Manipulating the Genome: Advanced Tools for CRISPR/Cas9
Genome Editing and Analysis
Montse Morell*, Ying Mao, Thomas P. Quinn, Mei Fong, Lily Lee, Tatiana Garachtchenko, Michael Haugwitz & Andrew Farmer
CRISPR/Cas9 technology is a powerful tool that allows gene manipulation in
virtually any cell line. We have developed a streamlined method and reagent
set for: 1) producing sgRNAs in vitro; 2) testing sgRNA cleavage efficiency in
vitro; 3) monitoring genome editing efficiency using a specific sgRNA; and 4)
identifying the spectrum of indels created in a cell population by NHEJ-based
repair. We developed a fast, comprehensive, and efficient method to transcribe
sgRNAs in vitro that uses a high-yield, lyophilized PCR master mix to generate
PCR amplicons that can be used as templates for in vitro transcription.To test the
cleavage efficiency of synthesized sgRNAs, we developed a highly optimized in
vitro cleavage assay by combining in vitro-transcribed sgRNA, recombinant Cas9
protein, and a PCR amplicon containing the target sequence; this assay allows
the efficiency of target-DNA cleavage by Cas9 to be assessed via densitometry.
Once the most optimal sgRNAs are identified, the in vitro-transcribed sgRNA
itself (or a mammalian expression vector encoding the selected sgRNA) and a
mammalian expression vector encoding Cas9 can be transfected into target cells
for gene editing. To screen for the presence of indel mutations, we developed a
mutation detection protocol that provides increased detection efficiency and is
less sensitive to PCR buffers than other commonly used techniques. Finally, the
variety of indel mutations in a cell population can be identified using a sequential
genomic-target-DNA cloning and sequencing approach.
1
2
4
Workflow for CRISPR/Cas9 Gene Editing
Choose
your
target
Produce
sgRNA
in vitro and
test its
efficacy
in vitro
Design
your
sgRNA
Deliver
your sgRNA
and
Cas9 to
your cells
Indentify
indels
Confirm
indels
Clontech Laboratories, Inc., 1290 Terra Bella Ave., Mountain View, CA 94043
A Streamlined Method for Characterizing Indels
A
CD81
B
Cells after
CRISPR/Cas9
Direct
PCR
Efficient Production and Efficacy Testing of sgRNAs
A
1
2
Design/purchase a 69 nt
forward primer
69 nt
forward
primer
Target specific
crRNA (20 nt)
T7 promoter
(22 nt)
3
Create template for in vitro
transcription of sgRNA
AcGFP1
Produce sgRNA by in vitro
transcription
Scaffold template
(tracrRNA)
sgRNA
Reverse primer
(pre-mixed with
scaffold template)
Homology to
scaffold template
(27 nt)
In-Fusion
Cloning
PCR (high yield PCR
EcoDry Premix)
sgRNA coding sequence
T7
Target
promoter specific
crRNA
B
Taken together, we present a complete experimental workflow for CRISPR/Cas9
gene editing, from in vitro sgRNA production to identification of the generated
indel mutations. We anticipate that these protocols and reagents will increase
the success rate of this exciting and powerful technology.
C
1
Introduction
tracrRNA
sgRNA sample:
1
2
3
4
5
6
total sgRNA yield (µg)
8
7
8.3
9.7
11
4
concentration (ng/µl)
410
350
450
475
580
200
2
Use PCR to generate a target
for cleavage
Colony
PCR
In vitro cleavage of target sequence by
recombinant Cas9 and synthesized sgRNA
Amplify by PCR
M
C1
N
N
R
sg
1
A
Cas9
sgRNA
5
Your favorite gene
Your favorite gene
CD81
Sequencing
PAM sequence
(5'-NGG-3')
PAM
sequence
CRISPR/Cas9 gene editing technology has revolutionized the field of genome
modification. This system is based on two key components that form a
complex: Cas9 endonuclease and a target-specific RNA (single guide RNA
or sgRNA) that guides Cas9 to the genomic DNA target site. Targeting to a
particular genomic locus is solely mediated by the sgRNA. Currently, for a
specific gene target, the available web-based tools, which analyze sequence
specificity and assess potential off-target cleavage events (1), provide a variety
of sgRNA sequences that could be used. However, not every sgRNA will
mediate equivalent cleavage of the intended target sequence. Because of this
lack of consistency and to ensure successful genome editing, it is necessary to
screen a number of different sgRNAs. To address this, we have established a
streamlined approach for production of sgRNAs followed by a highly-optimized
in vitro cleavage assay that allows estimation of cleavage efficiency for the
intended target. After identifying the most efficient sgRNA, Cas9 endonuclease
and the sgRNA are delivered to the cells, and genome editing activity at the
target locus can be assessed using an enzyme-based cleavage assay. To further
characterize insertions and deletions (indels) introduced at the target site due
to Cas9/sgRNA activity, we have developed a protocol for the amplification,
cloning, and preparation of modified target sites for DNA sequence analysis.
