PROTOCOL

Dharmacon Edit-R sgRNARNP Electroporation
A protocol for RNP electroporation of arrayedsynthetic sgRNAs for CRISPR gene knockout

Guidelines for using arrayed sgRNA plates
for gene knockout
Product description
Libraries of predesigned synthetic single-guide RNAs (sgRNAs) provide
an opportunity to use the CRISPR-Cas9 system for functional geneknockout analysis, in an arrayed format. Dharmacon sgRNA libraries
for functional gene knockout consist of Edit-R synthetic sgRNAs which
are predesigned using the proprietary Edit-R CRISPR RNA algorithm.
This algorithm was designed utilizing functional knockout data and has
demonstrated the ability to select guide RNA target regions more likely
to give functional knockout of the protein, not just create a cut. The
algorithm also includes specificity scoring using an internal alignment
tool for complete off-target identification.
Formats• Catalog libraries of predefined gene family collections.
» As a pool of three sgRNA per well, 1 nmol/well.
» Provided in NUNC Polystyrene 384-well plates(Thermo Scientific™ ABgene™ Cat #AB-0781) with outercolumns left open for inclusion of untreated cells andscreen-specific positive and non-targeting controls.
» ECHO qualified plates are available upon request.
• Cherry-pick sgRNA libraries based on a desired gene list.
» Multiple individual sgRNAs or a sgRNA pool of three constructs,
minimum 20 wells.
» Customizable plate layout; Edit-R catalog control sgRNAs may
be added to any wells within the plate(s).
» Provided in NUNC Polystyrene 384-well plates(Thermo Scientific™ ABgene™ Cat #AB-0781).
» ECHO qualified plates are available upon request.

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For phenotypic analysis with the arrayed synthetic sgRNAlibraries for gene knockout, we strongly recommend using
cell lines that constitutively express Cas9 nuclease. Whencells constitutively expressing Cas9 nuclease are not feasible,
such as for work in primary cells, transfection of sgRNAand Cas9 protein as a ribonucleoprotein (RNP) complex isrecommended. Successful electroporation of the Cas9 RNP
complex allows for downstream phenotypic analysis of theedited cell population.
Materials
Plates of synthetic sgRNAs, 1 nmol per well in 384-well plates.
Additional required materials are listed below and are not provided withsgRNA library purchase:• Cas9 Nuclease Protein NLS is available as:» 100µg, 500µg, and 5 × 500μg sizes• 10 mM Tris pH 7.4 nuclease-free buffer solution(Dharmacon, Cat #B-006000-100)• 384-well tissue culture plates• Assay for assessing cell viability such as CellTiter-Blue® Cell
Viability Assay (Promega Corp., Cat #G8081)• sgRNA controls» Positive control sgRNA and detection primers for assessmentof gene editing.
• Edit-R PPIB or DNMT3B positive controls and kits are designedand validated for mismatch detection assays to verify gene
editing experiments.
• Edit-R lethal synthetic sgRNA controls are universal positive
controls designed to induce cell death in a dose- andCas9-dependent manner by targeting multiple repeat
regions in the genome.
» Edit-R non-targeting control sgRNA.
» Assay-specific positive control sgRNA (defined bythe researcher).

• Cell maintenance medium: antibiotic-free cell culture medium(with serum and/or supplements) recommended for maintenanceof the cells of interest.
• Electroporation instrument and reagents» 384-well Nucleofector™ System» P3 Primary Cell 384-well Nucleofector™ Kit, or» SF, SE, SG Cell Line 384-well Nucleofector™ Kit(Lonza Cell Line Optimization Kit recommended for priordetermination of most suitable buffer for cell line of interest)

CRISPR sgRNA library nucleofection using RNPs
The protocol for RNP electroporation of arrayed sgRNA libraries
needs to be carefully optimized. Cell density will depend on thegrowth characteristics of specific cell lines and assay requirements.
Exact parameters for sgRNA electroporation in your cells of interestshould be empirically determined through careful optimization
before experimentation. Catalog sgRNA library plates are suppliedwith columns 1, 2, 23 and 24 empty to allow the addition ofresearcher-defined controls.
Optimization parameters
Editing efficiency will correlate directly with the transfection
efficiency, therefore, optimal delivery conditions will increaseknockout efficiency. Exact reagents, amounts and parameters forelectroporation should be empirically determined through careful
optimization in cells of interest, in accordance with the electroporation
instrument manufacturer’s recommendations.

Reagent/condition

Control type

Electroporated cells

Effect of electroporation
on readout

A. Positive control PPIB or DNMT3B, can be used to assess
editing efficiency by T7E1 and TIDE analysis
B. Lethal controls provide a quick assessment of electroporation
efficiency - when the nucleofection protocol is optimized andinduces minimal cell death with the NTC controls, the, editing
efficiency of the lethal controls correlates with cell death.
C. Custom control, a known gene that affects the phenotype
readout e.g., expression of a marker that can be assessed anddetected in the read-out assay.
4. Time course to establish the best readout timing after RNP delivery• This can be cell and assays specific and we suggest
examination between 2 to 7 days post electroporation.
Guidelines for resuspension of the sgRNAFor automated or high throughput electroporation workflows higher
higher stock concentrations of sgRNA are required (50–100µM) toallow for the dead volume in liquid handling or acoustic dispensing.
1.

The sgRNA libraries are shipped at ambient temperature as
dry pellets of RNA in each well and should be stored at–20 °C upon arrival in a manual defrost or non-cycling freezer.
If necessary, sgRNAs can be stored as dry pellets (unopened)at 4 °C for several weeks.

