Update on engineered Cas9 - lab meeting (DNA damage and repair research group)

1. Cas9 and its engineered
Lab meeting
31 August 2021
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2. Plasmid map of gRNA + Cas9 (TK6 system)
gRNA
Cas9
Compatible with mammalian expression
(addgene - plasmid #114365)
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3. Plasmid map of Cas9 expression(RNP system)
FspAI
(9243)
PpuMI
(9215)
pGEX 3'
(8578 .. 8600)
PflFI -Tth111I
(8478)
pRS-marker
(8418 .. 8437)
BspQI - SapI
(8340)
L4440
(8321 .. 8338)
pBR322ori-F
(8068 .. 8087)
AlwNI
(7814)
AhdI
(7335)
BsaI
(7269)
PstI
(7093)
PvuI
(6966)
Amp-R
(6766 .. 6785)
F1ori-F
(6259 .. 6280)
DraIII
(6195)
F1ori-R
(6049 .. 6068)
T7 Term
(5864 .. 5882)
6xHis
SV40 NLS
7 promoter
RBS
NLS
Compatible with E. coli expression
(addgene - plasmid #114365)
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4. Streptococcus pyogenes Cas9
158 kDa; 1368 aa; 4.1kb
PDB: 4OGC 10.1016/j.cell.2014.02.001
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5. Streptococcus pyogenes Cas9
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6. Streptococcus pyogenes Cas9
REC -- Recognition NUC -- Nuclease
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7. Streptococcus pyogenes Cas9
REC -- Recognition
Key features
- Not share structural similarity with other known proteins
- Cas9 has specific functional domain
- Least conserved regions across three Cas9 families within typeII
CRISPR system (IIA, IIB, IIC)
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8. Streptococcus pyogenes Cas9
PI – PAM interacting domain
Key features
- Adopt a novel protein fold
- Unique to the Cas9 family
- Recognize 5’-NGG-3’ PAM on the
noncomplementary strand
- Critical for Cas9 cleavage functions
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9. Streptococcus pyogenes Cas9
RuvC domains
Key features
- Contain RNase H fold (non-sequence-specific
endonuclease enzymes)
- Cleave the non-complementary strand of
target DNA
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10. Streptococcus pyogenes Cas9
HNH domains: contain three catalytic residues
Key features
- Has three catalytic residues (Asp839, His840,Asn863)
- Cleave the complementary strand of target DNA
- His840 is the critical residue to cleave complementary strand
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11. Streptococcus pyogenes Cas9
Stem loop 3
Stem loop 2
Stem loop 1
Tetraloop
Key features
- Tetraloop and stem loop 1 are critical for Cas9
function
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12. Streptococcus pyogenes Cas9
BH – bridge helix domain
Key features
- Arginine cluster – critical for sgRNA:DNA recognition
- Arg63, Arg66, Arg70, Arg71, Arg74, and Arg78
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13. 10.1016/j.sbi.2021.03.004
Streptococcus pyogenes Cas9 and its variants
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14. Streptococcus pyogenes Cas9 and its variants
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15. 10.1126/science.aad5227
Using gene
with know
on/off target
Generate
many mutant
of Cas9 --
reduce off
target
NGS
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16. 10.1126/science.aad5227
Generate
many mutant
of Cas9 --
reduce off
target
NGS
Obtain
candidate
Cas9
Using gene
with know
on/off target
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17. 10.1126/science.aad5227
Propose off-target cause; Neutralization of positively charged residues within
nontarget strand groove (nt-groove) could weaken nontarget strand binding
and encourage rehybridization between target and nontarget DNA strands
NGS
Trying with
different loci
and different
gRNA design
On-target
Off-target
Different DNA
sequence – Different
interaction between
nt-groove vs DNA
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18. 10.3390/ijms22168571
Last lab meeting
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