TECHNICAL MANUAL

Dharmacon™ Edit-R™
HDR plasmid donor
kit for fluorescentreporter knock-in

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

Contents
1 Introduction to the CRISPR-Cas9 system for gene engineering.....................................................................3
CRISPR-Cas: an adaptive immunity defense mechanism in bacteria and archaea.............................................................3
A CRISPR-Cas9 platform for mammalian genome editing...........................................................................................................3
2 Edit-R HDR plasmid donor kit for fluorescent reporter knock-in...................................................................5

Edit-R Cas9 Nuclease............................................................................................................................................................. 7
Edit-R synthetic guide RNA................................................................................................................................................ 7
Edit-R HDR plasmid donor kit............................................................................................................................................ 7
3

Guidelines for cloning HDR plasmid donors...................................................................................................8

Materials required for cloning HDR plasmid donors................................................................................................. 8Reagents to be supplied by researcher......................................................................................................................... 8
Cloning HDR plasmid donors............................................................................................................................................ 9
4

Guidelines for knock-in co-transfection using the Edit-R platform withHDR DNA plasmid donors............................................................................................................... 13
Materials required for knock-in co-transfection....................................................................................................... 13
Reagents to be supplied by researcher....................................................................................................................... 13
Co-transfection of HDR plasmid donor with Edit-R Cas9 Nuclease mRNAand Edit-R synthetic guide RNAs.....................................................................................................................................................14
Co-transfection of HDR plasmid donor with Edit-R Cas9 Nuclease protein
and Edit-R synthetic guide RNAs....................................................................................................................................................16

5

Appendix...........................................................................................................................................................18
Additional resources..................................................................................................................................................................................18
Gene editing assay recommendations...............................................................................................................................................18
Detect NHEJ-mediated gene editing with mismatch detection assay...........................................................................18
Detect integration of a fluorescent protein with several assays........................................................................................18

6 Frequently asked questions (FAQs)..................................................................................................................19
7 References..........................................................................................................................................................23
8 Limited use licenses...........................................................................................................................................232

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

1Introduction to the CRISPR-Cas9 system for gene engineering
CRISPR-Cas: an adaptive immunity defense mechanism in bacteria and archaea
The CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated proteins) system is anadaptive bacterial and archaeal defense mechanism that serves to recognize and silence incoming foreign nucleic acids.
Upon infection by bacteriophage or other foreign DNA elements, a host organism can incorporate short sequencesfrom the invading genetic material, called protospacers, into a specific region of its genome (the CRISPR locus) between
short palindromic DNA repeats of variable lengths. Multiple spacer-repeat units are clustered at the CRISPR locus toform the CRISPR array. The entire locus, including the CRISPR array, is transcribed by RNA polymerase into a primarytranscript, the pre-CRISPR RNA (pre-crRNA). The pre-crRNA is then processed into small, mature CRISPR RNAs (crRNAs)
such that they include sequences complementary to the foreign, invading DNA. crRNAs guide a multifunctional protein
or protein complex (the CRISPR-associated or Cas proteins) to cleave complementary target DNA that is adjacent toshort sequences known as protospacer-adjacent motifs (PAMs). Thus, the organism acquires a way to protect itself from
subsequent infection (Bhaya, 2011).

A CRISPR-Cas9 platform for mammalian genome editing
Many bacterial and archaeal CRISPR-Cas systems have been identified with diverse sets of mechanisms, Cas proteinsand multi-subunit complexes. In particular, the processes and key components of the Streptococcus pyogenes
CRISPR-Cas9 system have been well-studied and adapted for genome engineering in mammalian cells. In S. pyogenes,
only three components are required for targeted DNA cleavage at specific target sites adjacent to a protospacer
adjacent motiff (PAM; Jinek, 2012): (1) The endonuclease Cas9, programmed by (2) a mature crRNA processed fromtranscription of the CRISPR locus/array which complexes with (3) another CRISPR locus-encoded RNA, the trans-activating
CRISPR RNA (tracrRNA; Figure 1A; Deltcheva, 2011). Alternatively, the crRNA can be fused to the tracrRNA creating a
chimeric structure termed a single guide RNA (sgRNA; Figure 1B: Jinek, 2012). Upon site-specific double-stranded DNA
cleavage, a mammalian cell can repair such a break through either non-homologous end joining (NHEJ) or homologous
recombination (HR). NHEJ is often imperfect, resulting in insertions and deletions (indels) that can cause nonsense
mutations resulting in gene disruptions to produce gene knockouts (Mali, 2014; Sampson, 2014). This endogenous DNA
break repair process, coupled with the highly tractable S. pyogenes CRISPR-Cas9 system, allows for a readily engineered
platform to permanently disrupt gene function.

A.

B.
PAM

Genomic DNA

Genomic DNA

PAM

Cas9 Nuclease

crRNA

Cas9 Nuclease
3'

5'
3'

5'

tracrRNA

5'
3'

sgRNA

Figure 1. Illustration of CRISPR-Cas9 system. Cas9 nuclease (light blue), programmed by the crRNA (green):tracrRNA (blue) complex (A) or the sgRNA (B), cutting both strandsof genomic DNA 5' of the PAM (red).

3

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

Homologous recombination is the exchange of DNA sequence information using sequence homology. Homology-directed
repair (HDR) is a process of homologous recombination whereby a DNA template provides the homology necessary for
precise repair of a double-strand break (DSB). This DNA template can come from within the cell during late S phase andG2 phase of the cell cycle, when sister chromatids are available, prior to the completion of mitosis. Importantly, exogenous
repair templates can be delivered into a cell, most often in the form of a DNA plasmid donor or a synthetic single-stranded
DNA (ssDNA) donor oligonucleotide (oligo), to generate a precise change in the genome. (See Figure 2 for an illustration).

DNA double-strand breakgenomic DNA

Donor DNAEnd resection (5̒' to 3')

Strand invasion

Precise repair

Figure 2. Diagram of DSB repair using the homology-directed repair (HDR) pathway.

4

Edit-R HDR Plasmid Donor

Custom kit ordering

TECHNICAL MANUAL

Vector backbone

Fluorescent protein insert
Insert sequence

Custom homology arm primers

Colony PCR primers

5' homology arm 3' homology arm

fwd 1 rev 1

fwd 2 rev 2

backbone

insert

fwd

fwd

rev

rev

Homology arm
amplification

insertion site

fwd 2

fwd 1

5' homology arm

3' homology arm

rev 1

rev 2

Ligation-free DNA assemblyTransformation

Insert sequence
5' homology arm

3' homology arm
assembled plasmid donor

PCR from bacterial culture
rev

d

5'

fw

homology arm

Clone

1

Insert2

Pick & grow colonies

fwd
3'

homology arm

Agarose gel analysis of PCR amplicons
medium alone

Plasmid donor generation

vector backbone

3

4

5

v
re

Cloning the HDR plasmid donor

Amplify homology arms from gDNA

1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H

Prepare plasmid frompositive clones& confirm with
Sanger sequencing

A B A B A B A B A B

Expectedsize withhomology
arms

Figure 3. Diagram of the plasmid donor cloning workflow including component ordering, homology arm amplification, and plasmid donor
generation. This product supports the use of EGFP, mKate2, and custom sequence inserts. For illustrative purposes, mKate2 will be used. Colors
on the diagram indicate the origin of the DNA (dark blue = 5' homology arm, light blue = 3' homology arm, red = mKate2 fluorescent reporter
sequence, orange = plasmid backbone). medium alone = media-only negative PCR control.

