Bioinformatic Tools in CRISPR/Cas Platform

Ahmad, Aftab; Ashraf, Sidra; Majeed, Humera Naz; Aslam, Sabin; Aslam, Muhammad Aamir; Mubarik, Muhammad Salman; Munawar, Nayla

doi:10.1007/978-981-16-6305-5_3

Aftab Ahmad⁴,
Sidra Ashraf⁵,
Humera Naz Majeed⁶,
Sabin Aslam⁷,
Muhammad Aamir Aslam⁸,
Muhammad Salman Mubarik⁷ &
…
Nayla Munawar⁹

3150 Accesses
2 Citations

Abstract

CRISPR/Cas has emerged as a game-changing technology for genome editing with widespread applications ranging from human therapeutics to engineering bacterial genomes for beneficial purposes to editing plant genomes for agricultural purposes. Successful genome editing through CRISPR/Cas relies on two components: an appropriate Cas endonuclease and a 20-base-pair (bp), single-guide RNA (sgRNA). CRISPR/Cas is currently favored as a genome editing technique due to its simple design rules and efficient editing capabilities that do not necessarily involve adding any foreign DNA at the target site. Cas endonucleases can be programmed to target any site in the genome by changing the gRNA sequence, highlighting the importance of gRNA design for increased specificity and efficiency, and reduced off-targeting in CRISPR/Cas genome editing. The rapid rise in CRISPR/Cas genome editing and associated applications has led to the development of numerous computational tools for effective sgRNA design. In this chapter, we discuss the essentials of gRNA design and provide an overview of the design process. In addition to summarizing factors which affect gRNA specificity and CRISPR cleavage efficiency, we discuss predictions of target efficiency and off-target detection algorithms. Finally, we describe the application-specific (knockout, activation, repression, base editing, and RNA editing) requirements of gRNA design and different tools to facilitate gRNA design.

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Abbreviations

ABE:: Adenine base editor
BLESS:: Breaks labeling, enrichment on streptavidin
BWA:: Burrows-Wheeler aligner
CBE:: Cytosine base editor
CFD:: Cutting frequency determination
ChIP:: Chromatin immunoprecipitation
Cmr:: Cas module-RAMP
Cpf1:: CRISPR from Prevotella and Francisella 1
CRISPR/Cas:: Clustered regularly interspaced short palindromic repeats/CRISPR associated protein
CRISPRa:: CRISPR activation
CRISPRi:: CRISPR interference
CRISTA:: CRISPR target assessment
crRNA:: CRISPR RNA
DHS:: DNase I hypersensitivity
DSB:: Double-stranded break
GFP:: Green fluorescent protein
gRNA :: Guide RNA
HDR:: Homology-directed repair
HEK293:: Human embryonic kidney 293 cells
IDLV:: Integrase-deficient lentiviral vectors
KI:: Knock-in
KO:: Knockout
KRAB:: Krüppel-associated box
LSD1:: Lysine-specific demethylase 1
MMEJ:: Microhomology-mediated end joining
nCas9:: Cas9 nickase
NGS:: Next-generation sequencing
NHEJ:: Non-homologous end joining
PAM:: Protospacer adjacent motif
RAMP:: Repeat-associated mysterious proteins
RNP:: Ribonucleoprotein
sgRNA :: Single-guide RNA
SHERLOCK:: Specific high-sensitivity enzymatic reporter unlocking
TET1:: Ten-eleven translocation gene protein 1
TSS:: Transcription start site
WGE:: Wellcome Sanger Institute genome editing

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Authors and Affiliations

Department of Biochemistry/US-Pakistan Center for Advanced Studies in Agriculture and Food Security, University of Agriculture, Faisalabad, Pakistan
Aftab Ahmad
Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan
Sidra Ashraf
Department of Biochemistry, Government College Women University, Faisalabad, Pakistan
Humera Naz Majeed
Center for Advanced Studies in Agriculture and Food Security (CASAFS), University of Agriculture, Faisalabad, Pakistan
Sabin Aslam & Muhammad Salman Mubarik
Institute of Microbiology, University of Agriculture, Faisalabad, Pakistan
Muhammad Aamir Aslam
Department of Chemistry, College of Science, United Arab Emirates University, Al-Ain, UAE
Nayla Munawar

Authors

Aftab Ahmad
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Sidra Ashraf
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Humera Naz Majeed
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Sabin Aslam
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Muhammad Aamir Aslam
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Muhammad Salman Mubarik
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Nayla Munawar
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Correspondence to Nayla Munawar .

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Editors and Affiliations

Department of Biochemistry/US-Pakistan Center for Advanced Studies in Agriculture and Food Security, University of Agriculture, Faisalabad, Pakistan
Aftab Ahmad
Center of Agricultural Biochemistry and Biotechnology (CABB)/US-Pakistan Center for Advanced Studies in Agriculture and Food Security, University of Agriculture, Faisalabad, Pakistan
Sultan Habibullah Khan
Institute of Plant Breeding and Biotechnology, Muhammad Nawaz Sharif University of Agriculture, Multan, Pakistan
Zulqurnain Khan

Appendices

Appendix 1: List of Useful Bioinformatics Tools and Databases for Gene Modification Research

