Esercitazione 2 maggio pomeriggio - Unifem.docente.unife.it/francesco.bernardi/materiale... ·...

4. Selection of target sequence/ gRNA design:Plenty of webtools available:

• Predicted- on-target activity

• Predicted off-target activity

• … much more

http://crispor.tefor.net/

Selecting gRNA

https://omictools.com/genome-editing-category

Selecting gRNA4. Selection of target sequence/ gRNA design:Plenty of webtools available:

• Predicted- on-target activity

• Predicted off-target activity

• … much more

Selecting gRNA4. Selection of target sequence/ gRNA design:Plenty of webtools available:

• Predicted- on-target activity

• Predicted off-target activity

• … much more

Measuring Cas9 Efficacy

NON-HOMOLOGOUS END JOINING (NHEJ)

repairs with indels

How to measure the DSB efficacy of a CRISPR sgRNA and the nature of the mutations?

1. Sequencing based techniques

2. Denaturation based techniques

3. Alternative techniques

Measuring Cas9 Efficacy

1. Sequencing based techniques

Measuring Cas9 Efficacy

From Gelmini et al., Trends Biotechnol 2017

1. Sequencing based techniques: TIDE

Measuring Cas9 Efficacy

Tracking of Indels by DEcomposition

http://tide.nki.nl

Eva K. Brinkman, Tao Chen, Mario Amendola, Bas van Steensel

35%

32%

18%

15%

35%32%

15%18%

-4-3-2-10+2+1=

+

+

+

0

-1

-4

+1

R2 = 0.99

+3