits (donors and recipients) were used and nine founder KO kits were produced in as few as two months. Such high success rates and time efficiency make production of GTT rabbits technically and economically feasible for biomedical research. In the near future, Cas9 system will likely contribute to the generation of knock-in, multiplex GTT, conditional GTT rabbits too, as has been demonstrated in mice (Wang et al., 2013; Yang et al., 2013).

Taken together, we have established a highly effective Cas9-based system to produce KO rabbits. We achieved 100% success rates in all nine genes and generated four novel lines of KO rabbits (CD36, LDLR, RyR2, and APOE). Analysis on potential off-targets showed no effects in the Exon regions of KO rabbits, supporting the application of the Cas9 technology on generating GTT rabbits. Importantly, this success positions rabbits as a brand new animal model amendable to gene targeting in translational biomedical research arena.

[Supplementary material is available at Journal of Molecular Cell Biology online. This work was funded by grants from the National Institutes of Health (HL117491, HL088391, HL114038, and HL068878 to Y.E.C). This work utilized Core Services supported by Center for Advanced Models for Translational Sciences and Therapeutics (CAMTraST) at the University of Michigan Medical Center.]

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

Purpose: Recent studies have shown that the process of protein neddylation was abnormally activated in several human cancers. However, it is unknown whether and how UBE2F, a less characterized neddylation E2, regulates lung cancer cell survival, and whether and how NOXA, a proapoptotic protein, is ubiquitylated and degraded by which E3 and via which ubiquitin linkage.Experimental Design: Methods of immunohistochemistry and immunoblotting were utilized to examine UBE2F protein expression. The biological functions of UBE2F were evaluated by in vitro cell culture and in vivo xenograft models. The in vivo complex formation among UBE2F-SAG-CUL5-NOXA was measured by a pulldown assay. Polyubiquitylation of NOXA was evaluated by in vivo and in vitro ubiquitylation assays.Results: UBE2F is overexpressed in non-small cell lung cancer (NSCLC) and predicts poor patient survival. While UBE2F overexpression promotes lung cancer growth both in vitro and in vivo, UBE2F knockdown selectively inhibits tumor growth. By promoting CUL5 neddylation, UBE2F/SAG/CUL5 tri-complex activates CRL5 (Cullin-RING-ligase-5) to ubiquitylate NOXA via a novel K11, but not K48, linkage for targeted proteasomal degradation. CRL5 inactivation or forced expression of K11R ubiquitin mutant caused NOXA accumulation to induce apoptosis, which is rescued by NOXA knockdown. Notably, NOXA knockdown rescues the UBE2F silencing effect, indicating a causal role of NOXA in this process. In lung cancer tissues, high levels of UBE2F and CUL5 correlate with a low level of NOXA and poor patient survival.Conclusions: By ubiquitylating and degrading NOXA through activating CRL5, UBE2F selectively promotes lung cancer cell survival and could, therefore, serve as a novel cancer target. Clin Cancer Res; 23(4); 1104-16. ©2016 AACR.

/pdf/neddylation-e2-ube2f-promotes-the-survival-of-lung-cancer-35auexofuf.pdf

Neddylation E2 UBE2F Promotes the Survival of Lung Cancer Cells by Activating CRL5 to Degrade NOXA via the K11 Linkage.

CRISPR/Cas9 efficiently generates gene knock-out via nonhomologous end joining (NHEJ), but the efficiency of precise homology-directed repair (HDR) is substantially lower, especially in the hard-to-transfect human stem cells and primary cells. Herein we report a tube electroporation method that can effectively transfect human stem cells and primary cells with minimal cytotoxicity. When applied to genome editing using CRISPR/Cas9 along with single stranded DNA oligonucleotide (ssODN) template in human induced pluripotent stem cells (iPSCs), up to 42.1% HDR rate was achieved, drastically higher than many reported before. We demonstrated that the high HDR efficiency can be utilized to increase the gene ablation rate in cells relevant to clinical applications, by knocking-out β2-microglobulin (B2M) in primary human mesenchymal stem cells (MSCs, 37.3% to 80.2%), and programmed death-1 (PD-1) in primary human T cells (42.6% to 58.6%). Given the generality and efficiency, we expect that the method will have immediate impacts in cell research as well as immuno- and transplantation therapies.

/pdf/efficient-homology-directed-gene-editing-by-crispr-cas9-in-2f7cw2v3dp.pdf

Jie Xu

Papers

RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency.

Rabbit models for the study of human atherosclerosis: from pathophysiological mechanisms to translational medicine.

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

Neddylation E2 UBE2F Promotes the Survival of Lung Cancer Cells by Activating CRL5 to Degrade NOXA via the K11 Linkage.

Efficient homology-directed gene editing by CRISPR/Cas9 in human stem and primary cells using tube electroporation.