Technical Manual

Screening

Dharmacon™ Edit-R LentiviralsgRNA Pooled Libraries

Storage : -80°C

Version: 0001

Revision date: 012024

For research only. Not for use in diagnostic procedures.

Contents
1 Introduction ………………………………………………………………………………………………………… 2
A. CRISPR-Cas: An adaptive immunity defense mechanism in bacteria and archaea …………….… 2
B. Engineering a CRISPR-Cas9 platform for mammalian genome editing …………………………..….… 2

2 Edit-R Lentiviral sgRNA Pooled Screening platform …………………………………………..…. 2
A. Edit-R Lentiviral Cas9 Nuclease and sgRNA vectors ………………………………………………………….… 3
B. Overview of Edit-R A Lentiviral sgRNA Pooled Library screening workflow ………………….…….. 43 Laboratory protocols and calculation tracking worksheet ……………………………………. 5
4 Required materials for lentiviral sgRNA pooled library screening …………………..……. 6
A. Materials required …………………………………………………………………………………………………………..… 6
B. Additional materials required ……………………………………………………………………………………………. 6

5 Assay development and optimization ………………………………………………………………….. 6
A. Selection of the optimal promoter for Cas9 expression ……………………………………………..………. 6
B. Optimization of lentiviral transduction ………………………………………………………………………………. 6
C. Determination of functional titer ………………………………………………………………………………………. 7
D. Optimization of puromycin selection …………………………………………………………………………………. 8
E. Determination of assay-specific screening conditions ………………………………………………………… 8
F. Selection of average fold representation and number of biological replicates ………………….… 8
G. Calculation of number of cells needed for transduction ……………………………………………………… 9
H. Calculation of volume of lentiviral particles needed for transduction ……………………….……… 10

6 Primary screen …………………………………………………………………………………………………… 11
A. Cell transductions and selection screen ……………………………………………………………………………. 11
B. Genomic DNA isolation ……………………………………………………………………………….……….………….. 13
C. PCR amplification of constructs from genomic DNA …………………………………………………..…….. 13

7 Library prep and Illumina platform sequencing …………………………………………………. 16
8 Hit identification and follow up …………………………………………………………………………. 16
9 Appendix …………………………………………………………………………………………………………… 16
A. Multiplexing and Illumina platform sequencing of gDNA amplified with Edit-R Mouse PooledsgRNA Indexing PCR and Sequencing Primer Kits Stability and storage (for mouse and pre2024 libraries) …………………………………………………………………………………………………………………. 16
B. Stability and storage ……………………………………………………………………………………………………….. 19
C. Quality assurance and control …………………………………………………………………………………………. 19

10 Frequently asked questions ……………………………………………………………………………. 19
11 References ……………………………………………………………………………………………………… 21
12 Lentiviral particle product safety level information …………………………………………. 21
13 Limited use licenses …………………………………………………………………………………………. 22
1

1 Introduction
A. CRISPR-Cas: An adaptive immunity defense mechanism in bacteria and archaea
The CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated protein) system is an adaptive
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 sequences from theinvading genetic material, called protospacers, into a specific region of its genome (the CRISPR locus) between shortpalindromic DNA repeats of variable lengths. Multiple spacer-repeat units are clustered at the CRISPR locus to form the
CRISPR array. The entire locus including the CRISPR array is transcribed by RNA polymerase into a primary transcript, thepre-CRISPR RNA (pre-crRNA), which 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 (theCRISPR-associated or Cas proteins) to cleave complementary target DNA that is adjacent to short sequences known as
protospacer-adjacent motifs (PAMs). Thus, the organism acquires a way to protect itself from subsequent infection 1.