5

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

AAAA
Cas9 mRNAoror

mKate2

5' HA

1
day

Cas 9

3' HA

Synthetic guide RNA

Cas9 protein

HDR DNA plasmid donor

Co-transfection

Passage cells

10 20 30 40 50 60 70 80 90 10

Observe correct cellular localization & quantify HDR efficiency in the cell population

0

7
˜
days

Fluorescent microscopy

AutofluorescencebackgroundmKate2+

100

101

102

103

104

FACS analysis
Clonal cell line
isolation & expansion

8-28
days

Confirm precision editing in clonal cell lines

GAA TG TA TTTA G AAAAAT AAA C
GAA TG TA TTTA G AAAAATAAAC

Fluorescent microscopy

Sanger sequencing

Figure 4. Workflow to acquire desired mKate2 knock-in clonal cell line. A similar workflow will be followed if using EGFP or a custom insert sequence.

6

Edit-R HDR Plasmid DonorTECHNICAL MANUALThe Edit-R HDR plasmid donor kit contains the components to construct a plasmid donor for insertion of monomeric
EGFP (University of California, San Diego), mKate2 (Evrogen, Moscow, Russia), or a custom, user specified insert sequencein a desired endogenous genomic target location. The final plasmid donor (generated with the kit) will have the desired
insert flanked by two homology arms that are required for HDR-mediated knock-in. The workflow involves: (1) ordergene-specific homology arm primers and the Edit-R HDR donor kit (using the Edit-R HDR Donor Designer), (2) amplify
the homology arms surrounding the insertion site in your target gene, (3) assemble and verify the sequence of the HDR
plasmid donor (Figure 3), and (4) co-transfect the assembled plasmid donor with the Edit-R CRISPR-Cas9 components:
Cas9 mRNA or protein, synthetic guide RNA (crRNA:tracrRNA complex or sgRNA), followed by confirming the HDRmediated knock-in in a population as well as generating a clonal cell line (Figure 4).
This technical manual provides protocols and guidelines optimized for efficient plasmid donor generation and
HDR-mediated knock-in for precise genome engineering using the Edit-R CRISPR-Cas9 platform.

Edit-R Cas9 Nuclease
The Edit-R Cas9 Nuclease mRNA (Cat #CAS11195) contains a human codon-optimized version of the S. pyogenes
Cas9 (Csn1) gene with a 5' and 3' nuclear localization signal (NLS). The Cas9 mRNA is in vitro transcribed, 5' capped andpolyadenylated for translation and nuclear localization of the Cas9 protein. The Edit-R Cas9 Nuclease protein NLS(Cat #CAS11200) contains the S. pyogenes Cas9 (Csn1) gene with a C-terminal nuclear localization signal (NLS). The
Cas9 mRNA and Cas9 protein are ready for direct transfection or electroporation into cells.

Edit-R synthetic guide RNA
Edit-R trans-activiating CRISPR RNA (tracrRNA)
Edit-R tracrRNA is a synthetic, HPLC-purified, long RNA required for use with Edit-R crRNA to form the complex
that programs Cas9 nuclease. It is modified for nuclease resistance and can be used with modified or unmodified
Edit-R crRNA.
Edit-R CRISPR RNA (crRNA)
The Edit-R crRNA is a synthetic RNA, comprised of 20 nucleotides identical to the genomic DNA (gDNA) target
site, or protospacer, followed by a fixed S. pyogenes repeat sequence that interacts with the tracrRNA. The chosen
protospacer sequence in the target gDNA must be immediately upstream of a PAM sequence (NGG for S. pyogenes).
Edit-R Predesigned crRNAs are available for human, mouse and rat coding genes on and custom crRNA (forspecific applications including HDR) can be designed and ordered using the Dharmacon CRISPR Design Tool.
Edit-R synthetic single guide RNA (sgRNA)
Edit-R synthetic sgRNA is a 100 nucleotide chimera fusing the crRNA and tracrRNA sequences with a 4 nt tetraloop
sequence (Jinek 2012). It is modified for nuclease resistance on both 5’ and 3’ ends of the molecule. Edit-R synthetic
sgRNAs can be ordered through the Dharmacon CRISPR Design Tool.

Edit-R HDR plasmid donor kit
The Edit-R HDR plasmid donor kit contains all the DNA components to build and quality check a custom DNA plasmid
donor to introduce a fluorescent tag to any genomic location when coupled with Edit-R HDR DNA plasmid donor
homology arm (HA) custom primer pairs. Specifically, the kit comes with custom-designed HA PCR primers and threestatic components: the Edit-R HDR plasmid donor backbone, the Edit-R HDR plasmid donor insert, and Edit-R HDR
plasmid donor colony PCR primer pairs specific to verify the sequence of mKate2 fluorescent reporters. The HA PCR
primers and kit components are designed and ordered using the HDR Donor Designer. The HA PCR primers are designed
to amplify HAs with lengths ranging from 500 to 1000 bp, with an adapter sequence appended, which is necessary forcompatibility with the HDR plasmid donor backbone. The HDR Donor Designer additionally designs the HA primers
to introduce silent mutations to disrupt the intact CRISPR targeting sequence to avoid unintentional cutting of gDNA
repaired by HDR.
7

C

fwd

rev

fwd

rev

Amplify homology
armsHDR
from gDNA
Edit-R
Plasmid Donor
insertion site

amplification

Cloning the HDR plasmid donor
Homology
arm arm
Homology
Homologyarm
Customkit ordering
Customkit ordering
amplification Custom kit ordering
amplification
amplification
Homology arm

fwd 2 rev 2

Guidelines for cloning HDR plasmid donors

TECHNICAL MANUAL

fwd 2

fwd 1

5' homology arm

3' homology arm

rev 1
rev 2
To knock-in DNA sequence such as a fluorescent tag into specified gene target, an HDR plasmid donor
needs to beconstructed to contain the intended DNA sequence or fluorescent tag (EGFP/mKate2) flanked by two HAs of 500 to1000 bp in length. This protocol provides instructions for ligation-free cloning of the
HDR plasmid
Ligation-free
DNA donor.
assembly

Materials required for cloning HDR plasmid donors

Transfo

Insert sequence

Vector backbone

Fluorescentproteininsert5' homology arm
3' homology arm

Edit-R HDR EGFP, mKate2, or custom insert
Edit-R HDR plasmid donor backbone

»»
»»
»»
»»

Insertsequence
arm primers Colony PCR primers
Insertsequence
Edit-R colony PCR primer backbone forward, 5 nmoles Custom homology
fromarm
bacterial backbone
culturePick & gr
5' homology arm 3'PCR
homology
insertEdit-R colony PCR primer EGFP or mKate2 reverse, 5 nmolesrev
fwd
CustomCustomhomology
homologyarm
armprimers
primers Colony
ColonyPCR
PCRprimers
primers
Edit-R colony PCR primer EGFP or mKate2 forward, 5 nmoles
5'5'homology
homologyarmbackbone
inserthomologyarm
arm3'3'homologyarmbackbone
insert5'
3'
Edit-R colony PCR primer backbone reverse, 5 nmoles
Inserthomology
homologyarm
rev 2
fwd 1 rev 1
fwd 2 arm
fwd rev
fwd revdfw

1 2 3 4 5

Amplifyhomology
Amplifyhomologyarms
armsfrom
fromgDNAgDNA
Agarose gel analysis of PCR amplicons

Prepare plpositive cl& confirm
Sanger seq

insertion
insertionsite
site

Clone
fwd 1

1

42 5
3 fwd

2

A
B
C
D
E
F
G
H

fwd
fwd rev
rev

insertion site

3' homology arm primers:»» Edit-R custom HA primer forward 2, 20 nmoles»» Edit-R custom HA primer reverse 2, 20 nmoles

Reagents to be supplied by researcher

VectorVectorbackbone
backbone

Amplify
from gDNA
1 1 homology
22
fwd
1 1rev
fwd
fwd
rev
revarms
fwd
fwd2 2rev
fwd rev
rev

medium alone

5' homology arm primers:»» Edit-R custom HA primer forward 1, 20 nmoles»» Edit-R custom HA primer reverse 1, 20 nmoles

assembled plasmid donor

vectorbackbone
Insertsequence
Fluorescentprotein
Fluorescentproteininsert
insertv
reCustom PCR primers for homology arm amplification

