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