APPLICATIONRNACRISPR
Cas9 mRNA

Megan Basila, John A. Schiel, Travis Hardcastle, Melissa L. Kelley
Dharmacon, A Horizon Discovery Group Company, Lafayette, CO, USA

CRISPR-Cas9DNA
S. pyogenes Cas9RNADNA

1234EGFP
mKate2Edit-R Cas9 Nuclease mRNA
freemRNACas92

CRISPR-Cas9RNA
CRISPR-

Cas9FACS
5 6 7•

DNA-2A

Cas9 mRNA

Cas9mRNA
mKate2•

EGFP

Cas9•mKate2HDR

Cas9 mRNAmRNA
K-562
nuclease mRNA
tracrRNASLAMF74Cas9Edit-R mKate2 Cas9crRNA812244872mKate2DNAT7EI241A
1. mKate2

Cas9

A. mKate2 Cas9 mRNAB.
Cas9 mKate2
crRNA

241B

SLAMF7 crRNA tracrRNA
K-562
mKate2βmKate2 Cas9 mRNAhorizondiscovery.com

C. SLAMF7DNA

Cas9mKate2
4226
44

72

24

FACS

mRNA
24
K-562
mKate2 Cas9 mRNA
crRNA
tracrRNA24
mKate2PPIB

Edit-R

1048
36

33
mKate22A

1045

Nuclease mRNA
optimal

2B

2A
Cas9 mRNA
EGFP Cas9
less-than-optimalless-than-optimal

optimal
EGFP

optimal102B

less-than-optimal

EGFP

EGFP
38
optimal

9102C
EGFP18EGFP
16102EGFP

mRNA

38;

302C

2.

2C

A. mKate2 Cas9 mRNA
crRNA tracrRNAB. EGFP Cas9 mRNA
less-than-optimal
U2OS
Cas9 mRNA
U2OS

HDR

U2OS
DharmaFECT DuomKate2 Cas9 mRNA EMX1 crRNACas9 mRNA

tracrRNA

PPIB
mKate2
K-562
FACS

optimalC. EGFP

30nt

FLAG
EMX1 C
22424

3A
FACS

RFLP
HDRmKate2
915mKate2
mKate2 30

mKate248

3B

10NheI

163B

3. mKate2 Cas9 mRNA
HDR
A. EMX1CNheI
FLAG
B. mKate2
RFLP

horizondiscovery.com

HDR
HDR

2Edit-R Fluorescent Cas9 Nuclease mRNA

Cas9 mRNA

K-562

500 g 2
GE Healthcare Hyclone,
Cat#SH30264.01
500 x g2Edit-R Cas9 Nuclease mRNA–100 µL
VLonza, Cat #VCA-1003
5.36µM predesign Edit-R SLAMF7 crRNA Dharmacon Cat #CM017122-02-0020
5.36 µM Edit-R crRNA Non-targeting
Control 1 Dharmacon Cat U-007501-01-20
Edit-R
mKate2 Cas9 mRNA 5 µg, Dharmacon Cat #CAS11859
5.36 µM tracrRNA Dharmacon Cat #U-002000-120
DPBSCas9Cas924
Cas9mRNA

10610Lonza Nucleofector 2b10

optimalEdit-RFluorescent Cas9 Nuclease mRNA

FACS

10Cas9 mRNA

Cas9Cas9 mRNARNA

ssDNA
RFLP

10HDR

2Edit-R Fluorescent Cas9 Nuclease mRNA

Cas92mKate2EGFP
Edit-R Fluorescent Cas9 Nuclease mRNA
Cas910/K-562
ATCC, CCL-243
RPMI 1640
GEHealthcare Hyclone, Cat #SH30096.01
10FBS; GE Healthcare Hyclone Cat
SH30071 03
2 mM LFBS; GE Healthcare
Hyclone, Cat #SH30071.03
1 mM
GEHealthcare Hyclone, Cat #SH30239.01
NEAA
GE Healthcare Hyclone, Cat #SH30238.01
10mM HEPES GE Healthcare Hyclone, Cat #SH30237.01U2OS ATCC, HTB9610FBS 2 mM L-DNA
1 1055 µL Proteinase K Thermo Scientific, Cat #EO0492
RNase A
Thermo Scientific, Cat #EN0531
Phusion GC buffer
Thermo Scientific, Cat #F-549S)
56 C 198 C 100.5 µL
Phusion Hot Start II DNA
Thermo Scientific, Cat #F549S
5X Phusion HF
Thermo Scientific Cat F549S
200 µM dNTP Thermo Scientific, Cat #F-549S
0.5 µMSLAMF75‘-GCTGATGAAGATGGGGAGCA-3 ’
5‘GCCCTCAAAAGGAGAGAAGGC-3’; PPIB5‘ACCGTGTATTTTGACCTACGAAT-3 ’
5‘AAACATTCGTAGGTCAAAATACA -3 ’5 µL
50 µL
PCR
PCR
98 C 3/98 C 1072 C 15-1 C /72℃ 30
1098℃ 1062℃ 1572℃ 30
2572℃ 1095 C 1010 µL
PCR
5T7EINEBuffer 2 New England Biolabs, Cat #M0302L
37 C 25
3 µL 6X Orange Loading
Dye Thermo Scientific Cat R0631
T7EI
2Edit-R Synthetic crRNA Positive Controls Protocol
Image J(1-√(1(a+b)/(a+b+c)))*100 (9)5 105
1xGE Healthcare, Cat #80-650123