1. Optimize the number of cells for electroporation specific to yourcells of interest and assay requirements. A range between
105 -106 cells can be electroporated per well in a 384-wellelectroporation plate2.

Briefly centrifuge sgRNA plates to ensure that the sgRNA iscollected at the bottom of the well.

3.

Wipe adhesive foil cover with 70% ethanol orRNase-decontamination solution.

2. sgRNA:Cas9 protein amounts and ratio» Examine different amounts of sgRNA and Cas9 proteins withdifferent ratios of sgRNA to Cas9 protein. Test 30-150 pmol
sgRNA and 15-60 pmols of Cas9 protein per 20 µL ofelectroporation volume in different ratios of sgRNA to Cas9protein (4:1, 3:1, and 2:1)

4.

Carefully peel back the foil seal to gain access to wells.
Use caution and avoid shredding the seal.

5.

For a 384-well plate library with 1 nmol of sgRNAs per well,resuspend arrayed sgRNAs to 100 μM solution by adding10 μL of nuclease-free 10 mM Tris pH 7.4 buffer to each well.
Plan for any additional dead volume required for automationor other instrument. If necessary, increase the volume andmake lower sgRNA concentration, however, do not go below
50 μM sgRNA solution.

6.

Pipette solution up and down 3-5 times while avoiding theintroduction of bubbles.

7.

Seal the plate and place it on an orbital mixer/shaker for
70-90 minutes at room temperature.

8.

Briefly centrifuge plates to collect solution at the bottomof the wells.
sgRNA plates may now be used immediately or stored at–20 °C in a manual defrost or non-cycling freezer.

3. Optimization controls» The table below provides a list suggested control for
nucleofection optimization. We recommend assaying eachreagent/condition in triplicate.
Reagent/condition

Control type

PPIB or DNMT3B

Positive control to assess
editing efficiency

Non-targeting control

Effect of CRISPR reagents
without DNA cut

9.

Cas9 only

Non-specific Cas9 effect

Lethal control #1 or #2

Electroporation efficiency

10. For storage, seal plates with appropriate adhesive orheat seals.

Custom controlAssay specific control

Non-electroporated cells

Cell baseline response

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RNP nucleofection protocol
The protocol below is geared towards primary cells and provided
for illustrative purposes. Buffer type, cell density, pulse code, andtimelines may vary, and should be optimized specifically for thecells of interest.
1. Prepare electroporation buffer – For primary cells use 384-wellNuclefector kit (Lonza cat # V5SP-3010)—mix P3 buffer andSupplement 1 according to manufacturer’s recommendations
2. Retrieve the sgRNA library and Cas9 RNP from the -20° C freezer.
Thaw reagents on ice.
3. Prepare the RNP complex—transfer the CRISPR reagents(sgRNA and Cas9 protein) at the required amounts into a384-well electroporation plate (384-well Nucleocuvette™ plate)using automation. We recommend making replicate plates.
Note: Do not exceed 20% of electroporation volume (for 384-wellNucleocuvette™ plates, the electroporation volume is 20 µL, sothe RNP complex should not exceed 4 µL).
»For example, if it was determined that 120 pmols of sgRNA and60 pmols of Cas9 protein gave optimal editing in your cells, use
1 uL of Cas9 protein per well (Cat #CAS12207 at 61.8 uM) with
1.2 uL of 100 uM sgRNA per well for a total of 2.2 uL of RNP.

4. Briefly pulse spin the electroporation plate (up to 300 × g), add
P3 electroporation buffer to 10 uL/well and incubate the RNP
complex for 10-15 min at room temperature.

10. Top up the plate with 30 uL of pre-warmed cell maintenance media andtransfer desired amount of the electroporated cell suspensions into384-well tissue culture plates with prewarmed cell maintenance media
once the electroporation is complete, using automation.
11. Incubate the electroporated cells at 37 °C, 5% CO2 for 2 to 7 days
before performing genomic and/or functional analysis of thegene knockout.
Note: The editing timeframe will depend on parameters such as
the activation state of the cells or the half-life of the protein being
targeted. Therefore, multiple timepoints might be required toconfirm knockout.

Appendix
Variety of Cas9 protein quantities available under thesecatalog numbers.RNP nucleofection protocol
Concentration

VolumeQuantityµMµg/µLµLµgpmols

CAS12205
Catalog #CAS12206

61.8

1010100618

61.8

1050

5003090

CAS12207

61.8

105 × 50

5 × 500

5 × 3090

5. Prepare cell suspension containing a total number of cells
needed for your experiment (number of plates � number ofreplicates × number of cells/well).
Note: Cell numbers should be optimized for reagent amounts, but
a range between 105-106 ­—cells can be electroporated per well ina 384-well electroporation plate.
6. Pellet down the cell suspension by centrifugation (300 × g for5 min) and wash with PBS (this step helps remove RNAse activity
from trypsin or FBS in cell media).
7. Resuspend the cell pellet in complete P3 buffer and aliquot 10μLof cell suspension per well.
8. Pulse spin the 384 electroporation plate up to 300 × g. This step
helps removing any air bubbles in the wells prior to electroporation.
9. Electroporate the plate using the 384-well electroporator device
from Lonza.
The pulse code for best electroporation efficiency is cell
type dependent. It should be selected following optimisation
experiments recommended by the manufacturer.

For more information

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