Tool	Description	Link
AlleleID	“AlleleID^® is a comprehensive desktop tool designed to address the challenges of bacterial identification, pathogen detection or species identification”	http://premierbiosoft.com/bacterial-identification/index.html
Array Designer 2	It is an Oligo and cDNA Microarray Design Software. “It designs probes for SNP detection, microarray gene expression and gene expression profiling. In addition, comprehensive support for tiling arrays and resequencing arrays is available”	https://array-designer.software.informer.com/4.3/
AutoPrime	Autoprime is a very useful software for designing Reverse Transcription Real Time PCR (Q-RT-PCR) primers that are specific to the exon-intron boundaries	http://www.autoprime.de/
Beacon Designer	“Beacon Designer™ automates the design of real time primers and probes”	http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID=1
Biocomputing Tutorials	The site harbors a number of biocomputational online tools (Cleaner, Translator, NetPlasmit, Aligner, PatSearch, etc. for nucleotide and protein sequences) and half a dozen of software	http://datascience.unm.edu/intro-to-biocomputing/
BioEdit	“BioEdit is a biological sequence alignment editor written for Windows 95/98/NT/2000/XP/7.” One can download and then work with the molecular sequences for alignment, restriction mapping, RNA analysis, translation, graphical viewing of electropherogram, etc.	https://bioedit.software.informer.com/
BLAST	Basic local alignment search tool, provided by NCBI	https://blast.ncbi.nlm.nih.gov/
Cas-Database	Cas-Database is a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9)	http://www.rgenome.net/cas-database/
Cas-Designer	A bulge-allowed quick guide-RNA designer for CRISPR/Cas-derived RGENs	http://www.rgenome.net/cas-designer/
CINEMA 2.1	CINEMA stands for Color INteractive Editor for Multiple Alignments. It is a free software for sequence alignment with color editor	https://cinemahdapkapp.com/download/
Click2Drug	“Click2Drug contains a comprehensive list of computer-aided drug design (CADD) software, databases and web services. These tools are classified according to their application field, trying to cover the whole drug design pipeline”	http://www.click2drug.org/
Clustal Omega	The latest form of Clustal alignment program. It is online and command-line based. The distinguishing feature of Clustal-omega is its scalability, as several thousands of medium to large-sized sequences can be aligned simultaneously. It will also make use of multiple processors, where present. In addition, the quality of alignments is superior to the previous versions. The algorithm uses seeded guide trees and HMM profile-profile progressive alignments	https://www.ebi.ac.uk/Tools/msa/clustalo/
Clustal W	A very popular site for pairwise and multiple sequence alignment. It runs on Windows, Linux/Unix, and Mac operating systems	https://www.genome.jp/tools/clustalw/
CLUSTAL X	Latest version of ClustalX 2.0 is provided by “Plate-Forme Bio-Informatique de Strasbourge,” along with detailed instructions (help) for operating ClustalX. Besides, this site also provides online tools (viz. Actin-Related Proteins Annotation server, EMBOSS, Gene Ontology Annotation, SAGE experiment parameters, GPAT, etc,) and database (SRS, BAliBase, InPACT), Documentation (tutorials to elucidate the parameters of Clustal, GCG, EMBOSS, Bioinformatics protocols, etc.)	http://www.clustal.org/clustal2/
CODEHOP	“The COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP) program is hosted by the Fred Hutchinson Cancer Research Center in Seattle, Washington and designs PCR (Polymerase Chain Reaction) primers from protein multiple-sequence alignments”	https://4virology.net/virology-ca-tools/j-codehop/
Comparative RNA Website and Project	The Comparative RNA Web (CRW) Site disseminates information about RNA structure and evolution that has been determined using comparative sequence analysis	http://www.rna.icmb.utexas.edu/
Computational Biology at ORNL	The Computational Biology and Bioinformatics Group of the Biosciences Division of Oak Ridge National Laboratory provides data and bioinformatics tools for prokaryotic and some eukaryotic genome and related analysis. The tools are “Gene Channel,” “Generation Microbial Gene Prediction System,” “Microbial Gene Prediction System Internet Linked,” “Genome Analysis Pipeline,” etc.	https://www.ornl.gov/group/cbb
Computational Resources for Drug Discovery	“CRDD (Computational Resources for Drug Discovery) is an important module of the in silico module of OSDD. The CRDD web portal provides computer resources related to drug discovery on a single platform. Following are major features of CRDD”	http://crdd.osdd.net/
Compute pl/Mw	The tool “compute pI/Mw is a tool which allows the computation of the theoretical pI (isoelectric point) and Mw (molecular weight) for a list of UniProt Knowledgebase (Swiss-Prot or TrEMBL) entries or for user entered sequences”	https://web.expasy.org/compute_pi/
COSMID	A Web-based tool for identifying and validating CRISPR/Cas Off-target sites	https://crispr.bme.gatech.edu/
CPHModels 3.2 Server	“CPHmodels 3.2 is a protein homology modeling server. The template recognition is based on profile-profile alignment guided by secondary structure and exposure predictions”	http://www.cbs.dtu.dk/services/CPHmodels/
CRISPR gRNA Design tool	CRISPR gRNA Design tool lets you design gRNA(s) to efficiently engineer your target and minimize off-target effects using ATUM Scoring Algorithms	https://www.dna20.com/eCommerce/cas9/input
CRISPR multitargeter	CRISPR MultiTargeter is a web-based tool for automatic searches of CRISPR guide RNA targets	http://www.multicrispr.net/
CRISPRdb	It enables the easy detection of CRISPR in locally produced data and consultation of CRISPRs present in the data base	http://crispr.u-psud.fr/crispr
CrisprGE	CrisprGE is a central hub of CRISPR-based genome editing	http://crdd.osdd.net/servers/crisprge/
CSIR Informatics Portal	This page is maintained by CSIR and harbors the software/tools developed for bioinformatics analysis	http://crdd.osdd.net/info/
DAVID v. 6.7	The Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 “provides a comprehensive set of functional annotation tools for investigators to understand biological meaning behind large list of genes”	https://david.ncifcrf.gov/