50 µL

RIPA-80 C
13,300rpm

4C

10NuPAGE 4X LDSNuPAGE
10X
Thermo Scientific, Cat #NP0008,#NP0009
70 C 5
Novex 4-20 Tris
ThermoScientific, Cat #EC6025BOX
125 V 85
Invitrogen Xcell II
Thermo Scientific, Cat#EI0002
0.2 µm Amersham Protran
GE Healthcare, Cat #10600104
SuperBlock
PBS; Thermo
Scientific, Cat #37515
30
Cas911000
Novus Biologicals, Cat #NBP2-36440
TagRFP 1 1000
Thermo Scientific, Cat #MA5-15257

horizondiscovery.com

SuperBlock

14℃
PBS

2000

Abcam, Cat #6276

0.05 Tween 20 5IgG H + L
Thermo Scientific Cat 32430
SuperBlock
PBS
1 20,000

HRP
0.05 Tween 2072
PCR42
SuperSignal West Dura Substrate Thermo Scientific, Cat#34016
West Femto
Maximum Sensitivity Substrate Thermo Scientific, Cat #34095
Cas9mKate25FACSU2OSU2OSDNAGGAGCAGCTGGTCAGAGGGG-3 'GGGAAGGGGGACACTGGGGA-3'1U
FD0974PCR
FastDigest NheI
37 C 30

3/4

1.

E. Deltcheva, K. Chylinski, et al. CRISPR RNA maturation bytrans-encoded small RNA and host factor Nuclease III. Nature.
471(7340), 602-607 (2011).

2.

M. Jinek, K. Chylinski, et al. A Programmable Dual-RNA-Guided
DNA Endonuclease in Adaptive Bacterial Immunity. Science.
337(6096), 816-821 (2012).

3.

P. Mali, L. Yang, et al. RNA-guided human genome engineering
via Cas9. Science. 339(6121), 823-826 (2013).

4.

L. Cong, F. A. Ran, et al. Multiplex Genome Engineering Using
CRISPR/ Cas Systems. Science. 339(6121), (2013).

5.

K. Duda, L. A. Lonowski, et al. High-efficiency genome editing
via 2A-coupled co-expression of fluorescent proteins and zincfinger nucleases or CRISPR/Cas9 nickase pairs. Nucleic Acids
Res. 42(10), (2014).

6.

L. M. Grav, J. S. Lee, et al. One-step generation of triple
knockout CHO cell lines using CRISPR/Cas9 and fluorescentenrichment. Biotechnol. J. 10, 1446-1456 (2015).

7.

J. S. Lee, L. M. Grav, et al. Accelerated Homology-Directed
Targeted Integration of Transgenes in Chinese Hamster Ovary
Cells via CRISPR/Cas9 and Fluorescent Enrichment.
Biotechnol. Bioeng. 113, 2518-2523 (2016).

400 µL
Flow Cytometry Core Cancer Center
Support Grant P30CA046934
Moflo XDP 100
FBS
50 FBS

DNA48
Dharmacon Edit-R Fluorescent Cas9 Nuclease mRNA-U2OS
300,000/5 µg Edit-R
EGFP Cas9 Nuclease mRNA Dharmacon Cat CAS11160
25 nM tracrRNA Dharmacon Cat U-002000-120
25 nM
Edit-R PPIB Synthetic crRNA Control Cat U-007000-01-20
Edit-R crRNA Non-targeting Control 1 Dharmacon Cat U007501-01-20
3 µL less-than-optimal
9 µL
optimalDharmaFECT Duo
Dharmacon Cat T-2010-02
20
1637℃
245 CO2
HDR

10 nM

HDR
ssDNA–ssDNA

10 mM Tris-HCl

DNA
2
pH 7.5
1 µM

DNADNA
2100 µM

For more information

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or registered trademarks of their respective owners.
B027-2005-v3

PCR
500 ngFastDigest
Thermo Scientific Cat
2HDR
ImageJ

References

K-562

DNA2DNA

5'5'-