1.
Genome Engineering Using the
CRISPR-Cas9 System
(Ran FA et al Nature Protocols Vol.8 No.11 2013)
May 6th, 2014
Yudha Nur Patria

2.
CRISPR Cas System of Streptococcus pyogenes
Figure 1

3.
DSB-induced DNA-repair mechanisms
Figure 2

4.
http://tools.genome-engineering.org Step 1-2
Developing construct Step 3-5
Step 6-53
Step 54-126

5.
• CRISPR design tool: http://tools.genome-engineering.org
• Design two sgRNAs for each locus and check their efficiency in the
intended cell type
• What plasmid would we use?
• pSpCas9 (Cas9 alone)
• pSpCas9(BB) (Cas9 + invariant sgRNA scaffold and cloning sites)
• Do we need to do nicking to minimise the off target mutagenesis?

6.
Plasmid construct development strategies:
• sgRNA-encoding primers (PCR-based sgRNA)
• sgRNA-expressing plasmids
sgRNA-encoding primers (Step 5A) sgRNA-expressing plasmid (Step 5B)
• Append sgRNA to U6 promoter template
• The resulting amplicons can be co-transfected
with a Cas9 expressing plasmid (pSpCas9)
• Optimal for rapid screening of multiple
candidate sgRNAs (for generating large KO
libraries)
• Need over 100 bp long
• Easy and rapid in comparing sgRNA
efficiencies
• Oligo pairs encoding the 20-nt guide
sequences are annealed and ligated into a
plasmid (pSpCas9 or pSpCas9 (BB)) which has
sgRNA scaffold and cloning sites for inserting
a guide sequences)
• 20 bp long
• Higher cleavage efficiency
They also fused the Cas9 with GFP (2A-GFP or 2A-Puro for screening of transfected cells (pSpCas9(BB)-2A-GFP)
for cell screening and selecton.

7.
sgRNA construction and delivery
Figure 4

8.
CRISPR web tool results for SENP2 gene
• Best sgRNA sequence candidates:
• Exon 1 (1A): score 95 with 41 off target sites (2 are in genes)
• Exon 2 (2A): score 96 with 31 off target sites (2 are in genes)
Don’t forget to add G (guanine) if the first base of sgRNA sequence
does not start with G since the U6 RNA polymerase III promoter prefers
guanine as the first base of transcript.

9.
Exon 1 (1A)

10.
Exon 2 (2A)

11.
Design of repair template (optional)
• Single-stranded DNA oligonucleotides (ssODNs)
• For high HDR efficiency:
• ssODNs should contain at least 40 bp on each side which are homologous to
the target region

12.
Plasmid construct validations
• Step 5B xii

13.
• Functional assay:
• Surveyor nuclease assay (step 71-89)
• Sequencing
(the web page provides the primer for
those purposes or they can be designed
manually, the primers should be 18-25 bp
long with melting temp 600C)
• NHEJ detection:
• Primers should anneal at least 50 bp from
Cas9 target site to allow for longer indels
detection (Step 90-96).
• HDR detection:
• RFLP (step 97-108)
• The primers should anneal outside the
homology arms
• Sequencing: Sanger (step 109-117) or
deep sequencing (step 118-126)
• Transfection
• HEK 293FT (step 6-29)
• HUES 9 (step 30-53)
• Single cell isolation
• using FACS (step 54-65) or serial
dilutions (step 66-70), then followed
by specific clonal cell expansion

14.
Transfections
• What cells would we use?
• Methods: Lipofectamine 2000 or Amaxa SF cell line 4D-Nucleofector X
kit?
• Recommendation: triplicates + controls (eg. GFP plasmid)

15.
Clonal cell isolation and expansion
• FACS or serial dilution?

16.
Transfection efficiency validations
• Surveyor nuclease assay (step 71-89)
• NHEJ detection:
• Primers should anneal at least 50 bp from Cas9 target site to allow for longer
indels detection (Step 90-96).
• HDR detection:
• RFLP (step 97-108)
• The primers should anneal outside the homology arms
• Sequencing: Sanger (step 109-117) or deep sequencing (step 118-126)