B. Engineering a CRISPR-Cas9 platform for mammalian genome editing
Many bacterial and archaeal CRISPR-Cas systems have been identified with diverse sets of mechanisms, Cas proteins andmulti-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 threecomponents are required for targeted DNA cleavage at specific target sites adjacent to a PAM 2: (1) The endonuclease
Cas9, programmed by (2) a mature crRNA processed from transcription of the CRISPR locus/array which complexes with
(3) another CRISPR locus-encoded RNA, the trans-activating CRISPR RNA (tracrRNA, Figure 1A)3. Alternatively, the crRNA
can be fused to the tracrRNA creating a chimeric structure termed a single guide RNA (sgRNA, Figure 1B) 2.
Upon site-specific double-strand DNA cleavage, a mammalian cell can repair such a break through either nonhomologous
end joining (NHEJ) or homology directed repair (HDR). NHEJ is often imperfect, resulting in small insertions and deletions(indels) that can cause nonsense mutations resulting in gene disruptions to produce gene knockouts 4,5. This endogenous
DNA break repair process, coupled with the highly tractable S. pyogenes CRISPR-Cas9 system, allows for a readilyengineered platform to permanently disrupt gene function.

A.

B.

Genomic DNA

PAM

Genomic DNA

Cas9 Nuclease

crRNA

PAM
Cas9 Nuclease5'

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

2 Edit-R Lentiviral sgRNA Pooled Screening Platform
Edit-R Lentiviral sgRNA Pooled Libraries are pools of lentiviral constructs expressing sgRNAs targeting coding genes forknockout. Gene knockout pooled screening can be performed to identify the function of a coding gene in the regulation ofcellular responses and signaling pathways without the need for costly automation and equipment required for arrayed
screening. The Edit-R Lentiviral sgRNA Pooled Screening platform is a two-vector system that utilizes a lentiviral vector for
Cas9 expression and a gene-specific vector for sgRNA expression designed to the target site of interest. Edit-R LentiviralsgRNA Pooled Libraries are comprised of gene-specific sgRNAs and 50 or 100 non-targeting controls per pool for hit
normalization. Table 1 lists the pre-defined pooled lentiviral libraries that are available. All libraries are delivered with adata file containing complete library information including gene annotations, sgRNA target sequences, complete list of2

controls, and counts per millions of mapped reads obtained from high-throughput sequencing of library prior to lentiviral
packaging.
Table 1. Available Edit-R Lentiviral sgRNA Pooled Libraries

HUMAN

MOUSE

Edit-R Lentiviral sgRNA
Pooled Library1

Number of targeted
genes2

Number of pools

Number of targeted
genes

Number of pools x
number of constructs
per pool2,3Whole Genome

18890

1 pool19683

19 pools x 10 600
constructs

Druggable Genome

6411

1 pool9827

10 pools x 10 170
constructs

GPCR

470

1 pool498

1 pool x 5311
constructs

Ion Channel

581

1 pool340

1 pool x 3818
constructs

Protein Kinase1139

1 pool708

1 pool x 7451
constructs

Phosphatase282

1 pool269

1 pool x 3089
constructs

Protease689

1 pool533

1 pool x 5712
constructs

Ubiquitin Conjugate564

1 pool517

1 pool x 5564
constructs

1 Additional gene families are available by request (technical@horizondiscovery.com)
2

The number of genes and constructs per pool are subject to change at any time without notice.

3 Number of constructs per pool includes gene specific and negative control sgRNA constructs for mouse genes and human libraries pre-2024. Gene specificpositive controls: sgRNA constructs targeting up to 34 protein-coding genes including Actb, Gapdh, Lamb1, and Ppib; negative controls: 100 non-targetingsgRNA controls bioinformatically confirmed to not align with (target) any gene in the human, mouse and rat genomes.

