GeneArt CRISPR Nuclease mRNA Ready-to-transfect wild-type Cas9 mRNA for performing

USER GUIDE
GeneArt® CRISPR Nuclease mRNA
Ready-to-transfect wild-type Cas9 mRNA for performing
CRISPR-Cas9 mediated genome editing
Catalog Number A25640
Publication Part Number MAN0010804
Revision A.0
For Research Use Only. Not for use in diagnostic procedures.
For Research Use Only. Not for use in diagnostic procedures.
Information in this document is subject to change without notice.
DISCLAIMER
LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS
DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR
A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE
TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY
STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL
DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE
THEREOF.
IMPORTANT LICENSING INFORMATION
This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms
and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS
© 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and
its subsidiaries unless otherwise specified.
ii
Contents
Contents and Storage ................................................................................................................................................ iv
Introduction ....................................................................................................................................................... 1
Methods ............................................................................................................................................................. 3
General Guidelines ..................................................................................................................................................... 3
Target Sequences ........................................................................................................................................................ 4
Ready-to-transfect Protocol ....................................................................................................................................... 5
Complete RNA Protocol ............................................................................................................................................ 6
Transfection Guidelines ............................................................................................................................................. 8
Troubleshooting .........................................................................................................................................................10
Appendix A ......................................................................................................................................................12
Technical Support ......................................................................................................................................................12
Accessory Products ...................................................................................................................................................13
References ...................................................................................................................................................................14
Appendix B: Transfection ..............................................................................................................................15
®
™
Appendix C: Cloning GeneArt CRISPR Strings DNA ..............................................................................16
iii
Contents and Storage
The contents of the GeneArt® CRISPR Nuclease mRNA Kit (Cat. no. A25640) are
listed below.
Contents
Reagent
Composition
Amount
CRISPR nuclease mRNA (1.0 μg/μL)
Cas9 mRNA in 10 mM Tris buffer
1 × 15 µL
®
Required materials The following reagents are not included with GeneArt CRISPR Nuclease mRNA
Kit, but are necessary to perform CRISPR-mediated gene editing.
not supplied with
Additional details on GeneArt® CRISPR Strings™ DNA encoding custom ordered
kit
target-specific guide RNA (gRNA) are provided on page 4.
Product
Catalog No.
•
GeneArt CRISPR U6 Strings DNA (Ready-to-transfect
format) OR
•
In vitro transcribed gRNA (Complete RNA format)
®
o
o
™
GeneArt® CRISPR T7 Strings™ DNA,
Ready to use in vitro transcribed gRNA
(Contact customservices@lifetech.com)
Contact GeneArtSupport@lifetech.com
MEGAshortscript™ T7 Transcription Kit (Complete RNA format)
AM1354
MEGAclear Transcription Clean-Up Kit (Complete RNA format)
AM1908
Transfection reagent suitable for your cell line
—
GeneArt® Genomic Cleavage Detection Kit
A24372
™
Storage
GeneArt® CRISPR Nuclease mRNA is shipped on dry ice.
Upon receipt, keep mRNA at –80°C for long term storage. Working stock can be
stored at –20°C for short term.
iv
Introduction
Product
information
The GeneArt® CRISPR Nuclease mRNA Kit consists of ready-to-transfect wild-type
Cas9 mRNA for performing CRISPR/Cas9 mediated genome editing. The mRNA
format allows the experiment to proceed without the need for time consuming
cloning steps required when using CRISPR vector systems.
The Cas9 mRNA can be used in experiments through two methods:
•
Ready-to-transfect format
Cas9 mRNA is co-transfected directly with custom GeneArt® CRISPR U6
Strings™ DNA or other synthetic gRNA expression cassettes.
•
Complete RNA format
Cas9 mRNA is co-transfected with in vitro transcribed gRNA. In vitro
transcribed gRNA can be generated from GeneArt® CRISPR T7 Strings™
DNA or other custom templates.
Following transfection the Cas9 protein generated by the mRNA is directed by the
crRNA sequence of the gRNA to the encoded genomic locus to perform the desired
genome editing.
Using GeneArt® CRISPR Nuclease mRNA makes it easy to optimize the ratio of
Cas9 to gRNA for optimal genome targeting efficiency. Cas9 mRNA and in vitro
transcribed gRNA are especially useful to circumvent promoter restrictions or for
use in microinjection.
