Download Increasing gene editing efficiencies in eukaryotic cell lines by

Increasing gene editing efficiencies in eukaryotic cell lines
by selection of appropriate CRISPR-Cas9 reagents
Melissa L. Kelley, Žaklina Strezoska, Elena Maksimova, Hidevaldo Machado, Emily M. Anderson, Maren Mayer, Annaleen Vermeulen, Shawn McClelland, Anja van Brabant Smith
Dharmacon, now part of GE Healthcare, 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, US
Abstract
Lentiviral Cas9 combined with synthetic
crRNA:tracrRNA
Genetic engineering of living cells is critical for understanding gene function in normal
and diseased states. The CRISPR-Cas9 system is widely utilized because of its ease-ofuse compared to other gene editing methods. This system requires a complex of Cas9
protein with tracrRNA and a gene-targeting crRNA to introduce double-strand DNA
breaks at specific locations in the genome to disrupt protein translation and knockout
gene function. To achieve high gene editing efficiencies, it is essential to choose the best
CRISPR-Cas9 reagents for delivery and expression in the cells of interest.
Edit-R Lentiviral Cas9 Nuclease Expression vectors allow generation of a Cas9 stable
cell population for evaluating knockout phenotypes in a population (Figure 4). This
approach can be especially beneficial for experiments where reproducibility and
wider dynamic range of the phenotypic assay are highly desired, such as screening
applications. Additionally, integrated Cas9 and synthetic crRNA:tracrRNA results
in higher editing efficiency without the need to perform enrichment of plasmidtransfected cells using FACS or puromycin selection (Figures 5 and 6).
Choosing a CRISPR-Cas9 system for efficient
gene editing
CRISPR components can be delivered using transfection or viral transduction.
Plasmid-expressed Cas9 can be co-transfected with synthetic crRNA:tracrRNA for
efficient gene editing in cells amenable to lipid delivery, followed by isolation of
knockout clonal lines (Figure 1). Cas9 vectors packaged into lentiviral particles can
be used to generate a Cas9 stable cell line for transfections with crRNA:tracrRNA to
evaluate gene knockout phenotypes in a population (Figure 4).
Co-transfection of Cas9 plasmid and synthetic
crRNA:tracrRNA
A synthetic approach to crRNA:tracrRNA complex enables fast assessment of multiple
target sites per gene, or for multiple genes, without the requirement of any cloning
steps or in vitro transcription (Figures 2 and 3).
Transfect A549 cells
crRNA:tracrRNA
Cas9
expression
Transfect A549 cells
plasmid
crRNA:tracrRNA
Cas9 expression
plasmid
Lentiviral
transduction
1
day
Edit-R
Cas9
1
day
Target cells
6-15
days
Gene target
Puromycin selection
Gene target
PPIB
crRNA:tracrRNA
Puromycin selection
crRNA:tracrRNA
PPIB
+
-
-
-
+
+
UT
-
+
-
+
-
NA
+
+ +
CDKN1A
+ -
CDKN1A
UT
-
-
+
+
UT
NA
+
-
+
-
NA
-
+
+
UT
-
+
-
NA
At 24 hours split cells into a
At 24 puromycin
hours split cells
into a
containing
medium
Selection with
blasticidin
puromycin containing medium
3-6
weeks
Clonal cell isolation
and expansion
Expansion of
blasticidin-selected
cell population
Cas9 clonal cell line
Cas9 cell line
mixed population
Synthetic
crRNA:tracrRNA
transfection
M
Synthetic
crRNA:tracrRNA
transfection
C
1
2
21
11
1
17
% gene editing% gene editing
2
11
17
(PPIB)
(PPIB)
mismatch
detection
mismatch detection
assay using
T7E1 assay using T7E1
48 hours under
IncubateIncubate
48 hours under
antibiotic
antibiotic
selection selection
Figure 7. A549 cells were co-transfected with Edit-R Cas9-PuroR plasmid, tracrRNA and crRNAs
targeting PPIB or CDKN1 and selected with puromycin for 48 hours. A mismatch detection assay
using T7EI show increased editing in A549 cells after puromycin selection.
Mismatched
inhibitor target sites show that
3
position
of mismatch affects functionality
days
Enrichment of edited cells by FACS analysis
3
days
3
A.
mKate2 Expression in HEK293T
Negative
Low
Medium
High
B.
crRNA targeting PPIB in HEK293T
Unsorted cells
UT
Gene editing and phenotypic analyses
Figure 4. Lentiviral Cas9 and synthetic crRNA:tracrRNA workflow.
The optimal promoter for Cas9 expression
results in greater gene editing efficiency
Log of Fluorescence Intensity
hCMV-mKate2-Cas9 + tracrRNA:crRNA-PPIB
Untransfected control
To achieve the highest editing efficiency in a cell line of interest, it is important to select
the promoter most suitable for the cells of interest. Cas9 expression levels differ among
promoters, leading to varying levels of gene editing efficiency (Figures 5 and 6).
crRNA:tracrRNA
Cas9 expression
plasmid
Puromycin resistance marker allows rapid
selection and enrichment of edited cells
Sorted cells
US
Neg
Low
Med High
14
4
24
30
% Gated
Depending on the transfectablity of specific cells, CRISPR components can be delivered
using plasmid transfection or lentiviral transduction. Plasmid-expressed Cas9 can
be co-transfected with synthetic crRNA and tracrRNA for efficient gene editing in
cells amenable to lipid delivery. Cas9 that is packaged into lentiviral particles can be
transduced into cells that are refractory to transfection. Lentiviral Cas9 can also be used
to generate stable cell lines, which can then be transfected or transduced by synthetic
or lentiviral-based CRISPR RNA components; this is particularly useful for screening
applications. Importantly, expressing humanized Cas9 from different promoters (e.g.,
human and mouse CMV and EF1α, PGK, CAG) in different cell types results in varying
levels of Cas9 expression and consequently, varying efficiencies of gene editing. In
addition, cells transfected or transduced with gene editing reagents can be enriched by
antibiotic selection or FACS using reagents in which the Cas9 gene is co-expressed with
either an antibiotic resistance marker or a fluorescent protein reporter. This enrichment
facilitates the isolation of clonal cells containing the desired mutation. Presented here
are data demonstrating improvement of gene editing efficiencies in cells of interest by
using the most effective delivery and selection approaches with the optimal CRISPRCas9 reagents.
