CHOPCHOP

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.

Restriction enzymes:

Company preference:

Minimum size of restriction enzyme binding site:

Fasta input:

Color scoring should ignore one off-target without mismatches.

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

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

G20

5' requirements for sgRNA:

GN or NG

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

Extended backbone

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

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

G20

5' requirements for sgRNA:

GN or NG

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

Extended backbone

Custom backbone:

Distance between guides:

Max distance between offtargets:

sgRNA length without PAM:

5'-PAM:

TTN

TTTN

Custom PAM:

Self-complementarity (Thyme et al.):

Check for self-complementarity

Check for complementarity versus backbone:

Standard backbone

Extended backbone

Custom backbone:

Distance between TALENs:

Size of TALENs:

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

G repeat variable diresidue (RVD):

Design primers

Product size:

From: to:

Primer size:

From: to: Optimal:

Primer Tm:

From: to: Optimal:

Minimum distance from primer to target site: