Table of Contents
Genome Editing
Programmable nucleases - including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system - enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing.
CRISPR/Cas9 sgRNA Design
CRISPR design
A web tool crafted to simplify the process of CRISPR guide selection in an input DNA sequence by (i) discovering possible offtargets genome-wide, (ii) highlighting guides with high target specificity, and (iii) flagging guides with numerous or genic offtargets in target genomes. The CRISPR design tool allows users to enter a 23-1000bp DNA sequence of interest and will find all SpCas9 target sites within the input sequence. The result will contain a rank ordered list of target sites based on predicted specificity.
Publications
- (Hsu et al., 2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol.
Institution(s): Broad Institute of MIT and Harvard, Cambridge, MA, USA; McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
GuideScan
Allows for the design of CRISPR guide RNA libraries that can be used to edit coding and noncoding genomic regions. GuideScan produces high-density sets of guide RNAs (gRNAs) for single- and paired-gRNA genome-wide screens. Rather than using an alignment tool, GuideScan uses a retrieval tree (trie) data structure, which efficiently and precisely enumerates all targetable sequences present in a given genome. Traversals of the trie allow for the computation of sequence mismatch neighborhoods, which are used to construct databases of gRNAs whose target sites are unique in the genome up to a user-defined number of mismatches. The GuideScan website allows users to input coordinates of genomic features in batch, to choose between designing single internal gRNAs or pairs of flanking gRNAs, and retrieve for each genomic coordinate a pre-defined number of gRNAs or gRNA pairs.
Publications
- (Perez, 2017) GuideScan software for improved single and paired CRISPR guide RNA design. Nat Biotechnol.
Institution(s)
Computational Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
E-CRISP
A software tool to design and evaluate target sites for use with the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system.
Publications
- (Heigwer et al., 2014) E-CRISP: fast CRISPR target site identification. Nat Methods.
Institution(s)
Division of Signaling and Functional Genomics, German Cancer Research Center (DKFZ), Heidelberg, Germany; Department of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany
CRISPRscan
A predictive single guide RNAs (sgRNA)-scoring algorithm that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Based on a large scale analysis of sgRNA mutagenesis activity in zebrafish, we established rules to predict sgRNA activity in vivo and build the CRISPRscan model integrating these rules. We independently validated with success our predictions using sgRNAs different from the large scale analysis.
Publications
- (Moreno-Mateos et al., 2015) CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods.
Institution(s)
Department of Genetics, Yale University School of Medicine, New Haven, CT, USA
TALEN design
TALE-NT
Enables design of custom TAL effector repeat arrays for desired targets and prediction of TAL effector binding sites, ranked by likelihood, in a genome, promoterome or other sequence of interest.
Publications:
- (Doyle et al., 2012) TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res.
Institutions(s)
Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA, USA
TALEN Targeter
Aids researchers in adopting TAL effector nucleases (TALENs) as gene editing tools. TALEN Targeter is a web application that identifies paired monomer (typically heteromeric) binding sites oriented 50 –30 on opposite strands of the DNA and separated by a spacer. It also allows users to specify a range for the spacer length, and all possible combinations of repeat numbers and spacer lengths are considered.
Publications:
- (Doyle et al., 2012) TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res.
Institutions(s)
Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA, USA; Department of Entomology, Iowa State University, Ames, IA, USA; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA
ZFN Design
Zinc Finger Tools
This site provides several tools for selecting zinc finger protein (ZFP) target sites and for designing the proteins that will target them.
Publications:
- (Mandell and Barbas CF 3rd, 2006) Zinc Finger Tools: custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res.
Institutions(s)
Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA
ZifNN
Predicts high throughput of optimal zinc finger protein for 9 bp DNA sequences of choice. ZifNN is inspired from an ensemble machine learning approach, and incorporates the predictions made by 100 parallel neural networks. It assumes synergistic mode of binding, thus capturing the molecular interactions between the DNA sequence and the zinc finger protein (ZFP) helices in greater detail. The tool is capable of domain adaptation, which allows it to make predictions about data points from a domain other than the one used for training the model.