DeepView: SWISS PDBViewer v. 4.1	“Swiss-PdbViewer (aka DeepView) is an application that provides a user friendly interface allowing to analyze several proteins at the same time. The proteins can be superimposed in order to deduce structural alignments and compare their active sites or any other relevant parts. Amino acid mutations, H-bonds, angles and distances between atoms are easy to obtain thanks to the intuitive graphic and menu interface”	https://spdbv.vital-it.ch/download_prerelease.html
DNA/RNA GC Content Calculator	One can calculate the GC content of a nucleotide sequence	http://www.endmemo.com/bio/gc.php
Dotlet	Dotlet is a free online software used as a tool for diagonal plotting of sequences	https://myhits.sib.swiss/cgi-bin/dotlet
Dotplot(+)	Dot-plot(+) software is used to identify the overlapping portions of two sequences and to identify the repeats and inverted repeats of a particular sequence	http://bip.weizmann.ac.il/education/materials/gcg/dotplot.html
Dotter	Dotter is a graphical dotplot program for detailed comparison of two sequences. It runs on MAC, Linux, Sun solaris, and Windows OS	https://sonnhammer.sbc.su.se/Dotter.html
DRUG DESIGN APPS FOR SMART PHONE	A wonderful site that harbors a number of drug designing applications for smart mobiles	http://click2drug.org/directory_Mobile.php
Drug Designing	This webpage maintains several entries to drug designing. One can learn and make use of these software/links	https://www.hsls.pitt.edu/obrc/index.php?page=drugs_medical
Emboss Align	The European Molecular Biology Open Software Suite (EMBOSS) “is a free Open Source software analysis package specially developed for the needs of the molecular biology (e.g. EMBnet) user community.” Some of the applications are prophet (Gapped alignment for profiles), infoseq (Displays some simple information about sequences), water (Smith-Waterman local alignment), pepstats (Protein statistics), etc.	https://www.ebi.ac.uk/Tools/psa/emboss_needle/
Ensembl Genome Browser	“The Ensembl project produces genome databases for vertebrates and other eukaryotic species, and makes this information freely available online”	https://www.ensembl.org/
Ensembl Variant Effect Predictor	“This tool takes a list of variant positions and alleles, and predicts the effects of each of these on overlapping transcripts and regulatory regions annotated in Ensembl. The tool accepts substitutions, insertions and deletions as input”	https://www.ensembl.org/vep
E-RNAi	RNAi construct designer	http://e-rnai.org/
EsyPred3D	“ESyPred3D is an automated homology modeling program. The method gets the benefit of the increased alignment performances of an alignment strategy that uses neural networks”	https://www.unamur.be/sciences/biologie/urbm/bioinfo/esypred/
ExPASY Resource Portal	A resource portal supported by Expert Protein Analysis System and Swiss Institute of Bioinformatics for analyzing bioinformatics data	https://www.expasy.org/
Expasy-Translate tool	It is an online tool that “allows the translation of a nucleotide (DNA/RNA) sequence to a protein sequence”	https://web.expasy.org/translate/
Expert Protein Analysis System	“ExPASy is the SIB Bioinformatics Resource Portal which provides access to scientific databases and software tools (i.e., resources) in different areas of life sciences including proteomics, genomics, phylogeny, systems biology, population genetics, transcriptomics etc.”	https://www.expasy.org/
FASTA	This server is hosted by the University of Virginia, USA. It harbors a multiple online software for sequence (nucleic acid and amino acid) comparison, local and global alignment, hydropathy plotting, and protein secondary structure prediction	https://www.ebi.ac.uk/Tools/sss/fasta/
FastPCR	“FastPCR is an integrated tool for PCR primers or probe design, in silico PCR, oligonucleotide assembly and analyses, alignment and repeat searching.” This program can be downloaded and run on PCs	https://primerdigital.com/fastpcr.html
Galaxy Platform	“Galaxy is an open, web-based platform for data intensive biomedical research. Whether on the free public server or your own instance, you can perform, reproduce, and share complete analyses”	https://usegalaxy.org/
GAS	GAS is UNIX or DOS-based downloadable, command-line oriented “integrated computer program designed to automate and accelerate the acquisition and analysis of genomic data”	https://bioinformaticssoftwareandtools.co.in/bio_tools.php
Gel Compar II (Paid multimodule, stand-alone software)	It is a commercial product. “GelCompar II consists of the Basic Software and five modules: Cluster analysis, Identification & Libraries, Comparative Quantification and Polymorphism Analysis, Dimensioning techniques & Statistics, and Database Sharing Tools”	https://www.applied-maths.com/modules-and-features-gelcompar-ii
Gelcompar II V. 7.1	For analyzing 1D Gel	https://www.applied-maths.com/download/software
Gel-Quant software	The “Gel-Quant” software is used to analyze one-dimensional gel images. The gel image is saved in “bitmap” format, following electrophoresis and scanning the gel	http://biochemlabsolutions.com/GelQuantNET.html
GeneFisher	“GeneFisher is an interactive web-based program for designing degenerate primers.” The underlying assumption is “assumption that genes with related function from different organisms show high sequence similarity, degenerate primers can be designed from sequences of homologues genes.” This assumption “leads to isolation of genes in a target organism using multiple alignments of related genes from different organisms”	https://bio.tools/genefisher
GeneCopoeia	GeneCopoeia offers comprehensive tools for microRNA (miRNA) functional analysis so researchers can detect, express, validate, or knockdown microRNA of interest confidently. All known human, mouse, and rat microRNA in miRBase covered	https://www.genecopoeia.com/
geneid	“geneid is a program to predict genes in anonymous genomic sequences designed with a hierarchical structure”	https://genome.crg.cat/geneid.html
geneinfinity	This site contains description and links to various sites pertaining to Protein Secondary Structure. It is a hub for getting a quick look at several servers and metaservers that harbor databases and/or tools for prediction of protein secondary structures	http://www.geneinfinity.org/
GeneMark	GeneMark is a “family of gene prediction programs developed at Georgia Institute of Technology, Atlanta, Georgia, USA”	http://exon.gatech.edu/