N
C2
R
sg
N
2
A
3
ADBR2
A
N
R
sg
M
C
N
N
R
sg
4
A
N
R
sg
5
A
N
R
sg
6
A
N
R
sg
7
A
D
Efficient Knockout of CXCR4 using CRISPR/Cas9
Gene Editing
In vitro Cas9 cleavage assay
M NC
A
Cloning-free sgRNA production and in vitro estimation of sgRNA cleavage efficiency. Panel A. The Guide-it™
sgRNA In VitroTranscription System provides a cloning-free method for sgRNA production.The Guide-it scaffold
template is amplified using a 69 nucleotide sgRNA-specific primer and a provided scaffold template-specific
primer with EcoDry™ Premix, a high-yield lyophilized PCR master mix. This protocol allows generation of an
sgRNA template for in vitro transcription in less than 4 hr (steps 1 & 2). Panel B. For six different sgRNAs, RNA
yield was determined after in vitro transcription using a NanoDrop 2000 spectrophotometer; for all samples,
>4 µg of intact sgRNA was obtained. Panel C. The Guide-it sgRNA Screening Kit enables in vitro analysis of
cleavage efficiency of an sgRNA prior to Cas9-mediated gene editing in cells. With this system, a PCR fragment
containing an sgRNA target site (step 1) is combined with candidate sgRNAs and recombinant Cas9 (rCas9)
nuclease (step 2). The cleavage of the PCR product is assessed and quantified by agarose gel electrophoresis.
Panel D. Using this system, cleavage efficiency of different sgRNAs targeting CD81 (left) and ADBR2 (right)
was analyzed. For CD81, sgRNA1 targets a different region of the gene than sgRNA2 or sgRNA3. As a negative
control (NC), the DNA target fragment was incubated with only rCas9. After the in vitro cleavage reaction,
the cleavage products were resolved by gel electrophoresis. The best sgRNA for each loci was defined as the
sgRNA that resulted in the highest percentage of cleavage products (CD81: sgRNA3; ADRB2: sgRNA6).
1.Hsu, P. et al. (2013) Nature Biotechnology 31(9):827–832.
Indel identification. Panel A. This protocol uses direct PCR to amplify
a genomic DNA fragment (~500 to 700 bp) containing the target site
directly from crude cell lysates. The resulting amplified fragments
contain a pool of edited target sites from individual cells. These PCR
products are cloned directly into a pre-linearized pUC19 vector using
the In-Fusion® cloning system (a restriction digestion- and ligationfree cloning method). After transformation into an optimized E. coli
strain, colony PCR is used to amplify the target site from the plasmid.
DNA sequencing is then used to identify the different indels generated
at the targeted genomic site. Panel B. Indels generated after CRISPR/
Cas9 targeting of CD81 (top) and AcGFP1 (bottom) were identified from
several clones using the protocol in Panel A. The sequencing data for
each clone was aligned with the wild-type sequence, revealing a range
of deletions and insertions (highlighted in red).
C
2
3
4
sgRNA1
200
B
WT
M1
80
40
120
90
73.62%
160
M2
M1 2 3 4
sgRNA2
200
71.26%
160
120
1
Resolvase assay
M2
WT
M1
80
40
0
0
101
100
102
FL1-H
103
104
Knockout
101
100
102
FL1-H
103
104
Knockout
% (DNA cleavage or KO cells)
Abstract
*Corresponding Author
Counts
A Takara Bio Company
Counts
2036
Clontech Laboratories, Inc.
80
70
60
50
40
30
20
10
0
sgRNA1
• This sgRNA production method does not require cloning of the sgRNA
into an expression vector.
3
A PCR-Based Method to Confirm the Presence
of Mutations
A
1
2
Amplify genomic DNA from cells
using Terra PCR Direct Polymerase
3
Denature and reanneal
Imperfectly matched DNA is cleaved
by Guide-it Resolvase
Cleaved
fragment B
• Our indel identification protocol provides a quick workflow for identifying
the variety of indels introduced by genome editing in a cell population.
