lipofectamine® messengermax™ - a new transfection reagent ...€¦ · crispr-based genomic...
TRANSCRIPT
Reagent-DNAcomplexes
heparansulfate
endosome
nucleus
cytosol
Attachment to the cell
1.Endocytosis into cell
2.Escape fromthe endosome
3.Nuclear entry of DNA
4.
mRNAprotein
Introduction Induced pluripotent stem cells (iPSCs) hold immense promise for the future of neurodegenerative medicine and personalized therapeutic treatments. For example, one can create cellular disease models by deriving iPSCs from skin cells of an Alzheimer’s patient, correcting a mutation with CRISPR and finally differentiating these patient iPS cells into any desired neuronal specific cell model. However, one of the hurdles for iPSC generation is the delivery of large episomal DNA vectors, which encode for the reprogramming factors. This process is fairly inefficient and there is a risk that the DNA will integrate into the host cell genome. Alternatively to DNA, transfection of mRNA requires that the cargo enters only the cell cytoplasm, not the nucleus, and therefore mitigates the risk of integration and more importantly greatly improves the transfection efficiency. In the present study, we developed a new mRNA delivery reagent, Lipofectamine® MessengerMAX™, that significantly improves mRNA delivery in iPS cells and neural stem cells (NSC). In particular, we demonstrated more than 2-fold improvement in transfection efficiency across multiple difficult to transfect cell models, such as primary cortical neurons, NSCs and iPSCs with this new transfection reagent. The effectiveness of gene editing tools, such as clustered regularly interspaced short palindromic repeats (CRISPRs), is contingent upon maximum expression of the Cas9 protein and guide-RNAs in the cell of interest. These tools allow for precise cleavage of DNA at a specific locus, opening the door for endogenous repair mechanisms to insert new sequences or mutate existing ones. However, DNA based delivery in these cell models continues to be very difficult and this road block in hindering advancement. An mRNA based from of Cas9 with a guide-RNA results in more targeted cleavage of the host cell genome when compared to standard DNA based editing approaches. This improvement in delivery increases the yield of genetic editing in turn, simplifying downstream workflows, enabling easier and biologically relevant cell model manipulation, and enhancing site-specific insertion or deletion of transgenes in the cellular genome for the generation of knock-in or knock-out cell models and transgenic small animal models.
Nektaria Andronikou, Xin Yu, Sean Essex, Yue Geng, Natasha Roark, Bonnie Hammer, David Piper, Namritha Ravinder, Xavier de Mollerat du Jeu Thermo Fisher Scientific, Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008
Results • Lipofectamine® MessengerMAX™ mRNA Delivery
Conclusions • Lipofectamine® MessengerMAX™ is a new reagent that has shown to improve
delivery of mRNA in primary cells and difficult to transfect cells. • Improved gene editing with an mRNA based from of Cas9 + a gRNA and
Lipofectamine® MessengerMAX™ when compared to standard DNA delivery methods.
• mRNA delivery has become the “go-to” method for a variety of applications that have been difficult to execute in the past, and will help to propel many new and exciting applications and technologies forward.
Acknowledgments • Synthetic biology R&D team for their support in providing the required CRISPR
editing tools and expertise in design and genome engineering techniques. • R&D stem cell team for their support in providing all the necessary cell models for
many of the above referenced experiments.
Trademarks/Licensing ©2014 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
Lipofectamine® MessengerMAX™ - A new transfection reagent for improved CRISPR-based genomic editing in neural stem cells (NSCs) and induced pluripotent stem cells (iPSCs)
Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • lifetechnologies.com
Figure 5. Disease Model Development
CRISPR-based Genomic Editing Results • Reprogramming
A. B.
Materials & Methods • Reprogramming
iPSCs (wild type or
with mutations)
iPSCs (mutated or corrected)
Genome Editing
Differentiation
Differentiation
Phenotypic Comparison
-10 -9 -8 -7 -6 -5 -4 -30
10000
20000
30000
40000
50000
6 HydroxydopamineRotenone
PrestoBlue
Log [Compound]
Relat
ive F
luor
esce
nce
Units
Patient-derived iPSCs offer exciting potential in both cell therapy and in vitro disease modeling by enabling access to cell populations that are otherwise unavailable from living donors. With the recent discovery of site-specific gene editing, this true power is fast approaching. For example, an iPS cell can be genetically altered at a specific locus, using genome engineering tools such as CRISPR or TAL, differentiated to a neuronal cell type and be used for more targeted drug screening and design.