A. Edit-R Lentiviral Cas9 Nuclease and sgRNA vectors
The Edit-R Lentiviral Cas9 Nuclease expression vectors contain a human codon-optimized version of the S. pyogenes cas9(csn1) gene and the blasticidin resistance marker (BlastR). BlastR is placed upstream of the Cas9 coding region rather thanon the 3' end so that no extra amino acids are added to the C-terminus, thus preserving the full Cas9 endonucleaseactivity. Both are expressed as a bicistronic transcript with a 2A peptide sequence linker under the control of a single
promoter (Figure 2). Multiple promoter options are available (Figure 2A) so that the most active promoter driving Cas9
nuclease expression can be chosen for specific cells of interest. In the Edit-R Lentiviral sgRNA vector backbone (Figure 2B),
the gene-specific sgRNA is expressed under the control of a human U6 promoter, while expression of the puromycin
resistance marker (PuroR) is driven from the mouse CMV promoter and allows for rapid selection of cells with integrated
sgRNA. The optimized sgRNA scaffold used in the Edit-R Lentiviral sgRNA can further improve gene knockout efficiency.
The functional elements for both lentiviral vectors are listed and described in Table 2.
Each Edit-R Lentiviral sgRNA is specific to the gene or genomic site of choice. The crRNA region of the sgRNA is comprisedof 19-20 nucleotides identical to the genomic DNA target site, followed by the non-variable sgRNA scaffold containing the
tracrRNA sequence from S. pyogenes. The chosen genomic DNA target sequence must be immediately upstream of a PAM.
The predominant S. pyogenes PAM nucleotide sequence is NGG.
A

B

Figure 2. Schematic maps of the Edit-R Lentiviral Cas9 nuclease andLentiviral sgRNA vectors.

3

Table 2. Elements of the Edit-R Lentiviral Cas9 Nuclease Expression and sgRNA vectors.

Vectorelement

UtilityCas9

Human codon-optimized S. pyogenes Cas9 nuclease for cleavage of targeted DNA when programmed with a sgRNA

T2A

Self-cleaving peptide allows for simultaneous expression of puromycin resistance and Cas9 protein from a single transcript

PuroR

Puromycin resistance marker permits antibiotic selection of transduced mammalian cells

BlastR

Blasticidin resistance marker permits antibiotic selection of transduced mammalian cells

hCMV

Human cytomegalovirus immediate early promoter

mCMV

Mouse cytomegalovirus immediate early promoter

hEF1α

Human elongation factor 1 alpha short promoter

mEF1α

Mouse elongation factor 1 alpha short promoter

PGK

Mouse phosphoglycerate kinase promoter

CAG

Human cytomegalovirus, chicken β-actin hybrid promoter

U6

Human RNA polymerase III promoter U6

sgRNA

Optimized single guide RNA, a fusion of gene-specific crRNA with the tracrRNA scaffold

5' LTR

5' Long Terminal Repeat necessary for lentiviral particle production and integration of the construct into the host cell genomeψPsi packaging sequence allows lentiviral genome packaging using lentiviral packaging systems

RRE

Rev Response Element enhances titer by increasing packaging efficiency of full-length lentiviral genomes

WPRE

Woodchuck Hepatitis Post-transcriptional Regulatory Element enhances transgene expression in target cells

3' SIN LTR

3' Self-inactivating Long Terminal Repeat for generation of replication-incompetent lentiviral particles