Use of this kit for genome editing assumes that users are familiar with the
principles of the CRISPR system, handling RNA, transfection in mammalian
systems, and downstream processing of cells for detection of genomic cleavage.
GeneArt® CRISPR
Strings™ DNA
GeneArt® CRISPR Strings™ DNA are offered for custom order with either U6 or T7
promoters. They are 500 base pair synthetic DNA fragments designed to generate
the gRNA component of the CRISPR system. GeneArt® CRISPR Strings™ DNA
allow easy usage and screening of target specific gRNAs, while avoiding cloning
procedures.
The CRISPR system The CRISPR (clustered regularly interspaced short palindromic repeats) system is a
prokaryotic adaptive immune system that uses a RNA guided DNA nuclease to
silence viral nucleic acids (Jinek et al., 2012). In bacteria CRISPR loci are composed
of a series of repeats separated by segments of exogenous DNA (of ~30 bp in
length), called spacers. The repeat-spacer array is transcribed as a long precursor
and processed within repeat sequences to generate small crRNAs that specify the
target sequences (also known as protospacers) cleaved by Cas9 protein, the
nuclease component of CRISPR system. CRISPR spacers are then used to recognize
and silence exogenous genetic elements at the DNA level. Essential for cleavage is
a sequence motif immediately downstream on the 3’ end of the target region,
known as the protospacer-adjacent motif (PAM). The PAM is present in the target
DNA, but not the crRNA that targets it (Figure 1).
GeneArt® CRISPR Nuclease mRNA User Guide
1
Product Information, continued
Genome editing
Genome editing involves the use of engineered nucleases in conjunction with
endogenous repair mechanisms to insert, delete, or replace DNA sequences from a
specific location in genomic DNA. The CRISPR system has been shown to function
as a gene editing tool in various organisms including mammalian cells. (Mali1 et al.,
2013; Cong et al., 2013). The system simplifies genome editing, and has potential in
applications such as stem cell engineering, gene therapy, tissue and animal disease
models, and engineering disease-resistant transgenic plants (Andrew et. al., 2013;
Ota et. al., 2014; Ma et. al., 2014; Haoyi et. al., 2013; Qiwei et. al. 2013).
The CRISPR system consists of a short non-coding guide RNA (gRNA) made up of
a target complementary CRISPR RNA (crRNA), and an auxiliary trans-activating
crRNA (tracrRNA).
The gRNA guides the Cas9 endonuclease to a specific genomic locus via base
pairing between the crRNA sequence and the target sequence, and cleaves the
DNA to create a double-strand break (Figure 1).
Figure 1 Schematic representation of CRISPR/Cas9 mediated target DNA cleavage.
Following DNA cleavage, the break is repaired by cellular repair machinery
through non-homologous end joining (NHEJ) or homology-directed repair (HDR)
mechanisms.
2
GeneArt® CRISPR Nuclease mRNA User Guide
Methods
General Guidelines
Guidelines for
handling RNA
•
Always use RNase-free reagents and plasticware.
•
Clean workspace/benches/laminar hood surfaces using RNAse
decontaminating reagents like RNaseZap® Solution (see page 13).
•
Decontaminate pipets with RNaseZap® Solution or similar reagent.
•
Change gloves after touching any potentially contaminated object or surface.
Selecting a suitable Depending upon the requirements of your experiment, select a system from the
following table.
system
If you are using the GeneArt® CRISPR Nuclease mRNA with GeneArt® CRISPR U6
Strings™ DNA, follow the experimental protocol on page 5.
If you are using the GeneArt® CRISPR Nuclease mRNA with in vitro transcribed
(IVT) gRNA, follow the experimental protocol on page 6.
Requirements
GeneArt® CRISPR
Nuclease mRNA + U6
Strings™ DNA
GeneArt® CRISPR
Nuclease mRNA + IVT
gRNA
Ready to transfect
√
—*
High efficiency with broad cell type application
—
√
Avoid random integration associated with DNA
—
√
Suitability for microinjections**
—
√
* In vitro transcribed (IVT) gRNA can be prepared from a synthetic DNA template such as GeneArt®
CRISPR T7 Strings™ DNA, or a user-defined template. Ready to use IVT gRNA can also be ordered
through custom services by contacting customservices@lifetech.com.