Plasmid donor generation»»»»

CloningCloning
the HDR
HDR
plasmiddonordonor
theplasmid
HDR plasmid
Cloningthedonor

5' homologyarm
A B A arm
B A B A3'Bhomology
A B
rev 1

Expectedsize withhomology
arms

rev 2

fwd
fwd2 2

fwd
fwd1 1

5'5'homology
homologyarm
arm

3'3'homology
homologyarm
arm

Ligation-free DNA assemblyrev
rev1 1

Transforev
rev2 2This cloning protocol uses common molecular biology reagents and instruments.
The following protocol is provided
Insert sequence
for illustrative purposes, but substitutions with other similar products are acceptable. For further protocol information
5' homology arm
3' homology arm
Ligation-freeDNAassembly
Ligation-freeDNAassemblyor troubleshooting please consult the product’s manufacturer. The following additional
materials
arerequired:assembled
plasmid donor•

High-fidelity DNA polymerase [such as Thermo Scientific Phusion Hot Start
IIsequence
DNA Polymerase (Cat #F549S) or
Insertsequence
InsertNEB Q5® High-Fidelity DNA Polymerase (Cat #M0491S)]
5' 5'homologyarm
3' 3'
homologyarmhomologyarmhomologyarmassembled
plasmiddonor
assembled
plasmiddonor Pick & gro
PCRMixfromcultureTaq DNA polymerase [such as Thermo Scientific DreamTaq Green PCR Master2x bacterial(Cat #K0801)]•

1x TAE buffer•

1x PBS buffer•

PCR
PCRfrom
frombacterial
bacterialculture
cultureDNA Assembly cloning kit [such as NEB Gibson Assembly® Cloning Kit (Cat #E5510S)]•

rev
rev fwd
fwd as NEB 5-alphaChemically competent E. coli cells with a transformation efficiency of at least 1 × 109 cfu/µg[suchCompetent E. coli (High Efficiency) Cat #C2987I)]
5'5'
3'3'

vectorbackbone
vectorbackbone

rev

d
fw

fwd
3'

InsertInsertInserthomologyarmhomologyarm•

NZYM plus broth [such as Teknova (Cat #N1215)]•

LB Agar plates with carbenicillin (100 µg/mL)•

Thermal cycler•#740422.10)]
Endotoxin-free plasmid prep kit [such as Clontech NucleoBond® Xtra Midi Plus EFA (CatB A B A B A B A B•

Tris buffer: 10 mM Tris-HCl pH 7.4, nuclease-free buffer [such as 10
mM Tris-HCl
BufferpH 7.4ofofPCR
Agarosegel
Agarosegelanalysis
analysis
PCRamplicons
amplicons
Expectedsize with(Cat # B-006000-100)] or prepare with the recipe here
homology

Clone

medium alone

Agarose gel analysis of PCR amplicons
1

2

3

4

5

Prepare plpositive clo& confirm w
Sanger seq

Prep
Pre
posit
pos
&&con
co
Sang
San

arms
Clone
11 22 33 44 55
Clone
DNA extraction kit [such as Thermo Scientific™ GeneJET Gel Extraction
Kit(Cat #K0691)]•

Genomic DNA isolation kit [such as Thermo Scientific™ GeneJET Genomic DNA Purification
Kit(Cat
AAB BAAB BAAB BA
AB BA#K0712)]
AB B•

Blue light transilluminater [such as the Thermo Scientific™ Safe Imager
2.0 blue light transilluminator (Cat #G6600)]
Expected
Expected•

Fluorometric assay [such as Life Technologies Qubit® dsDNA HA Assay
Kit (Cat #Q32851)]
homology
homology

size
sizewith
with
arms
arms

Pick
Pic

1
A A
B B
C C
D D
E E
F F
G G
H H

medium alone
medium alone•

1 2 3 4 5
A
B
C
D
E
F
G
H

v
rev
re

homologyarmhomologyarmhomology arm

v
re

5'

homology arm

fw
fw
d
d

•

TrT

vector backbone

Plasmid donor
donor
generation
Plasmiddonor generation
Plasmid
generation3

fwd 1 rev 1

8

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

Cloning HDR plasmid donors
1.

Design and order custom HA primers and the Edit-R HDR plasmid donor kit
a. Use the Edit-R HDR Donor Designer webtool to custom design HA primers by defining the genomic locationfor HDR insertion.
b. Add all desired primers to the cart and ensure the static kit components are added to the cart as well.
c. The static kit components may also be ordered independently for use with previously designed HA primer sets.

2.

Generate homology armsa. Isolate gDNA following the manufacturer’s instructions to use as template for homology arm PCR.
b. Adjust concentration of isolated gDNA to 25 ng/µL.

gDNA from the cell line where will be performed is required, as cell line-specific SNPs can affect knock-in efficiencyc.

PCR amplify 5' and 3' homology arms
i. Resuspend Edit-R custom HA PCR primers to 50 µM. Verify the concentration using UV spectrophotometry
at 260 nm, and adjust volume accordingly.
ii. Set up a 50 µL PCR for each homology arm to be assembled into the plasmid donor(Tables 1 and 2) and perform PCR (Table 3). Conditions for Phusion Hot Start II High-Fidelity DNA polymerase
are shown (Table 3).

Multiple 50 µL reaction replicates (3-6) are recommended for each homology arm to acquire enough material after gel purification(minimum of 20 ng/µL purified homology arm).

Additives such as DMSO or high GC buffer may be necessary for difficult gDNA context as recommended by polymerase manufacturer.

Table 1. PCR setup for 5' homology arm for use generation of HDR plasmid donor

Reagent

VolumeFinal concentration

5x Phusion HF buffer
Edit-R custom HA primer forward 1 (50 µM)
Edit-R custom HA primer reverse 1 (50 µM)
dNTPs (10 mM)
Phusion Hot Start II High-Fidelity DNA polymerase (2 U/µL)
Water, nuclease freegDNA (25 ng/µL)

10 µL
0.5 µL0.5 µL
1 µL
1 µL
33 µL
4 µL

1x
500 nM
500 nM
200 µM each
0.04 U/µL
N/A
2 ng/µL

Reagent

VolumeFinal concentration

5x Phusion HF buffer
Edit-R custom HA primer forward 2 (50 µM)
Edit-R custom HA primer reverse 2 (50 µM)
dNTPs (10 mM)
Phusion Hot Start II High-Fidelity DNA polymerase (2 U/µL)
Water, nuclease freegDNA (25 ng/µL)

10 µL
0.5 µL0.5 µL
1 µL
1 µL
33 µL
4 µL

1x
500 nM
500 nM
200 µM each
0.04 U/µL
N/A
2 ng/µL

Table 2. PCR setup for 3' homology arm for generation of HDR plasmid donor

9Edit-R HDR Plasmid DonorTECHNICAL MANUAL
Table 3. Thermal cycling conditions

Reagent

Temperture

Time

Cycle(s)Initial denaturation

98 °C

30 s

1

Denature
Annealing
Extension

98 °C
Tm + 3 °C
72 °C

10 s
20 s
40 s

35

Final extension

72 °C

10 min1

When calculating melting temperature (Tm) the adapter sequences should not be included even though the Tm will change after the
first few cycles. Refer to Table 4 for the adapter sequences.
Table 4. Edit-R custom HA primer components

3.

HA primers

Adapter sequence (5' to 3')

Gene specificEdit-R custom HA primer forward 1

ACAGAGTGATATTATTGACACGCCC

customEdit-R custom HA primer reverse 1

TCGCCCTTGCTCACGCTGCCACCAGC (EGFP)ATCAGCTCGCTCACGCTGCCACCAGC (mKate2)

customEdit-R custom HA primer forward 2

GGACGAGCTGTACAAGGGAGGTAGC (EGFP)CAAACTGGGGCACAGAGGAGGTAGC (mKate2)

customEdit-R custom HA primer reverse 2

ATAACGGAGACCGGCACACTGGCCAT

customPurification of amplified homology arms
a. Load homology arm PCR, or pooled replicate reactions, on a 2% TAE agarose gel with SYBR green(1:10,000 dilution).
b. Use a blue light transilluminator to visualize and cut out the appropriate size DNA band.