Figure 1. Transfection – DNA vs mRNA
DNA transfection is a four step process that involves attachment to the cell, endocytosis into the cell, endosomal escape and nuclear entry for transcription to mRNA. The mRNA is then exported from the nucleus to the cytoplasm and translated into a protein of interest. In non-dividing primary cells, DNA entry to the nucleus tends to be the limiting step for many molecular biology experiments where a desired protein needs to be expressed by the cells. Delivery of mRNA bypasses the nuclear entry step and is present in the cytoplasm for direct translation into a protein of interest, regardless of the cell type being used. This process yields a much higher transfection efficiency and in turn a higher level of protein expression.
Materials and Methods • mRNA synthesis and Transfection
Figure 2. mRNA Workflow
- 1 - Prepare DNA
template
- 2 - Generate mRNA
- 3 - Transfect mRNA
For support, visit www.lifetechnologies.com/support.
Lipofectamine® MessengerMAX™ Reagent mRNA Transfection Protocol
-2-
Transfect cells according to the following table. Use the indicated volume of transfection reagent at the two recommended doses as a starting point for optimization.Volumes in each column are for a single well. Scale the volumes proportionally for additional wells.
Each reaction mix volume is for one well and accounts for pipetting variations.
Timeline Steps Procedure Details (Two Reaction Optimization)
Day
0
1
Seed cells to be 70–90% confluent at transfection
Component 96-well 24-well 6-well
Adherent cells 1–4 × 104 0.5–2 × 105 0.25–1 × 106
Day
1
2
Diluted MessengerMAX™ Reagent
Vortex 2–3 sec
Dilute MessengerMAX™ Reagent in Opti-MEM® Medium (2 tubes)
– Mix well
Opti-MEM® Medium 5 μL × 2 25 μL × 2 125 μL × 2
Lipofectamine® MessengerMAX™ Reagent 0.15 and 0.3 μL 0.75 and 1.5 μL 3.75 and 7.5 μL
3
Diluted mRNAPrepare Diluted mRNA master mix by adding mRNA to Opti-
MEM® Medium – Mix well
Opti-MEM® Medium 10 μL 50 μL 250 μL
mRNA (0.5–5 μg/μL) 0.2 μg 1 μg 5 μg
4
Add Diluted mRNA to each tube of Diluted MessengerMAX™
Reagent (1:1 ratio)
Diluted mRNA 5 μL 25 μL 125 μL
Diluted Lipofectamine® MessengerMAX™ Reagent 5 μL 25 μL 125 μL
5
Incubate Incubate for 5 minutes at room temperature.
6
Add mRNA-lipid complex to cells
Component (per well) 96-well 24-well 6-wellmRNA-lipid complex 10 μL 50 μL 250 μLmRNA 100 ng 500 ng 2500 ngLipofectamine® MessengerMAX™ Reagent 0.15 and 0.3 μL 0.75 and 1.5 μL 3.75 and 7.5 μL
Day
2–3
7
Visualize/analyze transfected cells
Incubate cells for 1–2 days at 37°C. Then, analyze transfected cells.
DNA
Preparation of mRNA is performed by first prearing a DNA template with a T7 promoter, then transcribing mRNA using an in vitro transcription kit. DNA used for transcription can be cloned into a T7 containing vector or PCR amplified with a T7-containing forward primer. This linearized DNA can then be used with the mMessage mMachine® T7 Ultra Transcription Kit for mRNA synthesis where an ARCA cap and poly(a) tail are also incorporated so as to mimic endogenous mRNA. The MEGAclear® kit is used to purify mRNA which can then be used directly for transfection with Lipofectamine® MessengerMAX™.
Figure 3. GFP mRNA Delivery
A. Primary cells
Neurons Keratinocytes Hepatocytes BJ Fibroblast
B. Human and mouse derived stem cells
iPSC H9 ESC hNSC mESC
C. Difficult to transfect cell lines
bEND3 SH-SY-5Y SK-N-SH L929
RAW 264.7 HT-29 A431 MCF7
Lipofectamine® MessengerMAX (0.75ul or 1.5ul per well) was used to deliver 500ng/well of GFP mRNA in a 24-well format in (A) primary cells, (B) stem cells and (C) difficult to transfect cancer cell lines. GFP expression was analyzed 24-hours post-transfection for transfection efficiency and intensity of expression.
Figure 4. Reagent Comparison
Fold
Impr
ovem
ent o
f Pro
tein
Exp
ress
ion
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
Mirus® TransIT®-mRNA Lipofectamine® 2000 Lipofectamine® MessengerMAX™Reagent
Results depicted for mRNA delivery are a summary from thirteen experiments, within thirteen different cell types (A549, BJ Fibroblast, HepG2, HT29, iPSC, L929, LnCap, MCF7, MDA-MB-231, N2a, Neurons, SH-SY-5Y, SK-N-SH) and multiple reagent doses. Transfections were performed with the indicated reagents and GFP mRNA (100ng/well) in 96-well plate format and GFP expression was determined 24-hours post-transfection via flow cytometry.
