Dataset Information

Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays.

ABSTRACT: Engineered zinc-finger nucleases (ZFNs) form the basis of a broadly applicable method for targeted, efficient modification of eukaryotic genomes. In recent work, we described OPEN (oligomerized pool engineering), an 'open-source,' combinatorial selection-based method for engineering zinc-finger arrays that function well as ZFNs. We have also shown in direct comparisons that the OPEN method has a higher success rate than previously described 'modular-assembly' methods for engineering ZFNs. OPEN selections are carried out in Escherichia coli using a bacterial two-hybrid system and do not require specialized equipment. Here we provide a detailed protocol for carrying out OPEN to engineer zinc-finger arrays that have a high probability of functioning as ZFNs. Using OPEN, researchers can generate multiple, customized ZFNs in approximately 8 weeks.

SUBMITTER: Maeder ML

PROVIDER: S-EPMC2858690 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays.

Maeder Morgan L ML Thibodeau-Beganny Stacey S Sander Jeffry D JD Voytas Daniel F DF Joung J Keith JK

Nature protocols 20090917 10

Engineered zinc-finger nucleases (ZFNs) form the basis of a broadly applicable method for targeted, efficient modification of eukaryotic genomes. In recent work, we described OPEN (oligomerized pool engineering), an 'open-source,' combinatorial selection-based method for engineering zinc-finger arrays that function well as ZFNs. We have also shown in direct comparisons that the OPEN method has a higher success rate than previously described 'modular-assembly' methods for engineering ZFNs. OPEN s ...[more]

PMID: 19798082

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Project description:BackgroundPrecise and efficient methods for gene targeting are critical for detailed functional analysis of genomes and regulatory networks and for potentially improving the efficacy and safety of gene therapies. Oligomerized Pool ENgineering (OPEN) is a recently developed method for engineering C2H2 zinc finger proteins (ZFPs) designed to bind specific DNA sequences with high affinity and specificity in vivo. Because generation of ZFPs using OPEN requires considerable effort, a computational method for identifying the sites in any given gene that are most likely to be successfully targeted by this method is desirable.ResultsAnalysis of the base composition of experimentally validated ZFP target sites identified important constraints on the DNA sequence space that can be effectively targeted using OPEN. Using alternate encodings to represent ZFP target sites, we implemented Naïve Bayes and Support Vector Machine classifiers capable of distinguishing "active" targets, i.e., ZFP binding sites that can be targeted with a high rate of success, from those that are "inactive" or poor targets for ZFPs generated using current OPEN technologies. When evaluated using leave-one-out cross-validation on a dataset of 135 experimentally validated ZFP target sites, the best Naïve Bayes classifier, designated ZiFOpT, achieved overall accuracy of 87% and specificity+ of 90%, with an ROC AUC of 0.89. When challenged with a completely independent test set of 140 newly validated ZFP target sites, ZiFOpT performance was comparable in terms of overall accuracy (88%) and specificity+ (92%), but with reduced ROC AUC (0.77). Users can rank potentially active ZFP target sites using a confidence score derived from the posterior probability returned by ZiFOpT.ConclusionZiFOpT, a machine learning classifier trained to identify DNA sequences amenable for targeting by OPEN-generated zinc finger arrays, can guide users to target sites that are most likely to function successfully in vivo, substantially reducing the experimental effort required. ZiFOpT is freely available and incorporated in the Zinc Finger Targeter web server (http://bindr.gdcb.iastate.edu/ZiFiT).

Project description:BackgroundCustomized zinc finger nucleases (ZFNs) form the basis of a broadly applicable tool for highly efficient genome modification. ZFNs are artificial restriction endonucleases consisting of a non-specific nuclease domain fused to a zinc finger array which can be engineered to recognize specific DNA sequences of interest. Recent proof-of-principle experiments have shown that targeted knockout mutations can be efficiently generated in endogenous zebrafish genes via non-homologous end-joining-mediated repair of ZFN-induced DNA double-stranded breaks. The Zinc Finger Consortium, a group of academic laboratories committed to the development of engineered zinc finger technology, recently described the first rapid, highly effective, and publicly available method for engineering zinc finger arrays. The Consortium has previously used this new method (known as OPEN for Oligomerized Pool ENgineering) to generate high quality ZFN pairs that function in human and plant cells.Methodology/principal findingsHere we show that OPEN can also be used to generate ZFNs that function efficiently in zebrafish. Using OPEN, we successfully engineered ZFN pairs for five endogenous zebrafish genes: tfr2, dopamine transporter, telomerase, hif1aa, and gridlock. Each of these ZFN pairs induces targeted insertions and deletions with high efficiency at its endogenous gene target in somatic zebrafish cells. In addition, these mutations are transmitted through the germline with sufficiently high frequency such that only a small number of fish need to be screened to identify founders. Finally, in silico analysis demonstrates that one or more potential OPEN ZFN sites can be found within the first three coding exons of more than 25,000 different endogenous zebrafish gene transcripts.Conclusions and significanceIn summary, our study nearly triples the total number of endogenous zebrafish genes successfully modified using ZFNs (from three to eight) and suggests that OPEN provides a reliable method for introducing targeted mutations in nearly any zebrafish gene of interest.

Project description:BackgroundMany biological laboratories that deal with genomic samples are facing the problem of sample tracking, both for pure laboratory management and for efficiency. Our laboratory exploits PCR techniques and Next Generation Sequencing (NGS) methods to perform high-throughput integration site monitoring in different clinical trials and scientific projects. Because of the huge amount of samples that we process every year, which result in hundreds of millions of sequencing reads, we need to standardize data management and tracking systems, building up a scalable and flexible structure with web-based interfaces, which are usually called Laboratory Information Management System (LIMS).MethodsWe started collecting end-users' requirements, composed of desired functionalities of the system and Graphical User Interfaces (GUI), and then we evaluated available tools that could address our requirements, spanning from pure LIMS to Content Management Systems (CMS) up to enterprise information systems. Our analysis identified ADempiere ERP, an open source Enterprise Resource Planning written in Java J2EE, as the best software that also natively implements some highly desirable technological advances, such as the high usability and modularity that grants high use-case flexibility and software scalability for custom solutions.ResultsWe extended and customized ADempiere ERP to fulfil LIMS requirements and we developed adLIMS. It has been validated by our end-users verifying functionalities and GUIs through test cases for PCRs samples and pre-sequencing data and it is currently in use in our laboratories. adLIMS implements authorization and authentication policies, allowing multiple users management and roles definition that enables specific permissions, operations and data views to each user. For example, adLIMS allows creating sample sheets from stored data using available exporting operations. This simplicity and process standardization may avoid manual errors and information backtracking, features that are not granted using track recording on files or spreadsheets.ConclusionsadLIMS aims to combine sample tracking and data reporting features with higher accessibility and usability of GUIs, thus allowing time to be saved on doing repetitive laboratory tasks, and reducing errors with respect to manual data collection methods. Moreover, adLIMS implements automated data entry, exploiting sample data multiplexing and parallel/transactional processing. adLIMS is natively extensible to cope with laboratory automation through platform-dependent API interfaces, and could be extended to genomic facilities due to the ERP functionalities.

Dataset Information

Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays.

Publications

Oligomerized pool engineering (OPEN): an 'open-source' protocol for making customized zinc-finger arrays.

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