SV40 pA

Simian virus 40 polyadenylation signal

pUC oripUC origin of replication

SV40 ori

Simian virus 40 origin of replication

AmpR

Ampicillin resistance gene

B. Overview of Edit-R Lentiviral sgRNA Pooled Library screening workflow
Once basic transduction and assay conditions have been optimized, Cas9-expressing cell lines can easily be generated with
the Edit-R Lentiviral Cas9 Nuclease Expression particles. Cells are transduced at a low multiplicity of infection (MOI) with alentiviral pooled library (see Figure 3 for a screening workflow diagram). Individual cells in the resulting transduced
population will contain single constructs integrated into their genomes. Following transduction and puromycin enrichment
for sgRNA-expressing cells, a selective pressure is applied such that those constructs involved in a specific biologicalresponse can be identified. As a result of the selective pressure, cells expressing the sgRNA construct are either enriched
or depleted in the cell population.
To identify “hits,” gDNA is isolated from the initial transduced cell population (reference sample) and from the transduced
cell population that remained following the application of selective pressure and/or phenotypic selection (experimental
sample). sgRNA constructs within the isolated gDNA are amplified using vector-specific NGS Hit Identification Primers thathave been designed and optimized to minimize amplification bias and introduce diversity into the high-throughput
sequencing run. Illumina P5 and P7 adapters and TruSeq indexes can then be added to the amplified library through a second
low amplification PCR step prior to high-throughput sequencing on an Illumina® platform. The differences in sgRNA
construct abundance between reference and experimental cell populations can then be determined to identify hits fromthe primary screen. Each step of the Edit-R Lentiviral sgRNA Pooled Library workflow, from transduction to hit identification,
has been empirically tested.
For libraries targeting mouse genes and human libraries ordered prior to 2024, sgRNA constructs within the isolated gDNA
are amplified using Edit-R Mouse Pooled sgRNA Forward and Reverse Index PCR primers that have been designed and4

optimized to minimize amplification bias. Following amplification, indexed reaction products can be directly loaded onto
Illumina flow cells and sequenced using Edit-R Mouse Pooled sgRNA Read 1 and Index Read Sequencing primers. The
differences in sgRNA construct abundance between reference and experimental cell populations can then be determined to
identify hits from the primary screen. Protocols for multiplexing and Illumina platform sequencing can be found in Appendix
A.

Figure 3. Gene knockout screening workflow using the Edit-R Lentiviral sgRNA Pooled Library platform.

3 Laboratory protocols and calculation tracking worksheet
The Pooled-sgRNA-screening-protocol-tracking-worksheet is a tool for the recording of key calculations and experimental
parameters. Please review all the detailed protocols provided in this manual prior to using the worksheet. An electronic
version of the worksheet can also be downloaded from the web page. The downloadable Excel worksheet allows users toincorporate specific input related to their screening and performs key calculations to simplify protocol planning. For libraries
targeting mouse genes and human libraries ordered prior to 2024, refer to the second tab of the worksheet.
54 Required materials for lentiviral sgRNA pooled library screening
A. Materials required•••

Edit-R Lentiviral sgRNA Pooled Library
Human libraries:o NGS Hit Identification Primers mCMV
Mouse libraries and pre-2024 human libraries:o Edit-R Mouse Pooled sgRNA Indexing PCR and Sequencing Primer Kit A for Illumina sequencing platformo Edit-R Mouse Pooled sgRNA Indexing PCR and Sequencing Primer Kit B for additional reverse indexing
PCR primers (if experimental design requires additional multiplexing capability)

B. Additional materials required
The following additional materials are required but not supplied:
•
•

•
•
•
•
•

•
•
•
•
•
•

Edit-R lentiviral positive and non-targeting sgRNA controls for gene editing validation and experimental
optimization
SMARTvector Non-targeting Control Particles with optimal promoter and fluorescent reporter (TurboGFP or
TurboRFP) for determining relative transduction efficiency and optimizing transduction conditions in desired cellsof interest.
Transduction enhancer such as LentiBOOST® (https://sirion-biotech.com/lentiboost)Base Medium: antibiotic-free cell culture medium (without supplements or serum)
Growth Medium: antibiotic-free cell culture medium (with serum and/or supplements) recommended formaintenance of the cells
Transduction Medium: the base cell culture medium containing lentiviral particles (with transduction additives
and serum (if necessary)
Live/Dead indicator such as Zombie Dyes (BioLegend®, https://www.biolegend.com/en-us/live-dead)Blasticidin S (Fisher Scientific, Cat #BP2647-25; InvivoGen, Cat #ant-bl-1)
HyClone™ Puromycin 2 HCl (Cytiva, Cat #SV30075.01 or similar)
Phusion Hot Start II High-Fidelity DNA Polymerase and 5x Phusion HF Buffer (Thermo Scientific, Cat #F-549L; NEB,
Cat #M0535L)
Genomic DNA purification kit (Blood and Cell Culture DNA Purification Kit, Qiagen, Cat #13362 or similar)
PCR purification kit (QIAquick™ PCR Purification Kit, Qiagen, Cat #28104 or similar)
Nucleotide mix dNTP set, 100 mM each (NEB, Cat # N0446S or similar)

