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HORIZON DISCOVERY
Enhancing the efficiency of rAAV and CRISPR gene knockins by DNA mismatch
repair suppression and negative selection
Ramu Mangena, Sally Clayton, Rohan Sivapalan, Eric Rhodes, Tom Henley
Horizon Discovery, Cambridge, UK
Introduction
Recombinant adeno associated virus (rAAV) is a precise and effective method to introduce
defined changes into endogenous genes and rAAV vectors can stimulate homologous
recombination (HR) up to 1000-fold over that seen using plasmids. rAAV incorporates targeted
changes without the requirement of a double strand DNA-break (DSB), however it has been
demonstrated that introduction of a DSB by nucleases such as CRISPR further increases the
rates of targeted incorporation of the rAAV donor.
We sought to test the improvement in targeting frequency in mammalian cells by inhibition of
the DNA mismatch repair pathway by RNA interference and the incorporation of negative
selection into rAAV donors. We tested if any improvements seen could be further enhanced by
using a combination of rAAV and CRISPR.
shRNA mediated negative selection vectors
shRNA expressions cassettes to knockdown genes required for cell survival, or the donors own
positive selection gene, are placed at one or both ends of the vector outside of the region
homologous to the target gene. Cells that have undergone HR will not integrate the shRNAs,
whereas cells in which the construct integrates randomly will retain them and be eliminated via
its expression.
We have found that incorporation of these negative selection elements in combination with
positive selection using a drug resistance marker increases the targeting frequency of rAAV
(A)
(B)
The combination of MSH2 suppression and rAAV vectors incorporating shRNA negative
selection has been used to engineer mutant isogenic cell lines which could not be isolated
without these improvements
No shRNA vector
No MSH2 suppression
Single shRNA vector
MSH2 suppression
Number of clones screened
by PCR
3680
762
Number of targeted clones
0
19
Targeting frequency
0%
2.5%
Gene editing with CRISPR and improved rAAV donors
The use of CRISPR with rAAV donors enables highly efficient gene editing. This combination can
be further improved using rAAV donors with shRNA negative selection.
Tools for measuring homologous recombination rates
To measure rates of HR and targeting frequencies, we generated cell lines with an integrated
GFP reporter gene inactivated by a nonsense mutation to prevent fluorescence. Donor
molecules could then be used to integrate the correct sequence to revert the mutation and
switch-on GFP, enabling rates of HR to be measured by flow cytometry.
Digital droplet PCR (ddPCR) was also used to measure the abundance of a mutation introduced
by a donor via HR in a pool of cells using locus specific primers and probes.
No donor
rAAV donor
(C)
Vector design
No. of colonies No. of correctly
targeted clones
screened
Targeting
frequency‡
Fold increase§
No ShRNA
286
6
2.1%
1
Single ShRNA
257
21
8.1%
3.9
Dual ShRNA
241
49
20.3%
9.7
Figure 6. rAAV targeting vectors incorporating
shRNA negative selection increase targeting
frequencies of the GFP reporter allele in HCT116
cells when combined with CRISPR.
Vectors with single or dual shRNA cassettes
targeting the Neomycin resistance marker were
infected in cells that had been transfected with
plasmids expressing Cas9 and gRNA to introduce
double stand breaks in the GFP exon. Cells were
selected for 2 weeks in the presence of G418 and
then analysed by FACS
(D)
Targeting frequency is the number of correctly targeted
colonies per 100 drug-resistant colonies screened.
§ The fold increase is the targeting frequency of the ShRNA
vectors divided by the targeting frequency of the no shRNA
vector (set at 1).
Note: Transfection efficiencies are also being
measured to determine if the increase might also
be related to expression levels of Cas9/gRNA
‡
GFP
Figure 1. GFP reporter for measuring homologous recombination. Schematic representation of the GFP reporter
inserted into the endogenous HPRT allele in HCT116 and HEK293 cells. GFP is split into exons and a nonsense
mutation is introduced in exon 3 to render the protein inactive. gRNA sites close to the mutated STOP codon
enable targeted cleavage of the exon by Cas9. Plasmid, ssODN and rAAV donors are designed to switch on GFP by
correcting the nonsense mutation and allow identification of cells undergoing correct HR by FACS.