** Potential applications of Cas9 mRNA include generation of transgenic model systems, but
microinjection and other in vivo delivery methods have not been tested using the GeneArt® CRISPR
Nuclease mRNA. However, a significant number of articles have described Cas9 mRNA use for in vivo
applications in a wide variety of organisms including mouse, rat, zebrafish and Drosophila (Haoyi et. al.,
2013; Ma et. al., 2014; Ota et. al., 2014; Andrew et. al., 2013).
GeneArt® CRISPR Nuclease mRNA User Guide
3
Target Sequences
GeneArt® CRISPR
Strings™ DNA
GeneArt® CRISPR Strings™ DNA (T7 or U6) are custom-made, 500 bp uncloned,
double-stranded linear DNA fragments, assembled from synthetic
oligonucleotides using the process developed for GeneArt® high-quality gene
synthesis.
GeneArt® CRISPR Strings™ DNA have the following features:
Ordering CRISPR
sequences
•
Designed to generate the gRNA component of the CRISPR system.
•
Delivered dried with ≥200 ng DNA, ready for resuspension.
•
Can be introduced directly into cells (U6) or used as template for in vitro
transcription (T7).
•
Can be multiplexed (using U6 Strings™ DNA or IVT gRNA with different
target sequences) so that multiple target gene sequences can be edited
simultaneously in a single transfection reaction.
•
Can be cloned into a suitable vector when more of the U6 or T7 gRNA
expression cassette is required.
GeneArt® offers support for designing and ordering CRISPR sequences. Obtain the
order form on the www.lifetechnologies.com/CRISPRmRNA.
To design and order your target specific GeneArt® CRISPR Strings™ DNA:
•
Select a 25– 500 bp target CRISPR DNA binding sequence from within
your gene of interest. Submit your completed order form to
GeneArtsupport@lifetech.com, and GeneArt® support will generate a set
of ranked CRISPR sequences which can be chosen for synthesis as either
T7 or U6 Strings™ DNA.
OR
•
Select a target specific CRISPR sequence (crRNA sequence). Submit your
completed order form to GeneArtsupport@lifetech.com, to order T7 or U6
Strings™ DNA.
Because cleavage efficiency of a CRISPR sequence at its target depends upon many
different factors, choose at least three CRISPR sequences against a gene of
interest to identify the CRISPR sequence with best cleavage efficiency.
Creating your own
synthetic gRNA
expression
cassette
4
If you have your own system for expressing gRNA, it is not necessary to order
GeneArt® CRISPR Strings™ DNA.
For details on creating your own synthetic gRNA expression cassette see
Appendix C (page 16).
GeneArt® CRISPR Nuclease mRNA User Guide
Ready-to-transfect Protocol
Experimental
outline for readyto-transfect format
Step
1
2
The ready-to-transfect protocol involves the direct co-transfection of GeneArt®
CRISPR Nuclease mRNA and GeneArt® CRISPR U6 Strings™ DNA (or other synthetic
gRNA expression cassette) into your cell line of interest.
Action
Select a target CRISPR sequence
and place an order for the
appropriate GeneArt® CRISPR U6
Strings™ DNA
Co-transfect The U6 Strings™ DNA
with GeneArt® CRISPR Nuclease
mRNA
Page
4
5, 8
Analyze sample for % gene
modification 48 or 72 hours post
transfection using:
3
•
GeneArt® Genomic Cleavage
Detection Assay (see page 13)
•
Sequencing (performed to
confirm the edited sequence)
Materials needed
Experimental
protocol
―
•
GeneArt® CRISPR Nuclease mRNA
•
GeneArt® CRISPR U6 Strings™ DNA or other synthetic expression cassette
•
Transfection reagent
•
Cell line of interest
1.
Pellet the lyophilized GeneArt® CRISPR Strings™ DNA by centrifugation at
room temperature for 30 seconds.
2.
Resuspend GeneArt® CRISPR Strings™ DNA in 20 µL of nuclease-free water.
3.
Centrifuge the resuspended DNA at high speed for 30 seconds to collect
contents at the bottom of the tube.
4.
Determine the DNA concentration using a NanoDrop™ spectrophotometer or
an equivalent system.
5.
Record the concentration and dilute to the required working concentration.
For assays using a 24-well plate format, we recommend diluting the U6
Strings™ DNA to ≥20–50 ng/µL.
6.
Proceed to transfection (page 8).
