CRISPR/Cas9 screening using unique molecular identifiers

Loss-of-function screening by CRISPR/Cas9 gene knockout with pooled, lentiviral guide libraries is a widely applicable method for systematic identification of genes contributing to diverse cellular phenotypes. Here, Random Sequence Labels (RSLs) are incorporated into the guide library, which act as unique molecular identifiers (UMIs) to allow massively parallel lineage tracing and lineage dropout screening. RSLs greatly improve the reproducibility of results by increasing both the precision and the accuracy of screens. They reduce the number of cells needed to reach a set statistical power, or allow a more robust screen using the same number of cells.Peer reviewe

Reply to "CRISPR screens are feasible in TP53 wild-type cells"

Haapaniemi et al address the issues raised by Brown et al and discuss several differences between the analyses performed by the two groups.Non peer reviewe

CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response

Here, we report that genome editing by CRISPR-Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells, leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR-Cas9.Peer reviewe

Identifying Fishes through DNA Barcodes and Microarrays

Background: International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings: This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of ‘‘DNA barcoding’’ and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the ‘‘position of label’’ effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (.90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance: Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products

Reply to "CRISPR screens are feasible in TP53 wild-type cells".

Haapaniemi et al address the issues raised by Brown et al and discuss several differences between the analyses performed by the two groups

Inhibition of p53 improves CRISPR/Cas - mediated precision genome editing

AbstractWe report here that genome editing by CRISPR/Cas9 induces a p53-mediated DNA damage response and cell cycle arrest. Transient inhibition of p53 prevents this response, and increases the rate of homologous recombination more than five-fold. This provides a way to improve precision genome editing of normal cells, but warrants caution in using CRISPR for human therapies until the mechanism of the activation of p53 is elucidated.</jats:p

CRISPR/Cas9 screening using unique molecular identifiers

Loss of function screening by CRISPR/Cas9 gene knockout with pooled, lentiviral guide libraries is a widely applicable method for systematic identification of genes contributing to diverse cellular phenotypes. Here, random sequence labels (RSLs) are incorporated into the guide library, which act as unique molecular identifiers (UMIs) to allow massively parallel lineage tracing and lineage dropout screening. RSLs greatly improve the reproducibility of results by increasing both the precision and the accuracy of screens. They reduce the number of cells and sequencing reads needed to reach a set statistical power, or allow a more robust screen using the same number of cells.</jats:p

Abstract 2718: Genomic and microRNAome subtraction identifies pathogenic viral sequences in pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is by far the most common type of pancreatic cancer. It constitutes about 90% of tumors of the exocrine pancreas. The aggressive nature of PDAC along with its poor diagnostic markers contributes to a high lethality of this disease. Studies from other cancer entities implicate the role of viruses in tumor development. However, there have been no established reports about virus(es) associated with pancreatic cancer. The present study identified a virus by genomic and digital microRNAome subtraction between healthy and PDAC patients, suggesting that this virus may play a role in carcinogenesis. Experimental procedures: Representational difference analysis (RDA) was utilized to perform an experimental genome-wide search for DNA sequences that occur in PDAC but not in normal tissues. Ten healthy and ten PDAC tissue DNA samples were used for the generation of representations and subtractive hybridization. The difference products (DPs) so obtained were paired end sequenced on the Illumina MiSeq platform. The sequencing data were aligned against the viral RefSeq database. We also did in silico subtraction of microRNA content (miRNA-seq) of PDAC samples. Eight PDAC samples were analyzed for viral sequences using relevant RefSeq databases. The two approaches (RDA and miRNA-seq) were pursued in parallel. RT-qPCR analysis and the in vitro functional characterization of viral sequences in PDAC cell lines were respectively performed using hydrolysis probes and second generation microRNA mimic systems. Stable cell lines expressing viral microRNA and luciferase were produced using lentivirus for mice in vivo studies. Results and Summary: We found DNA sequences of a virus in the difference products of RDA whose signature was also implicated in the microRNA analysis. Further, using RT-qPCR, we identified that a particular microRNA from this virus is expressed at significantly higher levels in PDAC samples than in normal tissue. This observation was also verified by droplet digital PCR analysis. Non-metastatic PDAC cell lines overexpressing viral microRNA showed significant invasion against relevant controls, interestingly with no significant change in proliferation. We are currently analyzing the functional consequences of these viral sequences and their involvement in carcinogenesis. Conclusion: From two different robust approaches, these findings strongly indicate viral sequences associated with PDAC that could affect pathways relevant to tumor development. Citation Format: Mohanachary Amaravadi, Agnes Hotz-Wagenblatt, Sandeep Kumar Botla, Pouria Jandaghi, Mehdi Manoochehri, Nathalia Giese, Markus W. Büchler, Andrea S. Bauer, Jörg D. Hoheisel. Genomic and microRNAome subtraction identifies pathogenic viral sequences in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2718. doi:10.1158/1538-7445.AM2015-2718</jats:p