Use of blue light (~470 nM) is required over ultraviolet (UV) light to avoid DNA damage and therefore improve overall cloning efficiency.

c.
d.

Extract DNA from the gel using a gel extraction kit.
i. Elute in Tris buffer for stability of PCR products through freeze-thaw cycles.
Quantify DNA, using an agarose gel or a fluorometric assay such as the Qubit® dsDNA
HA Assay Kit (Life Technologies Cat #Q32851).

Quantification using UV-Vis spectrophotometry is not recommended for this step, because accurate quantification of dsDNA is required.

4.

Ligation-free cloning of the Edit-R HDR plasmid donora. Resuspend the HDR insert and Edit-R HDR plasmid donor backbone components:
i. Resuspend insert to 20 ng/µL by adding 12.5 µL Tris buffer.
ii. Resuspend Edit-R HDR plasmid donor backbone to 30 ng/µL by adding 40 µL Tris buffer.
iii. Adjust concentration of PCR amplicons of homology arms to 20 ng/µL using Tris buffer.
iv. For all components (insert, backbone and PCR amplicons) verify concentration by agarose		
gel or fluorometric assay and adjust if necessary.
b. Set up a Gibson Assembly reaction according to manufacturer’s instructions. We have found that
0.2 pmol total reaction at a 1:1:1:1 molar ratio of (Edit-R HDR plasmid donor: backbone insert:5' homology
arm:3' homology arm) works well for this four-part assembly. Table 5 below contains example reaction setup
for 1000 bp homology arms. Adjust the volumes as necessary to accommodate different length homology
arms, while still maintaining the 0.2 pmol Gibson Assembly reaction.
i. Incubate at 50 °C for 60 minutes then store on ice until ready for transformation.
The Edit-R HDR plasmid donor kit contains material for 10 assembly reactions of 0.2 pmol total DNA as shown in Table 5. If the amountof total DNA for assembly is adjusted, the number of reactions will differ accordingly.

10Edit-R HDR Plasmid DonorTECHNICAL MANUAL
We recommend storing single use aliquots of the Gibson Assembly master mix to avoid multiple freeze-thaw cycles.

Table 5. Gibson Assembly reaction setup with total DNA input of 0.2 pmol using homology arms with length of 1000 bp and mKate2 insert.

Reagent

Size (kb)

Molecular Weight(g/mol)Concentration(ng/µL)DNA (pmol)

Volume (µL)

Edit-R HDR plasmid donor backbone

3.449

2,131,482

30

0.05

3.6

Edit-R HDR mKate2 insert

0.714

441,252

20

0.05

1.1

5' homology arm

1

618,000

20

0.05

1.5

3' homology arm

1

618,000

20

0.05

1.5

water, nuclease free

N/A

N/A

N/A

N/A

2.3

Gibson Master Mix

N/A

N/A

N/A

N/A

10

Total Volume

20

5.

Transformationa. Transform 1 × 109 competent cells.
i. Protocol for NEB® 5-alpha Competent E. coli (High Efficiency) (Cat # C2987I)—please refer to manufacturer’s
instructions for specific competent cells.
		 1. Warm LB Agar plates with Carbenicillin (100 µg/mL) in 37 °C incubator.
		 2. Thaw 50 µL of competent cells per assembly reaction on ice.
		 3. Add 2 µL Gibson Assembly reaction to the thawed cells.
		 4. Mix gently by pipetting the reaction 4-5 times.
		 5. Place on ice for 30 minutes.
		 6. Heat shock at 42 °C for 30 seconds without any mixing.
		 7. Return to ice for 5 minutes.
		 8. Add 100 µL SOC medium to each tube.
		 9. Spread up to 150 µL onto each prewarmed selection plate.
		 10. Incubate plates overnight at 37 °C.

6.

Colony PCRa. Any colony PCR protocol can be used based on typical lab protocols. Below is an optional and optimized fast		 colony PCR protocol that will allow for quick growing of colonies and colony PCR analysis in only one day.
		 i. Pick 10 colonies per assembly reaction and grow in 100 µL of NZYM plus medium for 6 hours at 37 °C.

After 6 hours, the 100 µL should be turbid by visual inspection. If not, continue growing at 37 °C for another 2 hours or until turbid.

		

ii. Set up colony PCR in a 96-well plate as shown in Table 6 below. Refer to Table 7 for specific primer pairs toamplify both the 5' and 3' regions for the intended insert. We recommend including a no template control
reaction (add NYZM plus medium alone instead of bacterial culture)

Table 6. Specific primer pairs needed for colony PCR QC of amplification regions, including the expected positive product sizes after electrophoresis.

Insert

EGFP

mKate2CustominsertPrimer pairs

Amplification region

Positive product size (bp)Edit-R Colony PCR Primer Backbone Forward—Universal and
Edit-R colony PCR primer EGFP reverse

5' EGFP homology arm

289 + 5' homology arm length

Edit-R Colony PCR Primer Backbone Reverse—Universal and
Edit-R colony PCR primer EGFP forward

3' EGFP homology arm

563 + 3' homology arm length

Edit-R Colony PCR Primer Backbone Forward—Universal and
Edit-R colony PCR primer mKate2 reverse

5' mKate2 homology arm

215 + 5' homology arm length

Edit-R Colony PCR Primer Backbone Reverse—Universal and
Edit-R colony PCR primer EGFP forward

3' mKate2 homology arm

415 + 3' homology arm length

Edit-R Colony PCR Primer Backbone Forward—Universal and
Edit-R colony PCR primer EGFP reverse

5' homology arm, insert, and3' homology arm

464 + 5' homology arm length, insert
length, and 3' homology arm length11

Edit-R HDR Plasmid DonorTECHNICAL MANUAL
Table 7. Example Colony PCR setup to detect the 5', mKate2 homology arms. (For EGFP or custom inserts, use the colony PCR primers specified in Table 6.)

		
		
		
		
		
		
		
		

Reagent

1 reaction volume (µL)

DreamTaq 2x master mix

12.5

Edit-R Colony PCR Primer Backbone Forward - Universal (10 µM)

2.5

Edit-R colony PCR primer mKate2 reverse (10 µM)

2.5

Water, nuclease free

4.25

DMSO (5%)

1.25

Template (diluted 4 fold)2

Total volume25

iii. Make a master mix of all components except the template according to Table 7.
iv. Add 23 µL of the master mix to each well of the 96-well plate.
v. Dilute growing cultures from step 6a. (template) into a new 96-well plate by adding 2 µL culture to 6 µL of
nuclease-free water and pipette up and down 3-4 times.
vi. Add 2 µL of the diluted template to one well in the colony PCR master mix plate. Add 2 µL NYZM plus
medium alone to one reaction for the no template control sample.
vii. Pipette up and down 3-4 times to mix the reactions.
viii. Seal the PCR plate carefully, spin down all volume to the bottom of the plate and run in a thermal cycler
according to these optimized conditions (Table 8). For a custom insert, adjust the PCR cycling conditions to 		match insert length, according to manufactures instructions.
ix. Store the remaining cultures at 4 °C to use as starter culture for an overnight growth and from positive
resulting colony PCR.

