CHOPCHOP

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CHOPCHOP

Target RefSeq/ENSEMBL/gene ID or genomic coordinates.

Target Paste fasta entry here.

In

In

Add new species.

Using Change default PAM and guide length in Options.

For Presets can be adjusted in Options.

General
Cas9
Cpf1
Cas13
Cas9 nickase
TALEN
Primers

Target specific region of gene:

Coding region

All exons (inc. UTRs)

Splice sites

5' UTR

3' UTR

Promoter

Only target exon(s):

Restrict targeting:

Search exons and immediate short flanking regions.

Only search within the exon.

Isoform consensus determined by:

Intersection (only searches regions present in all isoforms)

Union (searches all exons in all isoforms)

Pre-filtering:

Minimum required GC [%] content has to be between min: and max:

Self-complementarity has to be below:

Restriction enzymes:

Company preference:

Minimum size of restriction enzyme binding site:

Fasta input:

Color scoring should ignore one off-target without mismatches.

Displayed flanking sequence length in detailed view:

sgRNA length without PAM:

PAM-3':

NGG

NAG

NGA

NRG (R = A or G)

NNAGAAW (W = A or T)

NNNNGMTT (M = A or C)

NNGRRT (R = A or G)

Custom PAM:

Method for determining off-targets in the genome:

Off-targets with up to mismatches in protospacer (Hsu et al., 2013)

Off-targets may have no more than mismatches in the protospacer seed region (Cong et al., 2013)

Efficiency score:

Doench et al. 2014 - only for NGG PAM

Doench et al. 2016 - only for NGG PAM

Chari et al. 2015 - only NGG and NNAGAAW PAM's in hg19 and mm10

Xu et al. 2015 - only for NGG PAM, but can be used with other PAMs

Moreno-Mateos et al. 2015 - only for NGG PAM

G20

Repair profile prediction (Shen et al. 2018):

mESC (recommended when you don't know which cell type)

U2OS

HEK293

HCT116

K562

Don't calculate (saves time)

5' requirements for sgRNA:

GN or NG

GG

No requirements

Self-complementarity (Thyme et al.):

Check for self-complementarity

I intend to replace the leading nucleotides with "GG"

Check for complementarity versus backbone:

Standard backbone (AGGCTAGTCCGT)

Extended backbone (AGGCTAGTCCGT,ATGCTGGAA)

Custom backbone:

sgRNA length without PAM:

PAM-3':

NGG

NAG

NGA

NRG (R = A or G)

NNAGAAW (W = A or T)

NNNNGMTT (M = A or C)

NNGRRT (R = A or G)

Custom PAM:

Method for determining off-targets in the genome:

Off-targets with up to mismatches in protospacer (Hsu et al., 2013)

Off-targets may have no more than mismatches in the protospacer seed region (Cong et al., 2013)

Efficiency score:

Doench et al. 2014 - only for NGG PAM

Doench et al. 2016 - only for NGG PAM

Chari et al. 2015 - only for NGG and NNAGAAW PAM's in hg19 and mm10

Xu et al. 2015 - only for NGG PAM, but can be used with other PAMs

Moreno-Mateos et al. 2015 - only for NGG PAM

G20

5' requirements for sgRNA:

GN or NG

GG

No requirements

Self-complementarity (Thyme et al.):

Check for self-complementarity

I intend to replace the leading nucleotides with "GG"

Check for complementarity versus backbone:

Standard backbone (AGGCTAGTCCGT)

Extended backbone (AGGCTAGTCCGT,ATGCTGGAA)

Custom backbone:

Distance between guides: to:

Max distance between offtargets:

sgRNA length without PAM:

PFS:

5' end of target (like in Cpf1) e.g. Cas13a:

3' end of target (like in Cas9):

sgRNA length without PAM:

5'-PAM:

TTN

TTTN

TTCN (CasX)

Non-standard:

Efficiency score:

Kim et al. 2018

None (will display zeros in "Efficiency" column)

Self-complementarity (Thyme et al.):

Check for self-complementarity

Check for complementarity versus backbone:

Standard backbone (AGGCTAGTCCGT)

Extended backbone (AGGCTAGTCCGT,ATGCTGGAA)

Custom backbone:

Distance between TALENs: to:

Size of TALENs:

Number of mismatches to search for when finding off-targets:

G repeat variable diresidue (RVD):

NN

NH

Design primers

Product size:

From: to:

Primer size:

From: to: Optimal:

Primer Tm:

From: to: Optimal:

Minimum distance from primer to target site:

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