Table 1. Advantages of mRNA
Pros of mRNA Cons of mRNA
MUCH IMPROVED Transfection Efficiency Handling of mRNA
No genomic integration Preparation time
Faster protein expression Duration of protein expression
Homogeneity of protein expression Intensity of protein expression
DNA vs. mRNA Transfection Figure 6. Protocol Outline for generating iPSCs
§ Day (-1): Seed cells on Geltrex® Matrix coated dishes
§ Day 0: Transfect in Fibroblast Media for 24 hour
§ Day 1-14: Culture in N2B27 Medium
with 100ng/mL bFGF (change daily)
§ Day 15-20: Culture in E8 Medium (change daily)
§ Day 21+: Manually pick and
expand colonies
Day 0 Day 15 Day 21
Fibroblast Medium N2B27 Medium with bFGF (100ng/mL) Essential™ 8 Medium
Geltrex® Matrix
Overview of protocol outline for generating iPS cells with Epi5™ Episomal reprogramming kit and Lipofectamine® 3000 reagent. For a complete protocol detailing required materials and step-by-step instructions, please visit: www.lifetechnologies.com/3000
Somatic Cell Colony formation begins
Identify ESC-like colonies
Lipofectamine® 3000
Epi5™ Episomal Reprogramming Kit
• CRISPR Delivery Formats
DNA Format mRNA format
Lipofectamine® 3000 Lipofectamine® MessengerMAX™
Measure Cleavage Efficiency with Genomic Cleavage Detection (GCD)
Colony Screening and Expansion
6 Life Technologies™ | Lipofectamine® 3000 reagent
Genome editing of pluripotent stem cells
Figure 4. Lipofectamine 3000® improves cleavage effi cency of CRISPR vectors in iPSCs. A GeneArt® CRISPR vector targeting a specifi c locus was transiently transfected using Lipofectamine® 2000 or Lipofectamine® 3000 reagent. Cleavage effi ciency was assessed using the GeneArt® Genomic Cleavage Detection Kit.
Genome editing technology derived from clustered regularly interspaced short palindromic repeats (CRISPRs) allows precise cleavage of DNA at specifi c loci. However, the effectiveness of genome editing is contingent upon the intrinsic properties of the locus of interest, effi ciency of delivery, and the painstaking downstream processes of generating stable cell lines and knockout models to study the phenotypic effects of such genetic modifi cations. Here we demonstrate that Lipofectamine® 3000 can deliver plasmid DNA into various iPSC clones for genome engineering purposes using targeted CRISPR vectors. More than 2-fold increase in gene modifi cation effi ciency compared to Lipofectamine® 2000 reagent was observed using Lipofectamine® 3000 reagent, and with a lower lipid amount (Figure 4).
TK pA
U6 promoterCACCG
CAAAAPol IIIterm
tracrRNA
F1 origin
OFP
2A
Cas9 (with NLS1 and NLS2)
PCMV
Ampicillin
pUC origin
CRISPR Nuclease OFP Reporter 9,219 bp
Target-specific crRNA
Target genomic loci PAM
C G T A A A G C C A T A C G T A T A C T A C C
G C A T T T C G G T A T G C A T A T G A N G G
G G GGC C CA AA
AU U U UG
AA UU U GA U
tracrRNAA A A A A A
AAAAA
A
A
AAAAA
A AA
G
G
GG
G
G
G G G
G
G
GG
GGG
G
G
G C C C CC
C
C
C
CC
CC
UU
U U U
UU
U
U
UUUU
U
U U
UUU
AA
A AA
U
UU
U
Cas9
Indel in genomic DNA
500 bp400
300
200
100
Gene modifi cation effi ciency (%)
2.5 3.6 5.5 4.0 0.0
1 kb
ladd
er
Lipo
fect
amin
e®
2000
Lipo
fect
amin
e®
3000
GFP
cont
rol
vect
or
Dosage (µL) 0.3 0.5 0.15 0.3 0.3
Design insert for GeneArt® CRISPR vector
Deliver with Lipofectamine® 3000 reagent
Measure cleavage effi ciency using GeneArt® Genomic Cleavage Detection (GCD) Kit
Colony screening and expansion
6 Life Technologies™ | Lipofectamine® 3000 reagent
Genome editing of pluripotent stem cells
Figure 4. Lipofectamine 3000® improves cleavage effi cency of CRISPR vectors in iPSCs. A GeneArt® CRISPR vector targeting a specifi c locus was transiently transfected using Lipofectamine® 2000 or Lipofectamine® 3000 reagent. Cleavage effi ciency was assessed using the GeneArt® Genomic Cleavage Detection Kit.
