Web-based design and analysis tools for CRISPR base editing

Background: As a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as a genome editing tool. Recently, CRISPR base editors, which consist of deactivated Cas9 (dCas9) or Cas9 nickase (nCas9) linked with a cytidine or a guanine deaminase, have been developed. Base editing tools will be very useful for gene correction because they can produce highly specific DNA substitutions without the introduction of any donor DNA, but dedicated web-based tools to facilitate the use of such tools have not yet been developed. Results: We present two web tools for base editors, named BE-Designer and BE-Analyzer. BE-Designer provides all possible base editor target sequences in a given input DNA sequence with useful information including potential off-target sites. BE-Analyzer, a tool for assessing base editing outcomes from next generation sequencing (NGS) data, provides information about mutations in a table and interactive graphs. Furthermore, because the tool runs client-side, large amounts of targeted deep sequencing data (< 1 GB) do not need to be uploaded to a server, substantially reducing running time and increasing data security. BE-Designer and BE-Analyzer can be freely accessed at http://www.rgenome.net/be-designer/ and http://www.rgenome.net/be-analyzer /, respectively. Conclusion: We develop two useful web tools to design target sequence (BE-Designer) and to analyze NGS data from experimental results (BE-Analyzer) for CRISPR base editors

Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases.

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs

Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites

In silico design and analysis of targeted genome editing with CRISPR

CRISPR/Cas systems have become a tool of choice for targeted genome engineering in recent years. Scientists around the world want to accelerate their research with the use of CRISPR/Cas systems, but are being slowed down by the need to understand the technology and computational steps needed for design and analysis. However, bioinformatics tools for the design and analysis of CRISPR experiments are being created to aid those scientists. For the design of CRISPR targeted genome editing experiments, CHOPCHOP has become one of the most cited and most used tools. After the initial publication of CHOPCHOP, our understanding of the CRISPR system underwent a scientific evolution. I therefore updated CHOPCHOP to accommodate the latest discoveries, such as designs for nickase and isoform targeting, machine learning algorithms for efficiency scoring and repair profile prediction, in addition to many others. On the other spectrum of genome engineering with CRISPR, there is a need for analysis of the data and validation of mutants. For the analysis of the CRISPR targeted genome editing experiments, I have created ampliCan, an R package that with the use of ‘editing aware’ alignment and automated normalization, performs precise estimation of editing efficiencies for thousands of CRISPR experiments. I have benchmarked ampliCan to display its strengths at handling a variety of editing indels, filtering out contaminant reads and performing HDR editing estimates. Both of these tools were developed with the idea that biologists without a deep understanding of CRISPR should be able to use them, and at the same time seasoned experts can adjust the settings for their purposes. I hope that these tools will facilitate adaptation of CRISPR systems for targeted genome editing and indirectly allow for great discoveries in the future

CRISPy-web:An online resource to design sgRNAs for CRISPR applications

CRISPR/Cas9-based genome editing has been one of the major achievements of molecular biology, allowing the targeted engineering of a wide range of genomes. The system originally evolved in prokaryotes as an adaptive immune system against bacteriophage infections. It now sees widespread application in genome engineering workflows, especially using the Streptococcus pyogenes endonuclease Cas9. To utilize Cas9, so-called single guide RNAs (sgRNAs) need to be designed for each target gene. While there are many tools available to design sgRNAs for the popular model organisms, only few tools that allow designing sgRNAs for non-model organisms exist. Here, we present CRISPy-web (http://crispy.secondarymetabolites.org/), an easy to use web tool based on CRISPy to design sgRNAs for any user-provided microbial genome. CRISPy-web allows researchers to interactively select a region of their genome of interest to scan for possible sgRNAs. After checks for potential off-target matches, the resulting sgRNA sequences are displayed graphically and can be exported to text files. All steps and information are accessible from a web browser without the requirement to install and use command line scripts

WU-CRISPR: Characteristics of functional guide RNAs for the CRISPR/Cas9 system

The CRISPR/Cas9 system has been rapidly adopted for genome editing. However, one major issue with this system is the lack of robust bioinformatics tools for design of single guide RNA (sgRNA), which determines the efficacy and specificity of genome editing. To address this pressing need, we analyze CRISPR RNA-seq data and identify many novel features that are characteristic of highly potent sgRNAs. These features are used to develop a bioinformatics tool for genome-wide design of sgRNAs with improved efficiency. These sgRNAs as well as the design tool are freely accessible via a web server, WU-CRISPR (http://crispr.wustl.edu). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13059-015-0784-0) contains supplementary material, which is available to authorized users