Table 8. Optimized cycling conditions for colony PCR

Cycle step

Temperature

Time

Cycle(s)Initial denaturation

95 °C

10 min1

Denature

95 °C

15 s

Annealing

61 °C

50 s

Extension

72 °C

30 s*

Final extension

72 °C

5 min

35
1

Custom inserts will require a longer extension time. For DreamTaq polymerase an extension time of 30 s per kb is recommended.

		x. Load 5 µL on an agarose gel to confirm amplicon length is as expected for homology arm insertions into the
HDR plasmid donor.
		 xi. Expected sizes for negative controls are shown in Table 7. Product size for positive clones will run higher		
based on the size of the homology arms.
b. Grow 1-3 positive colonies (containing the expected sized 5'and 3' homology arm) and prepare the plasmids
		 using a plasmid DNA miniprep kit.
c. Sanger sequence plasmid DNA using the Edit-R HDR colony PCR and sequencing primers to confirm correct
		 insertion into the plasmid donor.
		 i. Align Sanger sequencing reads to either the provided EGFP or mKate2 insert sequence, or custom insert 		sequence as proved to us during the design process. Successful cloning is defined as in frame addition of the 				intended insert sequence between the defined homology arms.
d. Grow cultures containing sequence-confirmed plasmid and prepare using a transfection-quality plasmid DNA kit.
Our EGFP and mKate2 insert sequences contain a four amino acid flexible linker at the beginning of the fluorescent protein sequence,
and a three amino acid flexible linker at the end of the fluorescent protein sequence. See section 6 Frequently asked questions (FAQs)
for more detail.
12

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

4

Guidelines for knock-in co-transfection using the Edit-R platform
with HDR DNA plasmid donors
The following are example protocols for co-transfection of Edit-R components (Cas9 nuclease, synthetic guide RNA,and HDR plasmid donor) required for HDR-mediated knock-in in a 96-well format. The first protocol is for Cas9 nuclease
mRNA, synthetic guide RNA, and HDR DNA plasmid donor and the second protocol is for Cas9 nuclease protein, synthetic
guide RNA, and HDR DNA plasmid donor. See the Appendix for additional recommendations on cell plating andoptimizing lipid co-transfection conditions and HDR DNA plasmid donor design recommendations.
We recommend DNA-free reagents, Cas9 protein or Cas9 mRNA and synthetic guide RNA, to reduce the potential for unwanted
sequences integrating into the gDNA instead of your specific DNA donor.

Materials required for knock-in co-transfection
Edit-R CRISPR-Cas9 materials for gene editing can be ordered at https://dharmacon.horizondiscovery.com/.
•
Cas9 nuclease (choose one):•»»

Edit-R Cas9 Nuclease mRNA, 20 μg (1 μg/μL; Cat #CAS11195)»»orEdit-R Cas9 Nuclease protein NLS, 500 pmol (20 µM; Cat #CAS11200)

Synthetic targeting guide RNA (choose one):»»

Edit-R synthetic crRNA and tracrRNA oligos:
a. crRNA, predesigned for your gene of interest in a variety of sizes, or designed and ordered using theDharmacon CRISPR Design Toolb. tracrRNA, 5, 20 or 50 nmol (Cat #U-002005-XX)»»

orEdit-R synthetic sgRNA, custom ordering using the Dharmacon CRISPR Design Tool

For the most efficient knock-in, we recommend testing three to five guide RNA designs per knock-in experiment to identify
the one with highest editing efficiency. It has been demonstrated that knock-in is most efficient when the DSB is lessthan 10 bp from the desired insertion site, so choosing a crRNA close to the DSB with highest function is desirable.

•

Plasmid donor (constructed by the researcher from the Edit-R HDR plasmid donor kit) and utilizes the
Edit-R HDR Donor Designer webtool.

•

DharmaFECT™ Duo transfection reagent [Cat #T-2010-01 (0.2 mL); T-2010-02 (0.75 mL);
T-2010-03 (1.5 mL); or T-2010-04 (1.5 mL × 5 tubes)]

Additional reagents to be supplied by researcher
Transfection experiments require standard cell culture reagents and instruments appropriate for maintenance of cells.
The following additional materials are required but not supplied:•

Multi-well tissue culture plates or tissue culture dishes•

Antibiotic-free complete medium: Cell culture medium (including serum and/or supplements) recommended
for maintenance and passaging of the cells of interest without antibiotic•

Assay for assessing cell viability (such as Resazurin cell viability reagent)•

Assay(s) for detecting gene editing events in a cell population
13

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

Co-transfection of HDR plasmid donor with Edit-R Cas9 Nuclease mRNA and
Edit-R synthetic guide RNAs
The following is a general protocol using DharmaFECT Duo transfection reagent to deliver Edit-R Cas9 Nuclease mRNA,
synthetic guide RNA, and HDR plasmid donor into cultured mammalian cells. Exact reagent amounts and parameters
for co-transfection should be empirically determined through careful optimization in your cells prior to experimentation(see the transfection optimization section of the Edit-R CRISPR-Cas9 Genome Engineering with Cas9 Nuclease mRNA and
Synthetic RNAs technical manual. The protocol below describes delivery conditions in U2OS cells in one well of a 96-wellplate and is given for illustrative purposes only. Reagent volumes should be calculated to include replicate samples andpipetting overage as necessary.
Day 1
1. Plate cells in 96-well plates using growth medium at a cell density so that the cells are 70 to 90% confluent thenext day.
2. Incubate cells at 37 °C in a humidified CO2 incubator overnight.
Day 2
3. Prepare a 100 ng/μL Cas9 mRNA working solution by thawing Cas9 mRNA on ice and adding 2 μL of 1 μg/μL stock
solution of Cas9 mRNA to 18 μL of Tris buffer. Verify the Cas9 mRNA concentration using UV spectrophotometry at
260 nm and adjust the volume if necessary to obtain 100 ng/μL.
On initial use, aliquot remaining Cas9 mRNA stock or working solution into convenient volumes and store all aliquots at -80 °C tominimize the number of freeze-thaw cycles.

4. Prepare 100 ng/µL HDR plasmid donor working solution (e.g., Tris buffer).
5. Prepare 2 μM synthetic guide RNA transfection complex from previously prepared 10 µM stocks (see Appendix)»»»»

For crRNA:tracrRNA add 2 μL of 10 µM crRNA and 2 μL of 10 µM tracrRNA to 6 μL of Tris buffer(total volume is 10 μL).
or
For synthetic sgRNA add 2 μL of 10 µM synthetic sgRNA to 8 μL of Tris buffer (total volume is 10 μL).

6. In a 1.7 mL tube (or deep-well plate) prepare for each sample to be transfected as described in Table 9 (columns2-5) for a final 25 nM concentration of the synthetic guide RNA, 200 ng/well of Cas9 mRNA and 200 ng/well of
the HDR plasmid donor in the final transfection mixture.
We strongly recommend including three controls: 1) an untransfected control as a negative control and to assess cell viability, 2) a gene
editing control to confirm base editing and transfection efficiency, and 3) an HDR plasmid donor only control to normalize for potential
nonspecific-fluorescence from the plasmid donor alone.

We have observe background fluorescence from expression of the HDR DNA plasmid donor alone. This typically occurs when promoter
sequences or transcription initiation components are part of the 5' homology arm when the fluorescent protein insertion is at the 5'
end of the target gene.

7. Prepare a 30 μg/mL DharmaFECT Duo working solution by diluting 3 μL of 1 mg/mL stock DharmaFECT Duotransfection reagent in 97 μL serum-free medium and mix gently; this volume is sufficient for 10 wells with
0.3 μL/well in 96-well format. Incubate for 5 minutes at room temperature.