Genome Bioinformatics Research Lab	The site harbors “geneid” program which is used to “predict genes, exons, splice sites and other signals along a DNA sequence.” This site is also hyperlinked with “Gene prediction on whole genome” which is a “precomputed whole genome prediction data sets”	https://corelabs.ku.edu/genomics-and-bioinformatics-core
Genome Tools	“The GenomeTools genome analysis system is a free collection of bioinformatics tools (in the realm of genome informatics) combined into a single binary named gt. It is based on a C library named “libgenometools” which consists of several modules”	http://genometools.org/
GenomePRIDE 1.0	“GenomePRIDE is primer design program that designs PCR primers or long oligos on an annotated sequence”	http://pride.molgen.mpg.de/genomepride.html
GENSCAN	GENSCAN is a freely available software used for “identification of complete gene structures in genomic DNA.” Genscan can be used “for predicting the locations and exon-intron structures of genes in genomic sequences from a variety of organisms”	http://hollywood.mit.edu/GENSCAN.html
Glimmer	Glimmer (Gene Locator and Interpolated Markov ModelER) is a system for finding genes in microbial DNA, especially the genomes of bacteria, archaea, and viruses. Glimmer uses interpolated Markov models (IMMs) to identify the coding regions and distinguish them from noncoding DNA	http://www.cbcb.umd.edu/software/glimmer/glimmer2.jun01.shtml
GreenGenes (16srRNA sequence Alignment)	The greengenes web application provides access to the current and comprehensive 16S rRNA gene sequence alignment for browsing, blasting, probing, and downloading. The data and tools presented by greengenes can assist the researcher in choosing phylogenetically specific probes, interpreting microarray results, and aligning/annotating novel sequences	https://www.ccg.unam.mx/~vinuesa/Using_the_GreenGenes_and_RDPII_servers.html
HHpred	Homology detection and structure prediction by HMM-HMM: used for sequence database searching and structure prediction. It is fast enough and more sensitive in finding remote homologs. HHpred performs pairwise comparison of profile hidden Markov models (HMMs). It can produce pairwise query-template sequence alignments, merged query-template multiple alignments and 3D structural models calculated by the MODELLER software from HHpred alignments	https://toolkit.tuebingen.mpg.de/tools/hhpred
HMMgene 1.1 web server	“HMMgene is a program for prediction of genes in anonymous DNA.” “The program predicts whole genes, so the predicted exons always splice correctly. It can predict several whole or partial genes in one sequence, so it can be used on whole cosmids or even longer sequences”	http://www.cbs.dtu.dk/services/HMMgene/hmmgene1_1.php
IDT Antisense Design	To synthesize antisense oligos for a specific target sequence of interest	https://www.idtdna.com/pages/products/functional-genomics/antisense-oligos
I-TASSER Online	I-TASSER is an online bioinformatics platform for predicting protein structure vis-à-vis function. It has been developed by Zhang Lab (University of Michigan). It has topped in the CASP ranking of structure prediction during the years 2007–2010	https://zhanglab.ccmb.med.umich.edu/I-TASSER/
JALVIEW	It is a “multiple alignment editor written in Java.” It is used in EBI Clustalw, Pfam protein domain database; however, it is “available as a general purpose alignment editor and analysis workbench”	https://www.jalview.org/
LALIGN	Online free tool for finding local alignment between two sequences (provided in stipulated input format, viz. plain text without header line, Swiss-Prot ID, TrEMBL ID, EMBL ID, EST ID, etc.)	https://embnet.vital-it.ch/software/LALIGN_form.html
LAMP Designer	“LAMP Designer designs efficient primers for Loop-Mediated Isothermal Amplification assays, that amplify DNA and RNA sequences at isothermal conditions, eliminating the necessity of a PCR setup”	https://primerexplorer.jp/e/
MACAW	This link enables you to download Multiple Alignment Construction and Analysis Workbench (MACAW) software. This program is used for “locating, analyzing, and editing blocks of localized sequence similarity among multiple sequences and linking them into a multiple alignment”	http://en.bio-soft.net/format/MACAW.html
MAFFT version 6	“MAFFT is a multiple sequence alignment program for unix-like operating systems. It offers a range of multiple alignment methods, L-INS-i (accurate; for alignment of <~200 sequences), FFT-NS-2 (fast; for alignment of <~10,000 sequences)”	https://mafft.cbrc.jp/alignment/software/
Mapper	Java platform-based online software to map the RE sites on a target sequence	http://www.restrictionmapper.org/
Meth Primer	“MethPrimer is a program for designing bisulfite-conversion-based Methylation PCR Primers”	https://www.urogene.org/methprimer/
MethPrimer	It is a very useful site for designing primers for methylation PCR (Denatured, single-stranded DNA (ssDNA) is modified with sodium bisulfite “followed by PCR amplification using two pairs of primers, with one pair specific for methylated DNA; the other unmethylated DNA”)	https://www.urogene.org/methprimer/
mgene	“mGene is a computational tool for the genome-wide prediction of protein coding genes from eukaryotic DNA sequences”	http://mgene.org/
miRNa Body map (Human)	The microRNA body map is a repository of RT-qPCR miRNA expression data and functional miRNA annotation in normal and diseased human tissues	https://sites.google.com/site/mirnatools/mirna-databases
miRNA Target Gene Prediction	This website provides access to 2003 and 2005 miRNA-Target predictions for Drosophila miRNAs	http://www.mirbase.org/help/targets.shtml
miRNA Targets and Expression db	Predicted microRNA targets and target downregulation scores. Experimentally observed expression patterns	http://mirdb.org/
miRNAMap	miRNAMap 2.0 is a collection of “experimental verified microRNAs and experimental verified miRNA target genes in human, mouse, rat, and other metazoan genomes”	http://mirnamap.mbc.nctu.edu.tw/
Mobyle 1.5	This site maintains a number of online bioinformatics programs (assembly, database, display, hmm, phylogeny, protein, sequence, structure, etc.), workflows (alignment, db, phylogeny), and tutorial	http://www.mybiosoftware.com/mobyle-1-0-4-integration-bioinformatics-software-databanks.html