5 Assay development and optimization
Please use the Pooled-sgRNA-screening-protocol-tracking-worksheet as a guide.

A. Selection of the optimal promoter for Cas9 expression
Expression levels of Cas9 nuclease can have a significant effect on gene knockout efficiency. Therefore, select the lentiviralCas9 nuclease vector with the most active promoter in your cell line based on empirical testing or known promoter
activity.

B. Optimization of lentiviral transduction
While lentiviral particles exhibit broad cell tropism, the conditions for successful and efficient delivery can varysignificantly. It is essential to determine the optimal transduction conditions in each cell line or type of interest. Pleasekeep in mind that the conditions selected during these optimization steps must be compatible with primary screening
protocols and conditions.
Edit-R Lentiviral Cas9 Nuclease Expression and sgRNA vectors do not contain a fluorescent reporter. Therefore,
SMARTvector shRNA Non-targeting Control lentiviral particles (see Section 4.B. Additional materials required) containing
either TurboGFP or TurboRFP reporter genes can be substituted for optimization of transduction conditions.
Detailed protocols for transduction optimization are described in the SMARTvector Lentiviral shRNA Technical Manual(SMARTvector Lentiviral shRNA Technical Manual).
6

Use conditions that produce the lowest or no loss of cell viability. Parameters that may influence the efficiency of lentiviraltransduction include, but are not limited to:Transduction medium: When possible, the transduction of cells with lentiviral particles should be performed in a small
volume of low-serum (0.5-2%) or serum-free medium. For cells sensitive to low serum conditions, transduction
optimization can be performed in complete medium.
Transduction duration: Incubation time can vary between 4 and 24 hours and will depend on the cells of interest.
Transduction medium additives: Cationic polymers such as hexadimethrine bromide (Polybrene) or transduction
enhancers such as LentiBOOST® may be added to enhance lentiviral particle binding to the cell surface. We recommend
testing a range of concentrations for identification of optimal transduction efficiency with minimal or no cell toxicity.
Cell density at transduction: The density at which cells are seeded may also influence transduction efficiency. We
recommend seeding cells at a range of densities for optimization of transduction efficiency. Plate sizes for screening
should be chosen accordingly.

C. Determination of functional titer
The functional titers are found in the data file provided with all Dharmacon lentiviral pooled libraries. However, lentiviraltransduction efficiency varies widely from one cell type to another. After determining optimal lentiviral transduction
conditions, it is required to determine the functional titer in the experimental cell line of choice and calculate an MOI low
enough such that the majority of cells will have single integrations.
To determine the relative titer in the experimental cell line, perform a functional titer protocol such as limiting dilution
with cell viability assay by crystal violet staining. Alternatively, SMARTvector Non-targeting Control lentiviral particles (seeSection 4.B. Additional materials required) containing the optimal promoter and either TurboGFP or TurboRFP reporter
genes can be substituted for determining relative transduction efficiency in your desired cells. Detailed protocols fordetermining functional titer in your cells are described in the SMARTvector Lentiviral shRNA Technical Manual.
Once the functional titer has been determined, use the formula below to calculate the relative transduction efficiencyof your cell line of interest.
Functional titer of Edit-R GFP
Delivery Optimization Control inyour cell line (TU/mL)

÷

Titer of Edit-R GFP Delivery
Optimization Control reported
on the C of A (TU/mL)=