14

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

Table 9. Preparing transfection samples for gene editing experiment in a 96-well plate format

Serum-free
mediumSynthetic guide RNAtransfection complex(2 μM)

Cas9 mRNA
working solution(100 ng/µL)

HDR DNA
plasmid donor(100 ng/µL)

DharmaFECTDuo working
solution(30 µg/mL)

GrowthmediumTotal
volume per
well

Cas9 nuclease mRNA
with HDR plasmid donor
and gene-specific synthetic
guide RNA (HDR knock-in)

4.75

1.25

2

2

10

80

100

Cas9 nuclease mRNA withgene-specific synthetic guide
RNA (gene editing control)

6.75

1.25

2

0

10

80

100

HDR plasmid donor only
(No Cas9 mRNA and synthetic
guide RNA (nonspecificfluorescence control)

8

0

0

2

10

80

100

Untransfected(negative control)

20

0

0

0

0

80

100

SampleVolumes (µL) are for a single well of a 96-well plate of U2OS cells to be transfected. It is recommended to prepare sufficient sample volumes for the total number of
replicates and to account for pipetting error. Exact reagent amounts for co-transfection in other cell lines of interest should be empirically determined through careful
optimization prior to experimentation (see the Transfection optimization section in the Edit-R CRISPR-Cas9 Genome Engineering with Cas9 Nuclease mRNA and
Synthetic guide RNAs technical manual.

8. Add 10 μL DharmaFECT Duo working solution to each sample tube as shown in Table 9 (column 6); this will result in
0.3 μg/well final concentration. DO NOT add DharmaFECT Duo working solution to the untransfected control, whichshould contain serum-free medium only. This brings the total volume to 20 μL in each tube. Mix by pipetting gently
up and down and incubate for 20 minutes at room temperature.
9. Prepare transfection mixture by adding 80 μL antibiotic-free complete medium to each sample to bring the total
volume in each tube to 100 μL as shown in Table 9 (columns 7 and 8).
10. Remove medium from the wells of the 96-well plate containing cells and replace with 100 μL of the appropriate
transfection mixture to each well.
11. Incubate cells at 37 °C with 5% CO2 for 72 hours.
12. At 72 hours:		a. Image cells using fluorescent microscopy.

			 i. Observe cellular localization of the inserted fluorescent reporter in the HDR knock-in transfected sample.
			 ii. Confirm no fluorescence expression in the controls.
			
			
			
		

»» If the nonspecific-fluorescence control exhibits fluorescence, passage cells until the plasmid(and fluorescence) is diluted out of the cells. At this point, an aliquot of the HDR-transfected cells can beassayed for knock-in on a cell population level (see the Detect HDR integration of a fluorescent protein withseveral assays section in the Appendix).

b. Retain an aliquot of the gene editing control and negative control samples for analysis (see the Detect basegene editing with mismatch detection assay section in the Appendix).

13. Expand HDR-transfected cells for clonal cell line generation and assay clonal populations to confirm the desired
insertion (see Detect HDR integration of a fluorescent protein with several assays in the Appendix).

15

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

Co-transfection of HDR plasmid donor with Edit-R Cas9 Nuclease protein and
Edit-R synthetic RNAs
The following is a general protocol using DharmaFECT Duo transfection reagent to deliver Edit-R Cas9 Nuclease
protein, synthetic guide RNA, and plasmid donor into cultured mammalian cells. Exact reagent amounts and parameters
for co-transfection should be empirically determined through careful optimization in your cells of interest prior toexperimentation (see the Transfection optimization section of the Dharmacon Edit-R CRISPR-Cas9 Genome Engineeringwith Cas9 protein and synthetic guide RNAs technical manual. The protocol below describes delivery conditions in U2OS
cells in one well of a 96-well plate and is given for illustrative purposes only. Reagent volumes should be calculated toinclude replicate samples and pipetting overage as necessary.
Day 1
1. Plate cells in 96-well plates using growth medium at cell density so that the cells are 70 to 90%
confluent the next day.
2. Incubate cells at 37 °C in a humidified CO2 incubator overnight.
Day 2
3. Prepare a 2.5 µM Cas9 protein working solution by adding 0.5 µL of Cas9 protein (20 µM stock solution)
to 3.5 µL of serum-free medium.
It is advised that the minimum volume of Cas9 protein working solution required for the experiment be prepared, as storage of diluted
Cas9 protein is not recommended.

4. Prepare 100 ng/µL HDR plasmid donor working solution (e.g., Tris buffer).
5. Prepare 2 μM synthetic guide RNA, transfection complex from previously prepared 10 µM stocks (see Appendix).
»»»»

For crRNA:tracrRNA add 2 μL of 10 µM crRNA and 2 μL of 10 µM tracrRNA to 6 μL of Tris buffer(total volume is 10 μL).
or
For synthetic sgRNA add 2 μL of 10 µM synthetic sgRNA to 8 μL of Tris buffer (total volume is 10 μL).

6. In a 1.7 mL tube (or deep-well plate) prepare for each sample to be transfected as described in Table 10 (columns2-5) for a final 50 nM concentration of the synthetic guide RNA, 25 nM of Cas9 protein and 200 ng/well in the final
transfection mixture.
We strongly recommend including three controls: 1) an untransfected control as a negative control and to assess cell viability, 2) a gene
editing control to confirm base editing and transfection efficiency and 3) an HDR plasmid donor only control to normalize for potential
nonspecific fluorescence from the plasmid donor alone.

We have observe background fluorescence from expression of the HDR DNA plasmid donor alone. This typically occurs when promoter
sequences or transcription initiation components are part of the 5' homology arm when the fluorescent protein insertion is at the
5' end of the target gene.

16

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

Table 10. Preparing transfection samples for gene editing experiment in a 96-well plate format

Serum-free
mediumSynthetic guide RNAtransfection complex(2 μM)

Cas9 protein
working solution(2.5 µM)

HDR DNA
Plasmid donor(100 ng/µL)

DharmaFECT Duo
working solution(6 µg/mL)

Total volume
per well

Cas9 nuclease proteinwith HDR plasmid donor
and gene-specific synthetic
guide RNA(HDR knock-in)

45.5

2.5

1

1

50

100

Cas9 nuclease mRNA with andgene-specific synthetic guide
RNA (gene editing control)

46.5

2.

1

0

50

100

HDR plasmid donor only
(nuclease protein and synthetic
guide RNA) (nonspecificfluorescence control)

49

0

0

1

50

100

Untransfected(negative control)

100

0

0

0

0

100

SampleVolumes (µL) are for a single well of a 96-well plate of U2OS cells to be transfected. It is recommended to prepare sufficient sample volumes for the total number of
replicates and to account for pipetting error. Exact reagent amounts for co-transfection in other cell lines of interest should be empirically determined through careful
optimization prior to experimentation (see the Transfection optimization section in the Edit-R CRISPR-Cas9 Genome Engineering with Cas9 protein and synthetic
guide RNAs technical manual).

7. Prepare a 6 μg/mL DharmaFECT Duo working solution by diluting 3 μL of 1 mg/mL stock DharmaFECT Duotransfection reagent in 497 μL serum-free medium and mix gently; this volume is sufficient for 10 wells with
0.3 μL/well in 96-well format. Incubate for 5 minutes at room temperature.
8. Add 50 μL DharmaFECT Duo working solution to each sample tube as shown in Table 10 (column 6); this will result in
0.3 μg/well final concentration. DO NOT add DharmaFECT Duo working solution to the untransfected control, whichshould contain serum-free medium only. This brings the total volume to 100 μL in each tube. Mix by pipetting gently
up and down and incubate for 20 minutes at room temperature.
9. Remove growth medium from the wells of the 96-well plate containing cells and replace with 100 μL of theappropriate transfection mixture to each well.
10. Incubate cells at 37 °C with 5% CO2 for 14-18 hours; replace transfection mixture on the cells with growth medium
without antibiotics.
11. Continue to incubate cells at 37 °C with 5% CO2 until 48-72 hours post-transfection.
12. At 48-72 hours post-transfection:
		a. Image cells using fluorescent microscopy.

			 i. Observe cellular localization of the inserted fluorescent reporter in the HDR knock-in transfected sample.
			 ii. Confirm no fluorescence expression in the controls.
			