Modbase	It is a database for “comparative protein structure models.” The pipeline used is ModPipe	https://modbase.compbio.ucsf.edu/
MODELLER	The homology modeling of Protein 3D structures can be done using downloadable software “MODELLER.” It can also be used for the following protein structure-based applications: databases search for amino acid sequences, sequence and structural alignments clustering, de novo modeling of structural loops, model-optimization against user-defined objective function, and so on	https://salilab.org/modeller/
Mol. Modelling Database (MMDB)	It harbors “experimentally resolved structures of proteins, RNA, and DNA, derived from the Protein Data Bank (PDB), with value-added features such as explicit chemical graphs, computationally identified 3D domains (compact substructures) that are used to identify similar 3D structures, as well as links to literature, similar sequences, information about chemicals bound to the structures”	https://www.ncbi.nlm.nih.gov/Structure/MMDB/mmdb.shtml
Molecular Evolution Genetics Analysis (v. 5.1 beta)	A handy package for analyzing sequence data for pair-wise and multiple sequence alignment, phylogenetic tree (include neighbor-joining, maximum parsimony, UPGMA, maximum likelihood and minimum evolution based) construction, and estimation of evolutionary parameters	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203626/
MS Utils	Maintains links to several platforms, pipelines, libraries, software for visualization as well as software for proteomic data analysis	https://ms-utils.org/
NEB Cutter	This software is RE site mapper, hosted by New England Biolabs	http://nc2.neb.com/NEBcutter2/
NetPrimer	It is an efficient primer analysis software that can be used for determining the features of the secondary structures of the generated primer sequences	http://www.premierbiosoft.com/netprimer/
NRSP-8 Bioinformatics Online Tools	Explores and utilizes several bioinformatics tools	https://www.animalgenome.org/
Oligo Analyzer Version 3.1 (IDT)	The secondary structures produced by the primer(s) can be checked, and the Gibbs free energy required to produce these structures can be calculated using online Oligo Analyzer Version 3.1 (of IDT)	https://www.idtdna.com/pages/tools/oligoanalyzer
Oligo Tm Determination	Calculates the melting temperature of the oligos	https://worldwide.promega.com/resources/tools/biomath/tm-calculator/
Oligo.Net	“OLIGO Primer Analysis Software is the essential tool for designing and analyzing sequencing and PCR primers, synthetic genes, and various kinds of probes including siRNA and molecular beacons. Based on the most up-to-date nearest neighbor thermodynamic data, Oligo’s search algorithms find optimal primers for PCR, including TaqMan, highly multiplexed, consensus or degenerate primers. Multiple file batch processing is possible. It is also an invaluable tool for site directed mutagenesis”	https://www.oligo.net/
Oligonucleotide Properties Calculator	Calculates base-count, thermodynamic properties (ΔS and ΔH), Tm, and GC% values of a given oligo	http://biotools.nubic.northwestern.edu/
Oligos 6.2	“The program helps to design primer combinations given one fixed primer”	https://www.oligo.net/
ORF Finder	“The ORF Finder (Open Reading Frame Finder) is a graphical analysis tool which finds all open reading frames of a selectable minimum size in a user’s sequence or in a sequence already in the database”	https://www.ncbi.nlm.nih.gov/orffinder/
PCR PRIMER DESIGN AND REACTION OPTIMISATION	It is a very useful site to learn about the pros and cons of factors affecting PCR	http://www.mcb.uct.ac.za/mcb/resources/pcr/primer
PEDANT	“The pedant genome database provides exhaustive automatic analysis of genomic sequences by a large variety of bioinformatics tools”	http://pedant.gsf.de/
Peptide Mass	This online tool of ExPASy “PeptideMass cleaves a protein sequence from the UniProt Knowledgebase (Swiss-Prot and TrEMBL) or a user-entered protein sequence with a chosen enzyme, and computes the masses of the generated peptides. The tool also returns theoretical isoelectric point and mass values for the protein of interest”	https://web.expasy.org/peptide_mass/
Phylogeny Inference Package	“PHYLIP is a free package of programs for inferring phylogenies. It is distributed as source code, documentation files, and a number of different types of executables”	https://evolution.genetics.washington.edu/phylip.html
PHYRE2	Protein Homology/AnalogY Recognition Engine (PHYRE) is a non-commercial, very popular online protein structure prediction (homology modeling) server. The user friendly GUI is very helpful for the novice in the field of protein structure prediction	http://www.sbg.bio.ic.ac.uk/phyre2
Prediction of miRNA Targets (Mammals)	The tool “searches for predicted microRNA targets in mammals”	http://www.targetscan.org/
Primer Premier	Primer Premier is one of the “most comprehensive software to design and analyze PCR primers.” Primers can be designed for standard PCR, SNP genotyping assays, multiplexing assays, along with checking the secondary structures of the designed primers	http://www.premierbiosoft.com/primerdesign/
Primer3 (version 0.4.0)	It is a freely available online software for designing primers and probe from a DNA sequence. It is a very popular software due to availability of several parameters to design primers with high specificity and accuracy	http://bioinfo.ut.ee/primer3-0.4.0/
PrimerBLAST	Extensively used for designing primer and checking the specificity of a given primer	https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi
PrimerQuest	Online primer designing tool provided by IDT	https://www.idtdna.com/primerquest/home/index
Primo Degenerate3.4	“Primo Degenerate 3.4 designs PCR primers based on a single peptide sequence or multiple alignments of proteins or nucleotides. For degenerate primers, the probability of binding to the target is proportional to the effective concentration of the specific primer”	http://www.changbioscience.com/primo/primo.html
Primo Pro 3.2	It is another online primer designing software. Its notable feature is that it can reduce background noise by exercising check on mispriming on non-target DNA sequence. It also “introduces a batch mode option for high throughput PCR primer design”	http://www.changbioscience.com/primo/dihowto.html
Primo Pro 3.4	A java-enabled online primer designing tool	http://www.changbioscience.com/primo/primo.html