Publications:
- (Dutta, 2016) An ensemble micro neural network approach for elucidating interactions between zinc finger proteins and their target DNA. BMC Genomics.
Institutions(s)
Department of Biochemical Engineering and Biotechnology
ZFN-Site
ZFN-Site
A web interface that searches multiple genomes for ZFN off-target sites.
Publications:
- (Cradick et al., 2011) ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites. BMC Bioinformatics.
Institutions(s)
University of Iowa School of Medicine, Department of Internal Medicine, Iowa City, IA, USA
CRISPR/Cas9 Screen Analysis
CREATE | CRISPR-enabled trackable genome engineering
Links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. CREATE combines automated design of CREATE cassettes (modular guide RNA-editing oligos), arraybased CREATE cassette synthesis, and sequencing in a streamlined workflow for genome engineering. CREATE was applied to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria.
Publications:
- (Garst et al., 2017) Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering. Nat Biotechnol.
Institution(s)
Department of Chemical and Biological Engineering, University of Colorado Boulder, Boulder, CO, USA; Muse Biotechnology Inc., Boulder, CO, USA
MAGeCK | Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout
Identifies positively and negatively selected sgRNAs and genes in genome-scale CRISPR/Cas9 knockout experiments. MAGeCK consists of four steps: read count normalization, mean-variance modeling, sgRNA ranking and gene ranking. The software results are robust across different sequencing depths and numbers of sgRNAs per gene. It is also able to perform both positive and negative selection screens simultaneously, and identify biologically meaningful and cell type-specific essential genes and pathways.
Publications:
- (Li et al., 2014) MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol.
- (Li et al., 2015) Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR. Genome Biol.
- (Wu et al., 2018) Reducing False Positives in CRISPR/Cas9 Screens from Copy Number Variations. BioRxiv.
Institutions(s):
Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, MA, USA;
MAGeCK-VISPR
Allows users to estimate the effects of gene knockouts (KO) in clustered regularly interspaced short palindromic repeats (CRISPR) screens. MAGeCK-VISPR consists of an algorithm named “MAGeCK-MLE” that contains the following functions: (1) define a set of quality control (QC) measurements; (2) extend the MAGeCK algorithm to call essential genes under multiple conditions while considering single guide RNA (sgRNA) knockout efficiency; and (3) provide a web-based visualization framework (VISPR) for interactive exploration of CRISPR screen QC and analysis results.
Publications:
- (Li et al., 2015) Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR. Genome Biol.
- (Wu et al., 2018) Reducing False Positives in CRISPR/Cas9 Screens from Copy Number Variations. BioRxiv.
Institutions(s):
Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Harvard T.H. Chan School of Public Health, Boston, MA, USA
caRpools | CRISPR-AnalyzeR for pooled screens
An R package for exploratory data analysis that provides a complete workflow to analyze CRISPR/Cas9 screens. To further support the analysis of large-scale screens, caRpools integrates screening documentation and generation of standardized analysis reports. It is designed to be user-friendly for novice and expert users: caRpools’ open virtual appliance allows analysis without prior programming knowledge.
Publications:
- (Winter et al., 2016) caRpools: an R package for exploratory data analysis and documentation of pooled CRISPR/Cas9 screens. Bioinformatics.
Institutions(s):
German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics and Heidelberg University, Heidelberg, Germany
Genome Editing Database
Genome edition uses the properties of bacterial clustered regularly interspaced short palindromic repeat (CRISPR) to elicit targeted genetic modifications in cultured cells, as well as in whole animals and plants. Genome editing databases gather CRISPR/cas9 screening experiments, target sequences, and more, to help design gene editing experiments.
Find Meganuclease/CRISPR/Cas9/TALEN/ZEN database in HERE