GeneArt® CRISPR Nuclease mRNA User Guide
5
Complete RNA Protocol
Experimental
outline for
complete RNA
format
Step
The complete RNA protocol involves the co-transfection of GeneArt® CRISPR
Nuclease mRNA, and in vitro transcribed gRNA into your cell line of interest.
The format requires the production (or ordering) of gRNA using GeneArt® CRISPR T7
Strings™ DNA (or other gRNA encoding sequence) as a template.
Action
Page
1
Select a target CRISPR sequence
and place an order for the
appropriate GeneArt® CRISPR T7
Strings™ DNA
4
2
Prepare in vitro transcribed (IVT)
gRNA from T7 Strings™ DNA OR
Order ready-to-transfect in vitro
transcribed gRNA custom service
6–7
3
Co-transfect IVT gRNA with
GeneArt® CRISPR Nuclease mRNA
8
Analyze sample for % gene
modification 48 or 72 hours post
transfection using:
4
•
GeneArt® Genomic Cleavage
Detection Assay (see page 13)
•
Sequencing (performed to
confirm the edited sequence)
Materials needed
―
•
GeneArt® CRISPR Nuclease mRNA
•
Guide RNA (gRNA) ordered as either one of the following options
o
GeneArt® CRISPR T7 Strings™ DNA (template for IVT gRNA) OR
o
in vitro transcribed gRNA
•
Megashortscript™ T7 Transcription Kit (see page 13)*
•
Megaclear™ Transcription Clean-Up Kit (see page 13)*
•
Thermocycler or a heat block*
•
Transfection reagent
•
Cell line of interest
* Not required if using ready-to-use in vitro transcribed gRNA
6
GeneArt® CRISPR Nuclease mRNA User Guide
Complete RNA Protocol, continued
Experimental
protocol
If you have ordered ready-to-use in vitro transcribed gRNA, proceed directly to
transfection (page 8).
For a detailed protocol for generating IVT gRNA, refer to Megashortscript™ T7
Transcription Kit manual.
1.
Use 100 ng of DNA in a 20 µL reaction to set up an in vitro transcription
reaction according to the following table:
Component
GeneArt® CRISPR T7 Strings™ DNA
T7 10X Reaction Buffer
T7 ATP Solution (75 mM)
T7 CTP Solution (75 mM)
T7 GTP Solution (75 mM)
T7 UTP Solution (75 mM)
(optional) Labeled ribonucleotide
T7 Enzyme Mix
Water (Nuclease-free)
Amount
100 ng (≤8 μL)
2 μL
2 μL
2 μL
2 μL
2 μL
~1 μL
2 μL
to 20 μL final volume
2.
Incubate reaction at 37°C for 3–4 hours.
3.
Add 1 µL of Turbo DNase to the reaction and incubate at 37°C for 30 minutes.
4.
Perform purification of IVT gRNA using the Megaclear™ Transcription CleanUp Kit. For a detailed protocol, refer to kit manual.
5.
Prior to elution, preheat 110 µL of elution buffer to 95°C.
6.
Add 50 µL of heated elution buffer to the column and centrifuge to elute the
gRNA.
7.
(Optional) Add 50 µL of heated elution buffer to the column and centrifuge.
Combine the eluates for a total yield of 100 µL of eluate.
8.
Determine the concentration of the eluate using a NanoDrop™ spectrophotometer or an equivalent system. The typical yield of gRNA is 10–40 µg.
9.
Check the quality of the gRNA by performing electrophoresis on a denaturing
acrylamide gel. The expected gRNA transcript size is 100 bases. A discreet
band at 100 bases indicates intact RNA.
Quality can also be checked on a 4% agarose gel but a control RNA of known
size is required to compare the size of RNA bands with this method.
GeneArt® CRISPR Nuclease mRNA User Guide
7
Transfection Guidelines
Methods of
transfection
The delivery reagent is critical for transfection and gene editing efficiency, and
results will vary based on the cell type being used. Refer to our transfection
reagent recommendation guidelines at
www.lifetechnologies.com/us/en/home/life-science/cellculture/transfection/transfection-reagent-application-table.html
Additional details on cell lines and transfection reagents are listed in Appendix B
(page 15).
Lipofectamine®
MessengerMax™
reagent
CRISPR
transfection
guidelines
When performing transfection using GeneArt® CRISPR Nuclease mRNA we
recommend using Lipofectamine® MessengerMax™ reagent.