Promoter 2.0 Prediction Server	Promoter2.0 predicts transcription start sites of vertebrate PolII promoters in DNA sequences. It has been developed as an evolution of simulated transcription factors that interact with sequences in promoter regions. It builds on principles that are common to neural networks and genetic algorithms	http://www.cbs.dtu.dk/services/Promoter/
PROMOTERS & TERMINATORS	This site maintains links for different software and tools (viz. PromScan, SCOPE, Promoser, Arnold, WebGesTer) for scanning, predicting promoters and transcription terminators in Eukaryotes and Prokaryotes	https://molbiol-tools.ca/Promoters.htm
Protein Data Bank	PDB is an “information portal to biological macromolecular structure.” “The PDB archive contains information about experimentally-determined structures of proteins, nucleic acids, and complex assemblies”	https://www.rcsb.org/
Protein Tertiary Structure	This site contains links to several software for “calculating and displaying the 3-D structure of oligosaccharides and proteins. With the two protein analysis sites the query protein is compared with existing protein structures as revealed through homology analysis”	https://molbiol-tools.ca/Protein_tertiary_structure.htm
ProtParam	“ProtParam is a tool which allows the computation of various physical and chemical parameters for a given protein stored in Swiss-Prot or TrEMBL or for a user entered sequence. The computed parameters include the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (GRAVY)”	https://web.expasy.org/protparam/
Protscale	“ProtScale allows you to compute and represent the profile produced by any amino acid scale on a selected protein”	https://web.expasy.org/protscale/
QUARK Online	It is online software that applies QUARK algorithm for ab initio protein folding vis-à-vis structure prediction. It is another eminent online tool of Zhang lab that has secured esteemed ranking in CASP	https://zhanglab.ccmb.med.umich.edu/QUARK/
RaptorX	Another efficient protein structure prediction server that predicts the secondary and 3D protein structure. Besides, it also predicts solvent accessibility and disordered regions, and assigns the following confidence scores to indicate the quality of a predicted 3D model. It has been developed by Xu Group of Toyota Technological Institute at Chicago. RaptorX-Binding, another tool available in the homepage of RaptorX, is used for model-assisted protein binding site prediction	http://raptorx.uchicago.edu/
RASMOL	RasMol is a molecular visualization tool for protein in 3-dimension	http://www.openrasmol.org/
RASMOL Home page	“This site is provided for the convenience of users of RasMol and developers of open source versions of RasMol”	http://www.openrasmol.org/
RE specific primer designing	“PCR Designer for Restriction Analysis of Sequence Mutations”
ReadSeq-Sequence Format Conversion Tool	Online tool for conversion of sequence format	https://www.ebi.ac.uk/Tools/sfc/readseq/
RestrictionMapper	Online, freely available tool for mapping restriction endonuclease sites on a DNA sequence	http://www.restrictionmapper.org/
RNAfold	The RNAfold web server will predict secondary structures of single-stranded RNA or DNA sequences. Current limits are 7500 nt for partition function calculations and 10,000 nt for minimum free energy only predictions	http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi
RNAhybrid	RNAhybrid is a tool for finding the minimum free energy hybridization of a long and a short RNA	https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/
RNAi Atlas	RNAiAtlas provides a siRNA oligonucleotide data from different sources and companies like Dharmacon (ThermoFisher), Qiagen, and Ambion, esiRNA for humans, and visualizes interactions between siRNA oligo and predicted off-target	https://www.hsls.pitt.edu/obrc/index.php?page=rna_interference
RNAi Explorer-GeneLink-siRNA	A designing tool for siRNA	https://www.genelink.com/sirna/RNAicustomorder.asp
Robetta	Robetta (Beta Version) of Baker Lab, Washington, USA, is a full-chain protein structure prediction tool. It can be used both for ab initio and comparative approaches for protein structure prediction	https://robetta.bakerlab.org/
SANBI Tools	An array of online tools (dPORE-miRNA, TcoF, PROMEX, etc.) are available which are maintained by South African National Bioinformatics Institute	https://www.sanbi.org/resources/infobases/some-tools-developed-in-sanbi-for-use-in-biodiversity-research/
SDSC Biology Workbench	“The Biology WorkBench is a web-based tool for biologists. The WorkBench allows biologists to search many popular protein and nucleic acid sequence databases. Database searching is integrated with access to a wide variety of analysis and modeling tools, all within a point and click interface that eliminates file format compatibility problems”	http://workbench.sdsc.edu/
Secondary Structure Prediction Tools	“These are a collection of protein secondary structure analysis and information sites”	http://www.compbio.dundee.ac.uk/jpred/
Sequence Manipulation Suite-2	A suite available for almost all possible manipulation work that can be done with a given DNA or amino acid sequence, viz. Format change, Sequence splitting, CpG island detection, ORF finding, Pair-wise alignment, RE-Digestion, in silico mutation, etc.	https://www.bioinformatics.org/sms2/
sgRNA Designer	This tool ranks and picks candidate CRISPRko sgRNA sequences for the targets provided, while attempting to maximize on-target activity and minimizing off-target activity	http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design
sgRNAcas9	The BiooTools (Biological Online Tools) website is devoted to provide services to assist researchers design specific and efficient CRISPR sgRNA, primer pairs for detecting small ncRNA expression, such as miRNA, piRNA, and siRNA	http://www.biootools.com/
SIDDbase 1.0a.ws1	“SIDDbase-WS is a SOAP based Web Service” that “provides interoperable access to the SIDD software, and access to the repository of stored results from calculations previously performed on complete bacterial genomes”	https://bioinformaticssoftwareandtools.co.in/bio_tools.php
siDesign-Thermo Scientific	The siDESIGN Center is an advanced, user-friendly siRNA design tool, which significantly improves the likelihood of identifying functional siRNA. One-of-a-kind options are available to enhance target specificity and adapt siRNA designs for more sophisticated experimental design	http://www.thermofisher.com/order/genome-database/browse/sirna/keyword/siDESIGN+center
