4. Generate the oligo duplex, clone into the BsmBI predigested
plasmid MLM3636, sequence the resulting plasmid. Transform and amplify the guide expression plasmid in DH5 competent cells using a Midi or Maxi Prep kit depending on the
amount of plasmid required.
3.2 Delivery
of CRISPR Reagents
and Assessment
of Gene Editing
As knock-in using double-stranded donor template has relatively
low efficiency, successful HDR-mediated experiments require
experimental optimization. In particular, it is recommended to
control and optimize delivery of CRISPR reagents by evaluating
the levels of gene editing in absence of donor template. In some
cases, GFP fluorescence can be used to directly characterize and
optimize HDR events, but low to very low levels of fluorescenttagged proteins driven by endogenous promoters can be
GGCGAGAAGTTCGCGAAATCCTTT ACTACGAATC CGTCCC ATGAAGCAAGTTCTGGCCGCGAA...
CCGCTCTTCAAGCGCTTTAGGAAA TGATGCTTAG GCAGGG TACTTCGTTCAAGACCGGCGCTT...
P L F K R F R K
*
| | | | | | | | |
| | | | | | | | | | |
| | |
+
-
locus
5’ Homology Arm
3’ Homology Arm
PAM
Repair
donnor
5 Homology Arm
3’ Homology Arm
Cas9 cleavage site
GFP P2A Puro
sgRNA
+
-
Cas9
PAM
GGCGACAAGTTCTCTAAAGCCTTTCCACCACCG...
...GCCACGGACT ATGAAGCAAGTTCTGGC...
CCGCTGTTCAAGAGATTTCGGAAAGGTGGTGGC...
...CGGTGCCTGA TACTTCGTTCAAGACCG...
P L F K R F R K G G
G A *
TAGGAAATGATGCTTAGGCA
ATCCTTTACTACGAATCCGT
||||||||||||||||||||
UAGGAAAUGAUGCUUAGGCA
A
B
Fig. 3 Design of sgRNAs and HDR donor sequences. (a) Schematic representation of the Cas9 protein (gray)
bound to sgRNA (red) and targeted to genomic DNA (black). The PAM sequence (GGG with overbar), the Cas9
cleavage sites (black triangles), and the stop codon of the XPB gene (TGA, in cyan) are indicated. A ribbon
representation of the Cas9 nuclease from Streptococcus pyogenes bound to PAM-containing DNA target is
shown in the right panel (PDB code, 4UN3; [30]). (b) Repair donor designed to insert the eGFP-2A-Puro coding
sequence (all in frame with the last exon of the target protein) upstream of the XPB STOP codon (TGA).
Nucleotides targeted by the sgRNA are in bold. After integration of the tag, the sequence is significantly
modified (red line above the + strand), preventing cleavage of the repaired locus by Cas9
46
Sylvain Geny et al.
plasmid MLM3636, sequence the resulting plasmid. Transform and amplify the guide expression plasmid in DH5 competent cells using a Midi or Maxi Prep kit depending on the
amount of plasmid required.
3.2 Delivery
of CRISPR Reagents
and Assessment
of Gene Editing
As knock-in using double-stranded donor template has relatively
low efficiency, successful HDR-mediated experiments require
experimental optimization. In particular, it is recommended to
control and optimize delivery of CRISPR reagents by evaluating
the levels of gene editing in absence of donor template. In some
cases, GFP fluorescence can be used to directly characterize and
optimize HDR events, but low to very low levels of fluorescenttagged proteins driven by endogenous promoters can be
GGCGAGAAGTTCGCGAAATCCTTT ACTACGAATC CGTCCC ATGAAGCAAGTTCTGGCCGCGAA...
CCGCTCTTCAAGCGCTTTAGGAAA TGATGCTTAG GCAGGG TACTTCGTTCAAGACCGGCGCTT...
P L F K R F R K
*
| | | | | | | | |
| | | | | | | | | | |
| | |
+
-
locus
5’ Homology Arm
3’ Homology Arm
PAM
Repair
donnor
5 Homology Arm
3’ Homology Arm
Cas9 cleavage site
GFP P2A Puro
sgRNA
+
-
Cas9
PAM
GGCGACAAGTTCTCTAAAGCCTTTCCACCACCG...
...GCCACGGACT ATGAAGCAAGTTCTGGC...
CCGCTGTTCAAGAGATTTCGGAAAGGTGGTGGC...
...CGGTGCCTGA TACTTCGTTCAAGACCG...
P L F K R F R K G G
G A *
TAGGAAATGATGCTTAGGCA
ATCCTTTACTACGAATCCGT
||||||||||||||||||||
UAGGAAAUGAUGCUUAGGCA
A
B
Fig. 3 Design of sgRNAs and HDR donor sequences. (a) Schematic representation of the Cas9 protein (gray)
bound to sgRNA (red) and targeted to genomic DNA (black). The PAM sequence (GGG with overbar), the Cas9
cleavage sites (black triangles), and the stop codon of the XPB gene (TGA, in cyan) are indicated. A ribbon
representation of the Cas9 nuclease from Streptococcus pyogenes bound to PAM-containing DNA target is
shown in the right panel (PDB code, 4UN3; [30]). (b) Repair donor designed to insert the eGFP-2A-Puro coding
sequence (all in frame with the last exon of the target protein) upstream of the XPB STOP codon (TGA).
Nucleotides targeted by the sgRNA are in bold. After integration of the tag, the sequence is significantly
modified (red line above the + strand), preventing cleavage of the repaired locus by Cas9
46
Sylvain Geny et al.