DNA mismatch repair (MMR) suppression during HR
The DNA MMR pathway has been implicated in the inhibition of gene editing, potentially
through the suppression of homologous recombination between genetically different
sequences. We found an increase in targeting frequency of rAAV donors in cells with intact
MMR pathways in which expression of the important MMR gene MSH2 had been down
regulated by RNAi
Figure 2. MMR-proficient cells show
increased rates of rAAV gene editing
when MSH2 is suppressed by RNAi. The
targeting frequency of the BRAF V600E
mutation was measured by ddPCR 72
hours after infection. HCT116 and DLD1
cells known to be already MMR-deficient
showed marginal or no improvement in
the frequency of gene editing whereas
siRNA knockdown of MSH2 in MMRproficient MCF10a cells gave a more
significant increase.
siRNA
MCF10a
HCT116
-
-
+
Figure 5. Successful engineering of the
AKT E17K mutation in MCF10a cells using
rAAV vectors with single shRNA negative
selection
cassettes
plus
MSH2
suppression
+
MSH2
Actin
t + 44 (0)1223 655580
f + 44 (0)1223 655581
e [email protected]
w www.horizondiscovery.com
Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
DLD-1
-
+
Figure 3. rAAV vectors incorporating shRNA negative selection increase rates of gene editing. (A) Typical rAAV
vector incorporating shRNA cassettes (negative selection) outside the region of homology. (B) Single or dual
shRNA cassettes increase targeting frequencies of the GFP reporter allele in HCT116 cells. (C) and (D) Targeting
efficiency of the CDK2 D145N kinase-dead mutation in HCT116 cell line is increased using vectors with shRNA
cassettes against endogenous HPRT (selected against using HAT containing media).
The use of MSH2 suppression during CRISPR + rAAV targeting did not show any significant
increase in targeting frequencies in either the MMR-deficient HCT116 line (as expected) or the
MMR-proficient NCI-H838 lung carcinoma line. The same was observed when using both
ssODN and dsDNA plasmids as donors.
Combining MMR suppression and negative selection vectors
MSH2 suppression using siRNA followed by targeting with rAAV vectors incorporating shRNA
negative selection gave an increased rate of gene targeting in the MMR-proficient cell lines
MCF10a and NCI-H838. The improved rate of editing was larger than seen with either technique
when applied alone.
Figure 7. MSH2 suppression gives no significant benefit to targeting frequencies using CRISPR combined with
different donors for HR. HCT116 (left) and NCI-H838 (right) GFP reporter cells in which MSH2 expression had been
knockdown by siRNA were analysed for GFP positive cells 72 hours after transfection/infection with CRISPR and
indicated donor
Conclusions
We have shown the increase in gene editing efficiency of rAAV donors when incorporating
shRNA negative selection and supressing MSH2 gene expression during HR. When these
improvements are used in combination, the boost in targeting frequency has improved the
success of generating isogenic cell lines with defined genetic mutations.
Figure 4. Increase in gene targeting frequency by combination of MSH2 suppression and negative selection. Cells
were transfected with MSH2 siRNA, or left untransfected (NT) and cultured for 72 hours before infection with
either BRAF V600E (left) or EGFR T790M (right) virus. No shRNA vectors (white bars) and single HPRT shRNA
negative selection vectors (black bars) were tested. Cells were selected in G418 and HAT in combination for 2weeks, then analysed for targeted allele frequency by locus specific ddPCR.
The use of rAAV donors in combination with CRISPR further enhances gene targeting rates and
while MSH2 suppression of CRISPR/rAAV treated cells did not further elevate this, donors
incorporating shRNA negative selection did increase the proportion of correctly targeted cells
further