Lipofectamine® MessengerMax™ can be used for broad cell type applications and
difficult to transfect cell lines, and results in high genome editing efficiency when
used in conjuction with the complete RNA format.
Perform transfection using standard 24-well plates. This plate size is convenient
when screening different CRISPR sequences to identify the most suitable and
effective candidate for genome editing.
•
Seed cells 24 hours prior to transfection so that they are 70–90% confluent on
the day of transfection.
Note: Seeding density varies with cell type. See Appendix B (page 15).
•
Perform transfection using an appropriate transfection reagent. Use the
amounts of nucleic acid in the following table as a starting point
(optimization may be required).
For multiplex experiments, up to three target sequences can be combined at
the same time.
Component
GeneArt® CRISPR Nuclease mRNA
GeneArt® CRISPR U6 Strings™ DNA/IVT gRNA
•
Genomic cleavage
efficiency
8
Singleplex
0.5 μg
50 ng
Multiplex
1.0 μg
50 ng/target
If using different well formats, scale the recommended amounts based on the
transfection plate format. In cases where the amount of GeneArt® CRISPR
Strings™ DNA is limiting, the GeneArt® CRISPR Strings™ DNA can be cloned
into a plasmid vector. See Appendix C (page 16).
After transfection, perform a genomic cleavage detection assay. This technique
leverages mismatch detection endonucleases to detect genomic insertions or
deletions (indels) incorporated during cellular NHEJ repair mechanisms. The
GeneArt® Genomic Cleavage Detection Kit (see page 13) is recommended for
performing cleavage efficiency analysis.
GeneArt® CRISPR Nuclease mRNA User Guide
Transfection Guidelines, continued
Controls
Include controls in the experiment to evaluate your results. A negative control can
be mock/untransfected cells.
CRISPR target
positive control
For a positive control, use a gRNA whose efficiency at the genomic level is
already known. In the absence of such a control, the following CRISPR sequence
targeting human HPRT gene has been validated for cleavage efficiency using the
GeneArt® Genomic Cleavage Detection Kit in 293 FT cell lines.
5’–CATTTCTCAGTCCTAAACA–3’
The corresponding target in the genomic DNA for this CRISPR sequence is:
CATTTCTCAGTCCTAAACAGGG
The PAM (underlined) is a feature of the genomic locus and not included in the
CRISPR RNA coding sequence.
The primer sequences to perform PCR when using the GeneArt® Genomic
Cleavage Detection Kit are:
Forward:
5’–ACATCAGCAGCTGTTCTG–3’
Reverse:
5’–GGCTGAAAGGAGAGAACT–3’
Example of
transfection
This example protocol describes transfection of 293 FT cells using Lipofectamine®
MessengerMAX™ reagent. Optimization of conditions is required when using
other cell lines and transfection reagents.
1.
Seed 0.2 × 106 cells/well in 24-well tissue culture dish one day prior to the
experiment.
2.
Label and prepare tubes for appropriate experimental and controls samples.
3.
Dilute transfection reagent by adding 25 µL of Opti-MEM® Medium and
1.5 µL of Lipofectamine® MessengerMAX™ reagent. Mix gently.
4.
Dilute RNA by adding 25 µL of Opti-MEM® Medium and 50 ng of IVT gRNA
GeneArt® CRISPR U6 Strings™ DNA, and 0.5 µg of GeneArt® CRISPR
Nuclease mRNA. Mix gently.
5.
Add the diluted RNA to the diluted transfection reagent and mix gently.
6.
Incubate at room temperature for 5 minutes to allow formation of RNA-lipid
complexes.
7.
Add RNA-lipid complexes to the cells to be transfected. Shake the plates
gently to allow mixing of transfection mixture with the medium.
8.
Incubate the cells at 37° C for 48–72 hours.
9.
Harvest the cells and measure cleavage efficiency using the CRISPR by
GeneArt® Genomic Cleavage Detection assay.
GeneArt® CRISPR Nuclease mRNA User Guide
9
Troubleshooting
Issues with in vitro
transcription of
gRNA
When performing experiments using the complete RNA format, it is important to
follow proper RNA handling procedures (see page 3). Refer to the manual for the
Megashortscript™ T7 Transcription Kit for details on performing the IVT protocol.