			
			
		

»» If the nonspecific-fluorescence control exhibits fluorescence, passage cells until the plasmid (and fluorescence)is diluted out of the cells. At this point, an aliquot of the HDR-transfected cells can be assayed for knock-in on acell population level (see the Detect HDR integration of a fluorescent protein with several assays section in
the Appendix).

b. Retain an aliquot of the gene editing control and negative control samples for analysis (see the Detect basegene editing with mismatch detection assay section in the Appendix).

13. Expand HDR-transfected cells for clonal cell line generation and assay clonal populations to confirm the desired
17

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

5

Appendix
Additional resources
For general recommendations on cell plating and optimizing lipid co-transfection conditions, see the Edit-R CRISPR-Cas9
Genome Engineering with Cas9 Nuclease mRNA and Synthetic guide RNAs technical manual; and/or the Edit-R CRISPRCas9 Genome Engineering with Cas9 protein and synthetic guide RNAs technical manual. For a convenient benchtop
version of the HDR co-transfection protocol, see the Edit-R Cas9 mRNA, synthetic guide RNA and HDR donor templatetransfection protocol. We recommend optimizing lipid co-transfections with the Edit-R components (Cas9 nuclease andsynthetic guide RNA) before performing lipid co-transfection with a HDR plasmid donor. This maximizes the number of
DSBs available to the cell to repair through the HDR pathway.
For general HDR plasmid donor design recommendations see this useful guide . The tips included in the guide are used in
the HDR Donor Designer.
For a protocol to make 10 µM concentrated stock solutions of synthetic guide RNAs see (the Edit-R synthetic guide RNA
resuspension protocol.

Gene editing assay recommendations
Detect NHEJ-mediated gene editing with mismatch detection assay
The most commonly used method for detection of indels in a cell population is a DNA mismatch detection assay(Guschin, 2010; Reyon, 2012; Cong, 2013). This assay can be performed on either purified genomic DNA or whole cell lysate
using T7 Endonuclease I (T7EI) . For HDR-mediated knock-in experiments, mismatch detection should be performed on
the gene editing control (Cas9 nuclease and synthetic guide RNA) and compared to the negative control (untransfected)
sample to obtain the “base” editing or NHEJ-mediated editing of the transfection experiment. This is typically performed
at 72 hours post-transfection. Including this analysis is useful to confirm efficient editing in the transfection. If the
NHEJ-mediated editing on the control sample is significantly less than the previously optimized transfections, then theexperiment may need to be repeated or further optimized because the HDR-mediated knock-in efficiency is always lower
than the NHEJ-mediated editing (and might not be detectable when the transfection is low efficiency).
Detect integration of a fluorescent protein with several assays
The most commonly used methods for detecting HDR-mediated integration and expression of a fluorescent protein
in a population of cells are fluorescence microscopy and flow cytometry. Additionally, PCR-based assays [e.g., restriction
fragment length polymorphism (RFLP) assay] and Sanger sequencing are utilized. For more details about gene editing
assays to detect HDR-mediated knock-in on a population level, please see the following Application Note: Homologydirected repair with Edit-R CRISPR-Cas9 reagents and single-stranded DNA oligos.
After analysis at a population level we recommend isolating several fluorescently tagged clonal cell lines and performing
Sanger sequencing across the insertion site to verify correct insertion. For more details about clonal cell line generation
or detection of fluorescent protein knock-in in a clonal population, please see the following Application Notes:
A CRISPR-Cas9 gene engineering workflow: generating functional knockouts using Edit-R Cas9 and synthetic crRNA andtracrRNA and Fluorescent tagging of an endogenous gene by homology-directed repair using Dharmacon Edit-R CRISPRCas9 reagents.

18

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

6Frequently asked questions (FAQs)
Is the inserted florescent protein a direct fusion or is there a linker?
We have designed our donor template to contain a flexible linker of 4 and 3 amino acids, respectively, before and after the
fluorescent protein insertion.
Do I have to add in a start or stop codon for the fluorescent protein? No, this system is designed to utilize the startand stop codon of the endogenous gene transcript. When designing the custom homology arm PCR primers, the insertion
location should be chosen to be within the endogenous gene transcript. Ideally, the insertion location should be precisely
after the start codon for an N-terminal insertion, or conversely, immediately before the stop codon for a C-terminal insertion.
Please note that cells may express an alternative transcript from the one you design to and that may impact expression of theinserted fluorescent reporter.
Can I use the HDR donor kit with my own homology arms (without ordering the custom HA primers fromyour website)?
No, the HDR Donor Designer appends an adapter sequence to the homology arm primers that is necessary for compatibility
with the HDR plasmid donor backbone and the mKate2 kit components.
How should I choose the homology arm lengths?
The HDR Donor Designer will design optimal PCR primer pairs that are ranked based on a quality score provided by Primer3
with options of homology length ranging between 500 and 1000 bp. Depending on the chosen insertion site and thecorresponding sequence composition, the insert-adjacent primers may be non-ideal and require PCR optimization. For thisreason we suggest testing 2 or 3 pairs for each HA, representing different lengths, to ensure optimal success.
Do I need to gel purify the PCR-amplified homology arms or can I just do a column clean up?
We recommend gel purification of homology arms, because it results in the highest cloning efficiency. Gel purification isabsolutely required when PCR optimization is challenging (when multiple amplification bands or primer dimers are present).
Is there a way to check that the assembly reaction worked?
Yes, if you are using the Gibson Assembly method or another in vitro assembly method you can confirm assembly with
PCR; use 1 µL of the assembly reaction as template and the colony PCR primers provided in the kit in a PCR and then run it
out on a 2% agarose gel to confirm presence of the assembled junctions.
What should I do if I have multiple bands from my PCR-amplified homology arm reaction?
If there are multiple bands, including the desired amplicon length, the PCR should be optimized further to remove nonspecific product(s). PCR can be optimized by running a gradient thermal cycling protocol, adding DMSO for high GC
content, or using a different polymerase. If, after optimization, multiple bands still exist, separate on an agarose gel andextract the band of expected size. In this case, multiple PCRs can be pooled together and then purified to ensure enoughmaterial for assembly (at least 1.5 µL of 20 ng/µL).
What should I do if I have multiple bands from my colony PCR?
Because colony PCR is a crude method, multiple bands may result from potential contamination (inhibition from bacterial
cells or culture medium). If there is a band at the expected size of a positive clone and another band as well, in most cases
this indicates a positive clone and Sanger sequencing should be performed to confirm.

19

Edit-R HDR Plasmid DonorTECHNICAL MANUALCan I use the colony PCR primers for Sanger sequencing?
Yes, the colony PCR primers are intended to be used for both colony PCR and the Sanger sequencing.
What are the sequences of the colony PCR/Sanger sequencing primers?
Primer namePrimer sequence 5'- 3'