Genome editing technology derived from clustered regularly interspaced short palindromic repeats (CRISPRs) allows precise cleavage of DNA at specifi c loci. However, the effectiveness of genome editing is contingent upon the intrinsic properties of the locus of interest, effi ciency of delivery, and the painstaking downstream processes of generating stable cell lines and knockout models to study the phenotypic effects of such genetic modifi cations. Here we demonstrate that Lipofectamine® 3000 can deliver plasmid DNA into various iPSC clones for genome engineering purposes using targeted CRISPR vectors. More than 2-fold increase in gene modifi cation effi ciency compared to Lipofectamine® 2000 reagent was observed using Lipofectamine® 3000 reagent, and with a lower lipid amount (Figure 4).
TK pA
U6 promoterCACCG
CAAAAPol IIIterm
tracrRNA
F1 origin
OFP
2A
Cas9 (with NLS1 and NLS2)
PCMV
Ampicillin
pUC origin
CRISPR Nuclease OFP Reporter 9,219 bp
Target-specific crRNA
Target genomic loci PAM
C G T A A A G C C A T A C G T A T A C T A C C
G C A T T T C G G T A T G C A T A T G A N G G
G G GGC C CA AA
AU U U UG
AA UU U GA U
tracrRNAA A A A A A
AAAAA
A
A
AAAAA
A AA
G
G
GG
G
G
G G G
G
G
GG
GGG
G
G
G C C C CC
C
C
C
CC
CC
UU
U U U
UU
U
U
UUUU
U
U U
UUU
AA
A AA
U
UU
U
Cas9
Indel in genomic DNA
500 bp400
300
200
100
Gene modifi cation effi ciency (%)
2.5 3.6 5.5 4.0 0.0
1 kb
ladd
er
Lipo
fect
amin
e®
2000
Lipo
fect
amin
e®
3000
GFP
cont
rol
vect
or
Dosage (µL) 0.3 0.5 0.15 0.3 0.3
Design insert for GeneArt® CRISPR vector
Deliver with Lipofectamine® 3000 reagent
Measure cleavage effi ciency using GeneArt® Genomic Cleavage Detection (GCD) Kit
Colony screening and expansion
Various CRISPR editing formats are available based on editing needs. The GeneART® All-in-One CRISPR vectors, which contain reporters for enrichment, can be delivered with the Lipofectamine® 3000 transfection reagent kit. The mRNA formats, which contain a Cas9 mRNA and either a DNA gRNA format or an in vitro transcribed mRNA gRNA format, can be delivered with Lipofectamine® MessengerMAX™. Cleavage efficiency and recombination efficiency can be determined using the Genomic Cleavage Detection kit. The optimum transfection efficiency will help to minimize downstream clonal selection and expansion.
Figure 7. Available CRISPR formats and workflow
BJ HDFn HDFa
Lipofectamine® 3000 Reagent
Neon® Electroporation
System
Figure 8. Alkaline phosphatase stain for iPSC colony visualization of cells transfected with Epi5™ Episomal reprogramming vectors with Lipofectamine® 3000 or Neon® Electroporation System
Transfection performed in BJ fibroblast, neonatal human dermal fibroblast (HDFn) and adult human dermal fibroblast cells using Neon® Transfection System at recommended conditions and Lipofectamine® 3000, 3.6ul per well in a 6-well culture plate. Epi5™ Episomal Reprogramming vector was used. Media changes performed daily according to Life Technologies™ protocol: Generation of Human Induced Pluripotent Stem Cells (hiPSCs) from Fibroblasts using Episomal Vectors. A terminal stain was performed with red alkaline phosphatase at 18 days post-transfection.
• CRISPR gene editing
Transfection Reagent
Mes
seng
erM
AX
™
Lipo
fect
amin
e®
3000
Com
petit
or A
Com
petit
or B
pcD
NA
™
Con
trol
Nucleic Acid Format
mRNA DNA mRNA mRNA None
% cleavage efficiency
36.3 4.5 9.9 5.6 0.1
Figure 9. Improved gene editing with Lipofectamine® MessengerMAX™ and GeneART® CRISPR Nuclease mRNA
A.
B.
0
5
10
15
20
25
30
35
40
Messenger MAX™ Competitor A Competitor B Lipofectamine® 3000
Cells Only
% C
leav
age
Eff
icie
ncy
Cas9 mRNA + IVT gRNA
All-in-One CRISPR plasmid
Cleavage efficiency of various GeneART® CRISPR formats targeting the HPRT locus in Gibco iPS cells in a 12-well format. Lipofectamine® 3000 was used to deliver CRISPR Nuclease All-in-One plasmid DNA; Lipofectamine® MessengerMAX™ and two leading mRNA delivery reagents were used to deliver an all RNA CRISPR format (GeneART® CRISPR Nuclease Cas9 mRNA + IVT gRNA). Cleavage efficiency was determined using the GeneART® Genomic Cleavage Detection Kit 72-hours posttransfection.
Day -1