SIM4	A stand-alone program designed to run on Unix-based system. It is used for aligning an expressed DNA sequence with a genomic sequence, allowing for introns	http://nebc.nox.ac.uk/bioinformatics/docs/sim4.html
SIMPA96 Secondary Structure Prediction	An online tool to predict secondary structure of protein	https://npsa-prabi.ibcp.fr/NPSA/npsa_simpa96.html
SimVector	It is “an exceptional tool for drawing publication and vector catalog quality plasmid maps, carrying out restriction analysis and designing cloning experiments”	https://simvector.net/
siRNA Design: How to	A short introduction to siRNA Designing Steps	https://www.rnaiweb.com/RNAi/siRNA_Design/
siRNA Designing-BOCK-iT RNAi Designer	Online siRNA designing tool from Invitrogen	https://rnaidesigner.thermofisher.com/
siRNA Wizard v. 3.1	InvivoGen’s siRNA Wizard™ is a software designed to help you select siRNA/shRNA sequences targeting your gene(s) of interest. This program selects siRNA/shRNA sequences that match criteria suggested by studies of RNA interference and which will have the best expression rate in psiRNA vectors	https://www.invivogen.com/sirnawizard/
SOPMA	It is an online protein Secondary structure prediction tool	https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html
Splice Predictors	A method to identify potential splice sites in (plant) pre-mRNA by sequence inspection using Bayesian statistical models	http://www.phenosystems.com/www/index.php/links-to-various-tools-and-information/splice-prediction-tools
Statistical Analysis of Protein Sequences (SAPS)	It performs several statistical analysis of the physiochemical properties and other features of the protein sequence, viz. compositional analysis, charge distributional analysis, distribution of other amino acid types, repetitive structures, multiplets, periodicity analysis	https://www.ebi.ac.uk/Tools/seqstats/saps/
Structural Bioinformatics Group	This is the structural bioinformatics-related page maintained by Imperial College London. This site can be used for several purposes, viz. “analysis of protein structure and function with the aim of deriving evolutionary insights, modelling and comparison of biology networks to provide insights into Systems Biology, modelling of the activity and toxicity of small molecules as an aid to the design of novel drugs”	http://bioinformatics.charite.de/
Structural Biology Software Database	Harbors links to several software for docking	https://www.ks.uiuc.edu/Development/biosoftdb/
Swiss Institute of Bioinformatics	“The SIB Swiss Institute of Bioinformatics is an academic, non-profit foundation recognised of public utility.” SIB “provides high quality bioinformatics services to the national and international research community”	https://www.sib.swiss/
T-coffee	Tree-based Consistency Objective Function For alignment Evaluation (T-Coffee) is another popular multiple sequence alignment program, developed by Cedric Notredame, CRG Centro de Regulacio Genomica (Barcelona). It allows combining results obtained from several alignment methods. The URL is http://www.ebi.ac.uk/Tools/msa/tcoffee/. The default output format is Clustal, while it accepts sequences in PIR and FASTA format	https://www.ebi.ac.uk/Tools/msa/tcoffee/
The PCR Suite	It is an online primer designing software, hosted by UCSC, that allows users to design primers specific to various types of templates, viz. overlapping amplicons on a template, primers around SNP (in a GenBank), primers flanking exons and cDNA	http://pcrsuite.cse.ucsc.edu/
Translate a DNA Sequence	It is a Java-based free online software, to translate a given input DNA sequences and display one (at a time) of the six possible reading frames according to the selection made by the user. It also displays the graphical output for all the six reading frames together	https://web.expasy.org/translate/
UCSC Human Genome Browser	It is an interactive genome browser dedicated to human genome sequence	https://genome.ucsc.edu/
UnaFold	The likelihood of secondary structure formation by the single-stranded target is checked by UnaFold software of IDT (freely available online)	http://unafold.rna.albany.edu/
Uniprime2	It is a website for universal primer designing	https://bio.tools/uniprime2
User:Jarle Pahr/: Bioinformatics	This page harbors several “links and notes regarding bioinformatics.” This is a very useful link since a user can get link to almost all aspects of bioinformatics resources	https://openwetware.org/wiki/User:Jarle_Pahr/Bioinformatics
VBI resources	This site of Virginia Bioinformatics Institute maintains several tools for bioinformatics analysis, viz. “Analysis of Dynamic Algebraic Models,” “Complex Pathway Simulator,” “Genome Reverse Compiler,” etc.	https://www.thevillagefamily.org/content/vbi-resources
VLS3D	This page maintains a “list of in silico drug design online services, standalone and related databases. It is maintained by Dr. B. Villoutreix, research director at the French National Medical Research Institute (Inserm)”	https://www.vls3d.com/
Web Primer	A simple tool for primer designing for PCR or sequencing	http://www.candidagenome.org/cgi-bin/compute/web-primer
Webcutter 2.0	Another RE site detection software (online, free) for linear and circular DNA	https://www.hsls.pitt.edu/obrc/index.php?page=URL1043859576
Webgene	This site maintains several online “tools for prediction and analysis of protein-coding gene structure”	https://www.itb.cnr.it/webgene/
WGE	A website that provides tools to aid with genome editing of human and mouse genomes	http://www.sanger.ac.uk/htgt/wge/
WHAT IF	What If “is a versatile molecular modelling package that is specialized on working with proteins and the molecules in their environment like water, ligands, nucleic acids, etc.” The web interface provides a number of tools, viz. Structure validation, Residue analysis, Protein analysis, 2-D graphics, 3-D graphics, Hydrogen (bonds), Rotamer related, Docking, Crystal symmetry, mutation prediction, etc.	https://swift.cmbi.umcn.nl/whatif/WIF1_4.html
YASARA	Yet Another Scientific Artificial Reality Application (YASARA) is used for predicting the rotamers (protein side chain conformations) starting with single point mutations to complete homology models of proteins	http://www.yasara.org/

