CRISPR-Cas9 is a powerful tool for genome engineering. The document provides guidance on using CRISPR-Cas9 to modify genomes. It describes: 1) Designing single guide RNAs (sgRNAs) to target specific gene loci using online tools; 2) Constructing plasmids expressing Cas9 and sgRNAs; 3) Validating plasmid function using assays like Surveyor nuclease; and 4) Transfecting cells, isolating clones, and further validating genome edits through sequencing. The goal is to use this method to precisely modify genomes for research applications.

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.

10. Exon 1 (1A)

11. Exon 2 (2A)

12. 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

13. Plasmid construct validations
• Step 5B xii

14. • 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

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

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

17. 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)