Edit-R Colony PCR Primer EGFP Forward

AGGTGAACTTCAAGATCCGCCACA

Edit-R Colony PCR Primer EGFP Reverse

AACTTCAGGGTCAGCTTGC

Edit-R Colony PCR Primer mKate2 Forward

GGCCGACAAAGAGACCTACG

Edit-R Colony PCR Primer mKate2 Reverse

GTTGTTCACGGTGCCCTCCA

Edit-R Colony PCR Primer Backbone Forward

TCGCCCGGTTTATTGAAATG

Edit-R Colony PCR Primer Backbone Reverse

TTCGCCACCTCTGACTTGAGC

What are good stopping points during the plasmid donor cloning protocol?
We recommend that the PCR purification and assembly reaction are completed on the same day to avoid freeze-thaw
cycles of purified PCR products. Appropriate stopping points are before PCR (step 2b.) or gel purification (step 3c) where
DNA in agarose gel or PCRs can be frozen at -20 °C before proceeding with protocol.
How should I store my DNA assembly components?
Assembly components should be eluted and/or resuspended in 10 mM Tris-HCL pH 7.5, aliquoted into single-use tubes
and stored at -20 °C or -80 °C to minimize freeze-thaw cycles.
Can I use another bacterial growth medium instead of NYZM plus?
NZYM plus medium is a rich broth that supplies bacteria with amino acids, vitamins and metabolites that in turn do notneed to be synthesized by the bacteria allowing for faster growth. This is part of our optional, quick colony PCR protocolso that growth and colony PCR can be completed in one day. Other medium can be substituted for the quick protocol,
but growth time will need to be optimized for consistent colony PCR results.
What is the benefit to using Cas9 protein or Cas9 mRNA instead of Cas9 plasmid or a Cas9-integrated
cell line for HDR experiments?
Specifically, for HDR-mediated knock-in experiments, Cas9 protein and Cas9 mRNA are preferred as they typically givehigher gene editing efficiencies compared to Cas9 plasmid. This is important because HDR-mediated knock-in will
only occur in a fraction of DSBs at the target sites. Additionally, Cas9 protein and Cas9 mRNA are DNA-free options so
there is no concern of DNA integration resulting from the Cas9 source. Similarly, a Cas9-integrated cell line is not ideal
for HDR-mediated knock-in because continuous expression of the Cas9 nuclease increases potential for undesired
off-targets and mutations.
What is the stability of the Edit-R synthetic guide RNA?
Dried RNA oligonucleotide pellets are stable at room temperature for two to four weeks, but should be placed at -20 °C
or -80 °C for long-term storage. Under these conditions, the dried synthetic guide RNA will be stable for at least one year.
Maintaining sterile, RNase- and DNase-free conditions is always recommended as a critical precaution.
I see a fair amount of cell death after transfection of my cells. What can I do about this?
Extensive cell death following transfection is an indication that delivery conditions need to be further optimized. Basicparameters to consider when optimizing transfection include transfection reagent and cell-specific conditions such as theamount of transfection reagent, the lot/batch of transfection reagent, duration of transfection, cell passage number andcell density at transfection. Often decreasing the amount of lipid present during transfection and/or the total duration oftransfection will help minimize the toxic effect to the cells. Additionally, it is not uncommon to observe some variability
20

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

from one tube of transfection reagent to another, and this may also represent a source of experimental variability. If
the problem persists, we recommend that other transfection reagents be considered or contact Technical Support(dharmacon.horizondiscovery.com/service-and-support) for additional troubleshooting help.
How many freeze-thaw cycles can the Edit-R Cas9 Nuclease mRNA be subjected to?
We recommend not exceeding two to three freeze-thaw cycles for Cas9 mRNA. On initial use, thaw the Cas9 mRNA on
ice, aliquot remaining stock into convenient volumes and store all aliquots at -80 °C to minimize the number freeze-thaws
cycles prior to each experiment.
How many freeze-thaw cycles can the synthetic sgRNA, crRNA, or tracrRNA be subjected to?
We recommend not exceeding four to five freeze-thaw cycles for synthetic guide RNA to ensure reagent integrity.
How should I store my synthetic guide RNA?
RNA oligonucleotides should be stored at -20 °C or -80 °C in a non-frost-free freezer, either as a dried pellet or resuspended
in an RNase-free solution buffered to pH ~ 7.4 to help with stability during freeze-thaw cycles. We recommend that RNA
oligonucleotides be resuspended to a convenient stock concentration and stored in small aliquots to avoid multiple
freeze-thaw cycles. RNA oligonucleotides should not go through more than four to five freeze thaw cycles. If degradation
is a concern, the integrity of the RNA oligonucleotides can be evaluated on an analytical PAGE gel. For a resuspension
protocol see the Dharmacon crRNA and tracrRNA Resuspension protocol.

What is the formula for spectrophotometric quantification of synthetic crRNA, tracrRNA and sgRNA?
To quantify RNA, use Beer’s Law: Absorbance (260 nm) = (ε)(concentration)(path length in cm), where ε,epsilon, isthe molar extinction coefficient (provided on the Product Transfer Form supplied with the crRNA or synthetic sgRNAorder; the extinction coefficient for tracrRNA Cat# U-002005 is 757800). When solved for the unknown, the equation
becomes: Concentration = (Absorbance, 260 nm) / [(ε)(path length in cm)]. When a standard 10 mm cuvette is used, thepath length variable in this equation is 1. If a different size of cuvette is used, e.g., a 2 mm microcuvette, then the path
length variable is 0.2
Can I use a different transfection reagent other than DharmaFECT Duo to deliver the Edit-R components into
my cells for HDR experiments?
We have validated our protocols and demonstrated successful gene editing using DharmaFECT Duo transfection reagent
to efficiently deliver the Edit-R components for HDR (both ssDNA oligos and HDR DNA plasmids). We cannot predict theco-transfection ability of other transfection reagents, nor can we troubleshoot experiments performed with any reagent
other than DharmaFECT Duo transfection reagent for this application. However, other suitable transfection reagents could
be utilized provided co-transfection conditions are carefully optimized for each cell line.
Can the ploidy of my cell line affect the results of my CRISPR-Cas9 gene editing experiment?
When using a CRISPR-Cas9 system to disrupt gene function, it is important to know as much as you can about the gene
you are targeting and your cells. In particular, the ploidy of your cells, the gene copy number and the presence of any SNPs
are important considerations. In normal diploid cells, obtaining mutations on both alleles may be required for a complete
knockout and, an observable phenotype. Virtually all cancer lines and many immortalized cell lines exhibit aneuploidy
and, as such, mutation of multiple alleles may be necessary. The presence of SNPs and multiple genomic locations should
also be taken into account when designing crRNAs, as they can affect whether a complete knockout can be achieved.
Expansion of clones from single cells followed by sequence validation to verify the desired mutation on all alleles is themost rigorous approach for confirming complete gene knockout.

21

Edit-R HDR Plasmid DonorTECHNICAL MANUAL

7

References
1.
2.
3.
4.
5.
6.
7.
8.

8

D. Bhaya, M. Davison, et al. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation.
Annu. Rev. Genet. 45, 273-297 (2011).
L. Cong, F. A. Ran, et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 339 (6121), 819-823 (2013).
E. Deltcheva, K. Chylinski, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor Nuclease III.Nature.
471 (7340), 602-607 (2011).
D.Y. Guschin, A. J. Waite, et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol.Biol.
649, 247-256 (2010).
M. Jinek, K. Chylinski, et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity.Science.
337 (6096), 816-821 (2012).
P. Mali, L. Yang, et al. RNA-guided human genome engineering via Cas9. Science. 339 (6121), 823-826 (2013).
D. Reyon, C. Khayter, et al. Engineering designer transcription activator-like effector nucleases (TALENs) by REAL or REAL-Fast
assembly. Curr. Protoc. Mol. Biol. 100, 12.15.1-12.15.14 (2012).
T. R. Sampson, D. S. Weiss, et al. Exploiting CRISPR/Cas systems for biotechnology. Bioessays. 36 (1), 34-38 (2014).

Limited use licenses
The gene editing Products, use and applications, are covered by pending and issued patents. Certain Label licenses govern the useof the products; these can be found at http://dharmacon.horizondiscovery.com/uploadedFiles/dharmacon-licensing-statements.pdf.
It is each Buyer’s responsibility to determine which intellectual property rights held by third parties may restrict the use of Products
for a particular application. Please review the Label Licenses governing all use of the shRNA and gene expression products.

If you have any questions contact

Phusion is a trademark of Thermo Fisher Scientific, Inc. Q5 is a trademark of New England Biolabs, Inc. ©2018 Horizon Discovery Group
Company—All rights reserved. First published January 2017. UK Registered Head Office: Building 8100, Cambridge Research Park,
Cambridge, CB25 9TL, United Kingdom.

V4-0418

t +44 (0) 1223 976 000 (UK) or +1 800 235 9880 (USA); +1 303 604 9499 (USA)f +44 (0)1223 655 581
w horizondiscovery.com/contact-us or dharmacon.horizondiscovery.com/service-and-support
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom