USER GUIDE

Edit-R™ CRISPR Design Tool

The Edit-R™ CRISPR Design Tool allows you to quickly and easily generate
guide RNA sequences for ordering either synthetic and lentiviral guides.
There are several options for designing guide RNAs:• Gene Knockout: Input a gene ID or gene symbol design anywhere
within gene. Generate guide RNA designs to target a known geneof interest. (Figure 1) The gene target must be from a species that is
supported by the CRISPR RNA Design Tool (Table 1).
• Site-specific editing: Input a gene ID or gene symbol specificcleavage location for HDR. Generate guide RNA designs in aspecific region to facilitate HDR and knock-in experiments. Weighted
functionality and specificity scoring is applied. The gene target must befrom a species that is supported by
the Edit-R™ CRISPR Design Tool (Table 1).
• Provide a DNA region for design. Generate guide RNA designs totarget a particular DNA sequence; often used for non-standard species
or to target particular gene regions.
• Input my own guide RNA sequence. You can input your own
nucleotide target sequence for custom synthesis of synthetic sgRNA(s),crRNA(s), or generation of lentiviral sgRNA(s).
Edit-R™ CRISPR Design Tool Advanced Settings allow for customization ofcriteria such as nuclease, transcripts targeted, and specificity analysis. By
default, the Edit-R™ CRISPR Design Tool will select guide RNA designs thattarget all transcripts of the selected gene.
The Specificity Check performs a rigorous alignment that excludes resultsfrom any guide RNAs that have PAM-adjacent target sites with two or fewer
mismatches or gaps elsewhere in the selected genome.

Viewing results
Depending on the design option selected, results are presented as eithera List or Graphical view. Results are sorted by earliest to latest position in
the target gene or DNA region. Functionality (when using the default
S. pyogenes Cas9 nuclease) and Specificity rankings, as well as location
filters are available to aide in guide RNA design selection.
It is recommended to test 3-5 different designs to find one that is mostefficient. If you have an understanding of the functional domain(s) ofyour target gene, select designs across those exon(s). If not, choose targets
in more than one exon, but always including an early exon for a betterchance of disrupting translation.
ATG
Exon 1

Exon 2

Exon 3

Exon 4

Exon 55' GTGTATTTTGACCTACGAATTGG 3' Top and bottom strands of genomic DNA
3' CACATAAAACTGGATGCTTAACC 5' (top strand, target sequence and PAM;bottom strand, guide-bound sequence)

5' GTGTATTTTGACCTACGAAT 3'
5' GUGUAUUUUGACCUACGAAU 3'
5' GUGUAUUUUGACCUACGAAU
or

5' GUGUAUUUUGACCUACGAAU

20 nucleotide target sequence

DNA target sequence converted into guide
CRISPR Design Tool makes a guide RNA(either crRNA or sgRNA)

3'crRNA consisting of 20 nucleotide guidesequence and S. pyogenes repeat sequence

sgRNA consisting of 20 nucleotide guidesequence and S. pyogenes-derived repeat
and tracrRNA sequences

Figure 1. Example of how the Edit-R™ CRISPR Design Tool will select a 20nucleotide sequence targeting the human gene PPIB. The target sequence can belocated on either strand of the genomic DNA as long as it is in the 5' to 3' orientationand there is a NGG PAM (when using the default S. pyogenes Cas9) on the 3’ endof that strand. Cas9 nuclease will cut both strands of DNA at the position three
nucleotides upstream of the NGG PAM. It is suggested to choose a target site locatedentirely within an early constitutive exon of the coding gene, but the Edit-R™ CRISPR
Design Tool will return results from across the entire coding region so particular exonsor protein domains can be targeted, if desired.

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Table 1. Species with integrated gene identifiers and genome-wide alignment
capability in the Edit-R™ CRISPR Design Tool. The following species may be
selected in the “Organism” field when specifying the source species for a particulargene target, and for a genome-wide specificity check to ensure the resulting
guide RNA(s) only has a perfect match to the intended target, and has two or moremismatches to other regions of the genome.

Table 2. Default design parameters for Input a gene ID or gene symbol

Common nameScientific namemicroRNA

Human

Homo sapiens

Mouse

Mus musculus

RatRattus norvegicus

Zebrafish

Danio rerio

Western clawed frog

Xenopus tropicalis

Fruit flyDrosophila melanogaster

Chinese hamster

Cricetulus griseus

Pig

Sus scrofa

Cow

Bos taurus

Marmoset

Callithrix jacchus

Dog

Canis familiaris

Roundworm

Caenorhabditis elegans

Sea Squirt

Ciona intestinalis

HorseEquus caballus

CatFelis catus

Fugu

Takifugu rubripes

ChickenGallus gallus

Stickleback

Gasterosteus aculeatus

Opossum

Monodelphis domestica

FerretMustela putorius furo

Nile tilapia

Oreochromis niloticus

Platypus

Ornithorhynchus anatinus

RabbitOryctolagus cuniculus

MedakaOryzias latipes

SheepOvis aries

BonoboPan paniscus

Chimp

Pan troglodytes

BaboonPapio Anubis

Orangutan

Pongo pygmaeus abelii

RhesusMacaca mulatta

Alligator

Alligator mississippiensis

Purple sea urchin

Strongylocentrotus purpuratus

Zebra finch

Taeniopygia guttata

MacaqueMacaca fascicularis

GorillaGorilla gorilla

Japanese Rice

Oryza sativa subsp. Japonica

Maize

Zea mays

SoybeanGlycine max

AppleMalus domestica

CottonGossypium hirsutum

Green Alga

Chlamydomonas reinhardtii

Type of locus

TargetingProtein coding

Include all protein-coding transcripts and allow all
non-coding transcripts
Require cleavage in the coding DNA sequence (CDS)
Include all noncoding transcripts and exclude allprotein-coding transcripts
Cleavage allowed anywhere in the transcript

Long noncodingRNA

Include all noncoding transcripts and exclude all protein-coding transcripts
Cleavage allowed anywhere in the transcript

To find the contact information in your country for your technology of interest, please visit us at
horizondiscovery.com/contact-us
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom©2020 The Horizon logo and other trademarks are the property of Horizon Discovery Limited, unless otherwise stated. DHARMACON and EDIT-R are trademarks of Dharmacon Inc.

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