Relative transduction
efficiency of your cell line

Use the calculated relative transduction efficiency of your cell line to then calculate the relative functional titer for the
Edit-R Lentiviral sgRNA pooled library using the following formula:
Relative transduction efficiencyof your cell line

7÷

Titer of the pooled library
reported in data file (TU/mL)=

Relative functional titer ofthe pooled library in yourcell line (TU/mL)

Calculation example
If the functional titer for the Edit-R Lentiviral sgRNA Non-targeting Control particles in your cells of interest isdetermined to be 2.1 × 107 TU/mL and the titer on the C of A is listed as 5.0 × 107 TU/mL, then the relativetransduction efficiency of your cell type is calculated as follows:
2.1 × 107 TU/mL (functional titer in your cell line) ÷ 5.0 × 10 7 TU/mL (titer reported on the C of A) = 0.42
relative transduction efficiency
If the relative transduction efficiency of your cell line is 0.42 and the titer of an Edit-R Lentiviral sgRNA Pooled
Library, as indicated on the C of A, is 5.6 × 108 TU/mL, the relative functional titer of the pool in your cell line
would be determined as follows:
0.42 (relative transduction efficiency) × 5.6 × 108 TU/mL (titer reported on the C of A) = 2.35 × 10 8 TU/mL
relative functional titer in your cell line

D. Optimization blasticidin and puromycin selection
The Edit-R Lentiviral Cas9 Nuclease Expression vectors contain the blasticidin resistance marker while the Edit-R LentiviralsgRNA vector contains the puromycin resistance marker for selection of cells after transduction. Prior to treating cells withblasticidin or puromycin, determine the optimal concentration of each antibiotic required to kill non-transduced cells. This
concentration can be identified by generating an antibiotic kill curve. The blasticidin concentration range for many
mammalian cells is 2-20 µg/mL; for puromycin it is 0.5-10 µg/mL.
Day 1:
1. Plate cells in Growth Medium at a density appropriate for your cell type. Incubate overnight.
Day 2:
2. Change the medium to Selection Medium (Growth Medium supplemented with the antibiotic being tested) at a range ofconcentrations.
Days 4-15:
3. Approximately every 2-3 days replace medium with freshly prepared Selection Medium.
4. Monitor the cells daily and visually observe the percentage of surviving cells. Optimum effectiveness should be reachedin 2-6 days under puromycin selection or 5-15 days under blasticidin selection.
5. The minimum antibiotic concentration to use is the lowest concentration that kills 100% of the cells in 2-15 days fromthe start of antibiotic selection.

E. Determination of assay-specific screening conditions
The pooled screening workflow described here facilitates identification of genetic regulators of a range of biologicalprocesses. Variables to consider when planning screening conditions include, but are not limited to, assay duration,
conditions of selective pressure (for example, concentration or duration), and method of phenotypic selection (forexample, measuring viability, proliferation, surface marker expression or migration). We recommend that you optimize all
assay conditions prior to beginning a lentiviral sgRNA pooled library screen using, if possible, an Edit-R lentiviral sgRNA
construct targeting a gene known to be involved in the phenotype(s) of interest.

F. Selection of average fold representation and number of biological replicates
A critical consideration of lentiviral pooled screening is the extent to which any given construct in a pooled library will be
represented in the screen; in other words, the number of cells that contain an independent genomic integration of any
given sgRNA or the number of biological replicates of each sgRNA integration event and subsequent phenotypic selection.
High sgRNA fold representation results in improved reproducibility between biological replicates and ensures that there isa sufficient window for detection of changes in sgRNA abundance6.
8

Recommendations:• At least 200 independent integrations per sgRNA (i.e., at least 200-fold representation)• At least two biological replicates
We have observed that fold representation has a greater impact on the ability to identify hits than the number ofbiological replicates. Therefore, we recommend performing at least two biological replicates, while maintaining an
average sgRNA fold representation as high as is practical or technically feasible for your screen.