Observation
Reason
No or poor IVT gRNA RNase contamination
yield
Problem with the IVT reaction
10
Solution
•
Ensure that the water used to reconstitute
GeneArt® CRISPR Strings™ DNA is RNase
free.
•
Clean all the work areas with RNase
decontaminating agents like RNaseZap®
Solution .
•
Ensure all the tubes and tips used are
RNase free.
If using a PCR amplified or plasmid-based
template, please ensure these reagents are
clean and RNAse free. Plasmid
purification kits frequently introduce
ribonucleases into the purified plasmids.
•
Run the positive control reaction included
with the Megashortscript™ T7
Transcription Kit to ensure the kit is
performing well. The positive control
should generally yield >90 µg of RNA
however the yield of gRNA is not
expected to be high. Yield of gRNA is
typically 10–40 µg.
•
If the positive control worked but the
experimental sample does not produce
gRNA, collect small 1 µL aliquots at
different stages of the process (e.g., when
setting up the reaction, before and after
the DNase treatment, etc.) and run them
on a gel to identify the problematic step.
GeneArt® CRISPR Nuclease mRNA User Guide
Troubleshooting, continued
No detectable
cleavage of target
region
Observation
No detectable
cleavage of target
region
There are many factors that can result in no detectable cleavage in the target
region. Make sure to follow proper RNA handling procedures (see page 3). Refer
to the manual for the GeneArt® Genomic Cleavage Detection Kit for details on
performing the cleavage assay.
Reason
Suboptimal transfection
Solution
•
Check all the transfection conditions.
•
Check if the tranfection reagent used was
suitable for the cell line (Refer to
transfection guidelines at
http://www.lifetechnologies.com/us/en/h
ome/life-science/cellculture/transfection/transfection-reagentapplication-table.html
•
Perform a positive control reaction. A
commercially available mRNA for a
fluorescent protein (e.g., GFP) can be used
to assess transfection efficiency.
mRNA was degraded
•
Check the integrity by running 0.5–1.0 µg
of mRNA on a gel.
gRNA was degraded or
suboptimal amount used
•
Check the integrity of gRNA by
confirming it on a gel.
•
Ensure an adequate amount of gRNA is
transfected.
•
Make sure that PCR of the target region
generates a discreet (single) DNA band. If
it does not, optimize the PCR conditions.
Refer to the GeneArt® Genomic Cleavage
Detection Kit manual for more details.
•
Perform the positive control provided
with the GeneArt® Genomic Cleavage
Detection Kit. If no cleavage is observed
with the control, contact Technical
Support (page 12).
•
More than one CRISPR sequence may be
needed to identify the target that yields
the best CRISPR cleavage efficiency.
Little or no cleavage can be detected if the
Cas9/CRISPR complex cannot access the
target region. We recommend selecting at
least 3 different CRISPR target sequences
in the gene of interest to ensure that at
least one should work well.
Cleavage detection assay
protocol did not work
Target region is inaccessible to
Cas9/CRISPR complex
GeneArt® CRISPR Nuclease mRNA User Guide
11
Appendix A
Technical Support
Obtaining support
For the latest services and support information for all locations, go to
www.lifetechnologies.com
At the website, you can:
•
Access worldwide telephone and fax numbers to contact Technical Support
and Sales facilities
•
Search through frequently asked questions (FAQs)
•
Submit a question directly to Technical Support (techsupport@lifetech.com)
•
Search for user documents, SDSs, vector maps and sequences, application
notes, formulations, handbooks, certificates of analysis, citations, and other
product support documents
•
Obtain information about customer training
•
Download software updates and patches
Safety Data Sheets
(SDS)
Safety Data Sheets (SDSs) are available at www.lifetechnologies.com/support
Certificate of
Analysis
The Certificate of Analysis provides detailed quality control and product
qualification information for each product. Certificates of Analysis are available
on our website. Go to www.lifetechnologies.com/support and search for the
Certificate of Analysis by product lot number, which is printed on the box.
Limited product
warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as set
forth in the Life Technologies’ General Terms and Conditions of Sale found on
Life Technologies’ website at www.lifetechnologies.com/termsandconditions. If
you have any questions, please contact Life Technologies at
www.lifetechnologies.com/support.
12
GeneArt® CRISPR Nuclease mRNA User Guide
Accessory Products
Introduction
The products listed in this section may be used with the GeneArt® CRISPR Nuclease
mRNA. For more information, refer to our web site (www.lifetechnologies.com) or
contact Technical Support (see page 16).