Appendix 2: List of Commercial and Non-profit Sources of CRISPR/Cas Reagents

Resource	Description	Link
Addgene CRISPR plasmids	A collection of CRISPR plasmids and reagents	http://www.addgene.org/CRISPR/
Beam Therapeutics: Upleveling CRISPR’s Precision by Targeting Specific Bases	Beam Therapeutics, a company co-founded recently by leading CRISPR researchers Feng Zhang, David Liu, and J. Keith Young, is developing more precise versions of the CRISPR technology which can effectively swap one base for another in the genome without cutting the DNA or RNA	https://beamtx.com/
Caribou Biosciences: Using CRISPR to Impact Several Industries	Caribou Biosciences (@CaribouBio) is one of the companies using CRISPR technology developing tools that provide transformative capabilities to therapeutics, biological research, agricultural biotechnology, and industrial biotechnology	https://cariboubio.com/
CRISPR Kits	Synthego’s CRISPR kits offer economical access to fully synthetic RNA for high fidelity editing and increased precision in genome engineering	https://www.synthego.com/products/crispr-kits
CRISPRflydesign (Bullock Lab)	Offers Cas9 transgenic stocks	http://www.crisprflydesign.org/
Editas Medicine: Using CRISPR to Target Point Mutations in Serious Genetic Disorders	Editas Medicine (@editasmed) is targeting mutations that cause serious genetic diseases and hopes to modify and fix these gene mutations using CRISPR	https://editasmedicine.com/
eGenesis: Using CRISPR to Improve Organ Transplants	eGenesis (@eGenesisBio) is pioneering an especially interesting application of CRISPR-Cas9 technology in the field of human therapeutics. This company is reviving the idea of xenotransplantation, i.e., animal-to-human organ transplants	https://www.egenesisbio.com/
FlyCas9 (Ueda Lab)	Provides reagents, protocols, and online tools for genome engineering by the designer nuclease Cas9 in Drosophila	http://www.shigen.nig.ac.jp/fly/nigfly/cas9/index.jsp
flyCRISPR (O’Connor-Giles Lab, Wildonger Lab, and Harrison Lab)	Fly CRISPR resources	http://flycrispr.molbio.wisc.edu/
Goldstein Lab CRISPR	A genome engineering resource for the Caenorhabditis elegans research community	http://wormcas9hr.weebly.com/
Inari Agriculture: Using CRISPR to Develop “Customized Seeds”	Inari Agriculture is an agro-biotechnology company that is revolutionizing the agricultural industry through transformational plant breeding technology. Inari uses CRISPR technology to develop seeds with traits optimized to grow best in local conditions	https://www.inari.com/
Inscripta: Increasing CRISPR’s Reach	Inscripta (@InscriptaInc) is a Colorado-based CRISPR biotech company that is revolutionizing commercially available CRISPR-associated nucleases. Inscripta’s next-generation CRISPR nucleases include natural and synthesized versions of “MADzymes,” a nomenclature inspired by the biodiversity found on the island of Madagascar	https://www.inscripta.com/
Intellia Therapeutics: Using Genome Editing for Personalized Disease Treatment	Intellia Therapeutics (@intelliatweets) aims to produce a new class of therapeutic products using a simplified manufacturing process. The company develops CRISPR-based solutions for personalized and curative treatments, and its current in vivo studies are focused on the use of Lipid Nanoparticles (LNPs) for delivery of the CRISPR/Cas9 complex to the liver	https://www.intelliatx.com/
Joung Lab CRISPR	A genome engineering resource for zebrafish research community	http://www.crispr-cas.org/
Ligandal: Establishing the CRISPR Delivery System	Ligandal (@ligandal), one of the companies using CRISPR based in San Francisco, has developed new technology which streamlines the in vivo delivery mechanisms for CRISPR, RNA, and other genetic tools. Ligandal has developed next-generation, non-viral protein-based biomaterials to effectively deliver gene therapy materials	https://www.ligandal.com/
Mammoth Biosciences: Using CRISPR to Advance Clinical Diagnostic	Mammoth Biosciences (@mammothbiosci) has capitalized on CRISPR’s unique ability to accurately find and bind to specific sequences of DNA. This company has created the first CRISPR-mediated platform for human disease detection. Their innovative point of care test allows for easy and affordable multiplexed detection of RNA/DNA sequences associated with disease	https://mammoth.bio/
NTrans: Helping CRISPR Edit All Cell Types	NTrans Technologies (@NtransTech), a CRISPR technology company based in the Netherlands, is working to ensure genome engineering can be performed in all cell types. NTrans pioneered a cellular uptake mechanism which circumvents the problems with delivery of CRISPR components for therapeutic purposes	https://www.ntranstechnologies.com/
OxfCRISPR (Liu Lab)	Oxford Fly CRISPR Resources	http://www.oxfcrispr.org/
Pairwise Plants: Using CRISPR to Grow New Varieties of Crops	Pairwise Plants (@PairwisePL) intends to create new crops and modify existing ones using gene editing technology such as CRISPR. The goal is to also assist farmers by providing them with new varieties of crops that require less resources to grow	https://pairwise.com/
Plantedit: Increasing the Worldwide Food Supply using CRISPR	Plantedit (@plantedit) is an Ireland-based CRISPR startup company aiming to produce “DNA-free” non-transgenic sustainable plant products in an attempt to introduce genome editing to food supply enhancement in a regulatory-free manner. The company focuses on creating modified plants that do not contain any foreign genetic material with a goal to meet the ever-increasing demand for “designer” crops while circumventing both the general aversion to ingesting non-plant-based DNA or RNA and the regulatory fences around traditional “GMO.”	http://plantedit.com/
Synthetic Genomics: Harnessing CRISPR to Create Sustainable Energy	Synthetic Genomics (@SynGenomeInc) manipulates microalgae for sustainable oil production. Partnering with Exxon Mobil, Synthetic Genomics identified 20 transcription factors thought to be negative regulators of lipid production in microalgae. The company then applied CRISPR-Cas9 to insert loss of function mutations in 18 of the 20 genes. They report a 200% increase in oil production from one of the modified microalgae species with little effect on growth, marking a key advancement in renewable energy biofuels	https://syntheticgenomics.com/
transOMIC	transOMIC offers reagents for CRISPR Cas9 gene editing, shRNA constructs, and cDNA and ORF clones	https://www.transomic.com/cms/home.aspx/
Zhang Lab Genome Engineering	CRISPR genome engineering resources website	http://www.genome-engineering.org/

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Ahmad, A. et al. (2022). Bioinformatic Tools in CRISPR/Cas Platform. In: Ahmad, A., Khan, S.H., Khan, Z. (eds) The CRISPR/Cas Tool Kit for Genome Editing. Springer, Singapore. https://doi.org/10.1007/978-981-16-6305-5_3

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DOI: https://doi.org/10.1007/978-981-16-6305-5_3
Published: 01 January 2022
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