B
2
3
4
5
Guide-it
Resolvase
assay
M2
WT
M1
80
FACS
40
0
Knockout
101
102
FL1-H
103
104
100
101
102
FL1-H
103
104
Knockout
D
Surveyor
6
M
1
2
3
4
5
6
468–
*
**
**
291–
177–
Abbreviations
120
sgRNA4
Guide-it Resolvase cleaves
imperfectly matched DNA
Guide-it Resolvase
M 1
80
100
• We designed a robust in vitro system and reagent set that provides a rapid
method for cleavage analysis of multiple sgRNAs, allowing identification
of the most effective sgRNA prior to transfection.
bp
WT
M1
0
Cleaved
fragment A
• Our mutation detection assay (Resolvase assay) is faster, more sensitive,
and less prone to non-specific cleavage than a CEL-I based assay
(Surveyor).
120
M2
sgRNA3
In vitro
screening
74.9%
160
40
Uncleaved
Region of interest is amplified
(mutation is marked in blue)
11.50%
160
sgRNA4
200
Counts
• Our in vitro sgRNA-production method generates high yields of functional
sgRNA, sufficient for multiple in vitro screening reactions and/or cell
transfections.
sgRNA3
200
Counts
Conclusions
sgRNA2
1
2
3
4
5
6
transfected
100
80
60
40
20
0
nontransfected
0
20
40
60
80
100
% cleavage
59
46
28
15
<10
0
Note: Smearing is due
to non-specific cleavage
CRISPR—Clustered Regularly Interspaced Short Palindromic Repeat
sgRNA—single guide RNA
indel—insertion and deletion
NHEJ—non-homologous end joining
crRNA—CRISPR RNA
tracrRNA—trans-activating crRNA
For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Clontech®, the Clontech logo, EcoDry, Guide-it,
In-Fusion, Terra, and Xfect are trademarks of Clontech Laboratories, Inc. Takara and the Takara logo are trademarks of TAKARA HOLDINGS,
Kyoto, Japan. All other marks are the property of their respective owners. Certain trademarks may not be registered in all jurisdictions.
©2015 Clontech Laboratories, Inc.
Comparison of mutation detection assays for detecting NHEJ-based repair in mammalian cells. Panel A. The
Guide-it Mutation Detection Kit can be used to confirm the presence of mutations in the genomic DNA of
cells in a population. With this method, the target sequence is amplified directly from cells, without genomic
DNA extraction/purification, using Terra™ PCR Direct Polymerase (step 1). The PCR product is melted and
rehybridized, forming mismatched targets between wild-type and mutant sequences that can be cleaved
by the Guide-it Resolvase (steps 2 and 3). Panel B. The Guide-it Resolvase assay and the Surveyor assay
were used to detect indels in mammalian cells after CRISPR/Cas9 targeting. HEK 293T cells were transfected
with plasmids encoding Cas9 and an sgRNA targeting the AAVS1 locus. Cells were harvested 48 hr posttransfection and mixed with untransfected cells at varying ratios (Panel B, table) to determine detection limits.
A fragment containing the AAVS1 target sequence was generated directly from the cell population by PCR.The
PCR products were purified and cleaved with either Guide-it Resolvase or the CEL-I enzyme (Surveyor assay).
Mutations were easily discernible using the Guide-it kit (*: uncleaved; **: cleaved). In contrast, the Surveyor
assay showed considerable smearing, making it difficult to determine cleavage efficiency and reducing the
ability to detect low levels of mutation.
Successful knockout of CXCR4 using the Guide-it system. Panel A. The in vitro cleavage efficiency of
four different sgRNAs (sgRNA1-4) targeting the CXCR4 gene were tested using the methods described
in Figure 2. A negative control reaction that lacked an sgRNA was included for comparison (NC). Of all
of the candidate sgRNAs, sgRNA3 had the lowest in vitro cleavage activity. Panel B. HeLa cells were cotransfected with plasmids encoding Cas9 and one of the four different sgRNAs using Xfect™ transfection
reagent. Six days after transfection, the cell population was assayed for the presence of mutations at the
CXCR4 target site using the Guide-it Resolvase assay (as described in Figure 3). Cleavage fragments were
present for all sgRNAs except sgRNA3, further indicating low guide efficiency of this sgRNA. Panel C. The
efficiency of CXCR4 gene disruption was assessed by flow cytometry using a FITC-labeled antibody against
CXCR4 (left). There was a clear correlation between the efficiency predicted by the in vitro Cas9 cleavage
assay and by the Resolvase assay and functional knockout of CXCR4 (right). As predicted by the other
assays, sgRNA3 was the least efficient in disrupting the CXCR4 gene. Panel D. For sgRNA2, mutations at
the target site were identified using the protocol described in Figure 4. The alignment shows individual
indels generated at the CXCR4 locus in eight clones (highlighted in red).
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