Ordering
oligonucleotides
Custom oligonucleotides for use with the GeneArt® CRISPR Nuclease Vectors can
be ordered from Life Technologies. For additional details, visit our web site at
www.lifetechnologies.com/oligos
Additional products Many of the reagents suitable for use with the mRNA, and tissue culture of
mammalian cells are available separately from Life Technologies. Ordering
information for these reagents is provided below.
Item
Quantity
Catalog No.
DNase/RNase-free Microfuge Tubes
500 tubes
AM12400
RNaseZap Solution
250 mL
AM9780
Lipofectamine 2000 Reagent
1.5 mL
11668-019
Lipofectamine® RNAiMAX Reagent
0.75 mL
13778075
Lipofectamine MessengerMAX Reagent
0.3 mL
LMRNA003
GeneArt Genomic Cleavage Detection Kit
20 reactions
A24372
Zero Blunt TOPO Cloning Kit
25 reactions
K2800-20
One Shot® Top10 Competent E. coli
20 reactions
C40404-03
Purelink Quick Plasmid Miniprep Kit
50 preps
K2100-10
Megashortscript T7 Transcription Kit
25 reactions
AM1354
Megaclear™ Transcription Clean-Up Kit
20 preps
AM1908
®
®
®
™
®
®
®
™
™
GeneArt® CRISPR Nuclease mRNA User Guide
13
References
Andrew R. B., Charlotte T., Chris P. Pontingand Ji-Long Liu. (2013). Highly Efficient Targeted
Mutagenesis of Drosophila with the CRISPR/Cas9 System. Cell Rep. Jul 11, 2013; 4(1): 220–228.
Chen, C., and Okayama, H. (1987). High-Efficiency Transformation of Mammalian Cells by Plasmid
DNA. Mol. Cell. Biol. 7, 2745–2752.
Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the Efficient Transfection of Mammalian
Cells with DNA. Nucleic Acids Res. 15, 1311–1326.
Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.-P., Hawley-Nelson, P., Evans, K., Roy, L., and Bennett, S.
(1999). LipofectamineTM 2000 Reagent for Rapid, Efficient Transfection of Eukaryotic Cells. Focus 21, 54–
55.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A.,
Zhang, F. (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:6121, 819–823.
Felgner, P. L., Holm, M., and Chan, H. (1989). Cationic Liposome Mediated Transfection. Proc. West.
Pharmacol. Soc. 32, 115–121.
Felgner, P. L. a., and Ringold, G. M. (1989). Cationic Liposome-Mediated Transfection. Nature 337, 387–
388.
Fu, Y., Foden, J.A., Khayter, C., Maeder, M.L., Reyon, D., Joung, J.K., Sander, J.D. (2013) High-frequency
off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology 31, 822–
826.
Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F., and Jaenisch, R. (2013). Onestep generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome
engineering. Cell May 9;153(4):910-8.
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., Charpentier E. (2012) A Programmable
Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337:6096, 816–821.
Kunkel, G. R., Maser, R. L., Calvet, J. P., and Pederson, T. (1986). U6 Small Nuclear RNA is Transcribed by
RNA Polymerase III. Proc. Natl. Acad. Sci. USA 83, 8575-8579.
Kunkel, G. R., and Pederson, T. (1988). Upstream Elements Required for Efficient Transcription of a
Human U6 RNA Gene Resemble Those of U1 and U2 Genes Even Though a Different Polymerase is
Used. Genes Dev. 2, 196-204.
Ma, Y., Shen, B., Zhang, X., Lu, Y., Chen, W., Ma, J., Huang, X., and Zhang, L. (2014). Heritable multiplex
genetic engineering in rats using CRISPR/Cas9. PLoS One. 2014 Mar 5;9(3):e89413.
Mali, P., Aach, J., Stranges, P.B., Esvelt, K.M., Moosburner, M., Kosuri, S., Yang L., Church Church, G.M.
(2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative
genome engineering. Nature Biotechnology 31, 833–838.
Mali, P., Yang, L., Esvelt, K.M., Aach, J., Guell, M., DiCarlo, J.E., Norville, J.E., Church, G.M. (2013) RNAGuided Human Genome Engineering via Cas9. Science .339:6121, 823–826.