Enrichment of cell populations for increased
gene editing events
Highly efficient gene editing in Cas9-expressing mouse
NIH/3T3 cells using synthetic PPIB crRNA:tracrRNA
% editing:
27
mismatch detection assay using T7E1
Figure 8. Edit-R mKate2-Cas9 bicistronic plasmids allow enrichment of gene edited cells by FACS
analysis. HEK293T cells were transiently co-transfected with mKate2-Cas9 expressing plasmids,
synthetic crRNA targeting PPIB, and synthetic tracrRNA. Cells were sorted 72 hours post-transfection.
Percent editing was determined with a mismatch detection assay using T7EI.
Conclusions
best
2-3 Days
Analyze editing in
cell population
3-4 Days
Enrichment of edited
cells by FACS
Mismatch
detection assay
Edit-R (mKate2)
Nuclease Expression
Plasmids
Enrichment of edited cells
by antibiotic selection
Edit-R (PuroR)
Nuclease Expression
Plasmids
Isolate clonal knockout lines for further analysis
Figure 1. Plasmid Cas9 and synthetic crRNA:tracrRNA co-transfection and enrichment workflow.
Rapid assessment of multiple crRNAs with
Dharmacon™ Edit-R™ CRISPR-Cas9 Gene Engineering system
crRNAs targeting VEGFA M
U2OS cells
96-well format
T7EI endonuclease
No enrichment
% editing
VCP cr1
Cas9 promoter
crRNA:tracrRNA
-
hCMV
+ + +
mCMV
- + + +
-
hEF1α
+ + +
-
mEF1α
+ + +
-
PGK
+ +
+
-
CAG
+ +
+
• Synthetic crRNA:tracrRNA with a Cas9 nuclease allows fast, high efficiency gene
editing without cloning steps.
• The ability to quickly evaluate multiple crRNAs leads to the selection of the best target
sequence and, therefore, most effective knockout of the gene of interest.
% gene editing
(Ppib)
27 32 29
43 44 44
52 56 55
46 52 51
45 46 47
48 54 53
A
Figure 5. NIH/3T3 cells were transduced with Edit-R Lentiviral Cas9 Nuclease Expression particles
for stable expression of Cas9 and a blasticidin resistance gene driven by the indicated promoters.
The cell populations were selected with blasticidin for a minimum of 10 days before crRNA;tracrRNA
transfections. Cells were transfected with 50 nM synthetic crRNA:tracrRNA targeting Ppib using
DharmaFECT 1 Transfection Reagent. After 72 hours, the relative frequency of gene editing was
calculated from a mismatch detection assay using T7EI.
• Applying a highly efficient lentiviral Cas9 nuclease approach allows researchers to
establish stable cell populations or clonal lines for improved reproducibility and a
further increase in editing frequency.
• Choosing the best promoter is recommended to achieve maximum Cas9
expression in the cell line of interest and therefore better editing efficiency.
- cr1 - cr2 cr3 - cr4 cr5
Figure 2. Edit-R Cas9-mKate2 plasmid,
synthetic tracrRNA and five different
synthetic crRNAs targeting VEGFA were
co-transfected into U2OS cells. Various
levels of gene editing were achieved
as assessed by a mismatch detection
assay using T7 Endonuclease I (T7EI).
22
VCP cr2
21 14
14 16
PSMD7 cr2
PSMD14 cr4
• Antibiotic resistance gene or fluorescent marker on the plasmid expressing
Cas9 enable enrichment of edited cells, thus improving phenotypic readout and
facilitating clonal selection.
Efficient gene editing in U2OS cells
expressing Cas9 from different promoters
gelifesciences.com/dharmacon
best
Cas9 promoter
crRNA:tracrRNA
-
hCMV
+ + +
mCMV
- + + +
-
hEF1α
+ + +
mEF1α
- + + +
-
PGK
+ +
+
-
CAG
+ +
+
PSMD cr5
% gene editing
(PPIB)
Figure 3. Disruption of proteasome function by crRNAs targeting components of the 26S proteasome
pathway: VCP, PSMD7 and PSMD14. A recombinant U2OS ubiquitin-EGFP proteasome cell line was
co-transfected with Cas9 plasmid and crRNA:tracrRNA. The accumulation of ubiquitin-EGFP was
measured 72 hours post-transfection demonstrating disruption of proteasome function by knockout
of functional proteins.
25 24 24
24 24 24
28 28 27
29 26 30
17 17 19
39 34 28
Figure 6. A recombinant U2OS ubiquitin-EGFP proteasome cell line with stably integrated Cas9 was
transfected with 50 nM synthetic crRNA:tracrRNA using DharmaFECT 3 Transfection Reagent to
target PPIB. After 72 hours, the relative frequency of gene editing was calculated from a mismatch
detection assay using T7EI.
TM