Ota, S., Hisano, Y., Ikawa, Y., and Kawahara, A. (2014). Multiple genome modifications by the
CRISPR/Cas9 system in zebrafish. Genes Cells. Jul;19(7):555-64.
Qiwei Shan, Yanpeng Wang, Jun Li, Yi Zhang, Kunling Chen, Zhen Liang, Kang Zhang, Jinxing Liu,
Jianzhong Jeff Xi, Jin-Long Qiu,& Caixia Gao (2013).Targeted genome modification of crop plants using a
CRISPR-Cas system. Nature Biotechnology Aug 31(8), 686-688
14
GeneArt® CRISPR Nuclease mRNA User Guide
Appendix B: Transfection
Seeding densities
for tested cell lines
Cell Line
The GeneArt® CRISPR Nuclease mRNA system has been optimized for 24-well
tissue culture plates. If using different well formats, the seeding density must be
scaled accordingly.
Seeding Density
Transfection Reagents Tested
Viability (at time
of seeding)
•
293 FT
0.2×106 cells/well
>90%
•
•
•
HeLa
0.1×106 cells/well
U2OS
0.1×10 cells/well
>85%
•
•
•
6
>90%
•
A549
0.1×106 cells/well
HCT116
0.1×10 cells/well
6
>85%
0.1×10 cells/well
•
Lipofectamine® MessengerMAX™
Reagent
Lipofectamine® RNAiMAX Reagent
>90%
>90%
•
•
Mouse Neuro2A (N2)
0.8×105 cells/well
GeneArt® CRISPR Nuclease mRNA User Guide
>85%
Lipofectamine® MessengerMAX™
Reagent
Lipofectamine® RNAiMAX Reagent
Lipofectamine® MessengerMAX™
Reagent
•
Hep-G2
Lipofectamine® MessengerMAX™
Reagent
Lipofectamine® RNAiMAX Reagent
Lipofectamine® 2000 Reagent
•
•
6
Lipofectamine® MessengerMAX™
Reagent
Lipofectamine® RNAiMAX Reagent
Lipofectamine® 2000 Reagent
•
•
Lipofectamine® MessengerMAX™
Reagent
Lipofectamine® RNAiMAX Reagent
Lipofectamine® MessengerMAX™
Reagent
Lipofectamine® RNAiMAX Reagent
Lipofectamine® 2000 Reagent
15
Appendix C: Cloning GeneArt® CRISPR Strings™ DNA
Generation of
sequence verified
gRNA expression
plasmid
GeneArt® CRISPR Strings™ DNA fragments can be cloned into a vector if a
sequence verified expression plasmid is desired. This procedure can also be used
in cases where the amount of GeneArt® CRISPR Strings™ DNA become limiting
for example when larger transfection well or plate formats are desired.
•
Use the Zero Blunt® TOPO® Cloning Kit to clone GeneArt® CRISPR Strings™
DNA fragments.
•
Sequence the resulting clones using the SP6 primer from the vector (we do not
recommend using M13 primers for sequencing GeneArt® CRISPR Strings™
DNA).
•
Sequence verified clones can be used with the following primers to amplify
the appropriate gRNA cassette and promoter.
o
T7 Strings™ DNA primers:
Forward:
5’–AGAGGCGGTTTGCGTATTG–3’
Reverse:
5’–AAAAAAGCACCGACTCGGTG–3’
o
U6 Strings™ DNA primers:
Forward:
5’–AATTAAGGTCGGGCAGGAAG–3’
Reverse:
5’–ACAGCTATGACCATGATTACGCC–3’
Note: If you already have your CRISPR sequence of interest cloned in a
GeneArt® CRISPR Nuclease vector (A21177 or A21178), the same set of U6
Strings™ DNA primers can be used to perform PCR and generate a U6
expression cassette.
To generate a T7-based expression template, the following primers can be
used with these vectors.
Forward:
5’–TAATACGACTCACTATAGGNNNNNNNNNNNNNNNNNNN–3’
Reverse:
5’–AAAAAAGCACCGACTCGGTG–3’
In the forward primer, the T7 promoter sequence is underlined. The
subsequent run of 19–20 nucleotides should be replaced by the CRISPR target
sequence.
16
GeneArt® CRISPR Nuclease mRNA User Guide
For support visit www.lifetechnologies.com/support or email techsupport@lifetechn.com
www.lifetechnologies.com
11 July 2014