G. Calculation of number of cells needed for transduction
Once transduction optimization experiments have been performed and the assay-specific screening parameters
determined, cells need to be prepared for transduction. The calculations below outline how to determine the number ofcells required at the time of transduction.
Multiplicity of infection (MOI) is defined as the ratio of transducing units of lentiviral particles to target cells. At high MOIs,
each cell will likely be transduced by more than one lentiviral particle. Conversely, at low MOIs, the probability that any
cell is transduced with more than one lentiviral particle is lower (Table 3). For lentiviral pooled screens, each cell will
ideally express a single sgRNA. This ensures that the knockout of a single gene in a particular cell is responsible for the
resulting phenotype. Therefore, we recommend performing lentiviral transductions at an MOI ≤ 0.3.
Table 3. Poisson distribution showing the distribution of cells with the indicated number of lentiviral integrations at various MOIs.

0.2

0
0.90
0.82

Number of lentiviral integrants per cell
1
2
3
0.09
0.00
0.00
0.16
0.02
0.00

4
0.00
0.00

0.3

0.74

0.22

0.03

0.00

0.00

0.4

0.67

0.27

0.05

0.01

0.00

0.5

0.61

0.30

0.08

0.01

0.00

0.6

0.55

0.33

0.10

0.02

0.00

0.7

0.50

0.35

0.12

0.03

0.00

0.8
0.9

0.45

0.36

0.14

0.04

0.01

0.41

0.37

0.16

0.05

0.01

1.0

0.37

0.37

0.18

0.06

0.02

MOI
0.1

At each MOI, the portion of cells having 0, 1, 2, 3, or 4 lentiviral integrants is indicated. For example, at an MOI of 0.3, only 3% of
cells are predicted to contain more than one lentiviral integrant.

To calculate the number of lentiviral integration events required to achieve a given sgRNA foldrepresentation, use the following formula:

Number of lentiviralsgRNAs in the pool

Desired foldrepresentation

×

Desired number of cells withlentiviral sgRNA integrations=

Use the following formula to calculate the number of cells you will need per sample at the time oftransduction:
Desired number of cells withlentiviral sgRNA integrations

9

÷

MOI

=

Number of cells requiredat the time of transduction

Calculation example
If you have a pool of 76000 sgRNAs and you wish to achieve a 200-fold average representation, the desired
number of lentiviral integration events would be calculated as follows:
76000 (number of sgRNA) × 200 (average representation) = 1.52 × 107 lentiviral integration events
Then calculate the number of cells needed at the time of transduction as follows:
1.52 × 107 (lentiviral integration events required) ÷ 0.3 (maximum recommended MOI) = 5.1 × 107 cells
needed at the time of transduction

Plate sizes should be chosen such that the required number of cells can reach the optimum density for transduction, as
determined above, at the time of transduction. Prepare additional plates of cells for each pool and biological replicate thatyou wish to transduce. The calculations below outline how to determine cell plating requirements.
To determine the number of plates required for each biological replicate, use the following formula:

Number of cells
required at the timeof transduction

Cell density attransduction÷÷

Size of plate (mm2)=

Number of plates
required per sample

To determine the total number of plates to seed, use the following formula:

Number of plates
required per sample

×

Number of
biologicalreplicates=

Number of plates
required

Table 4 provides examples of cell and plate numbers and plate sizes for various targeted sgRNA fold representations,assuming a pool size of 76000 sgRNAs and a cell line with optimal transduction density of 250 cells/mm 2.
Table 4. Number of cells required at the time of transduction and cell plating recommendations for indicated average fold representation assuming a wholegenome pool of 76000 sgRNAs, MOI of 0.3, optimal transduction density of 250 cells/mm2 and an effective surface area on 100 mm plates of 7800 mm2.

Fold representation

Number of lentiviral
integrations required

Number of cells required attransduction

Recommended cell plating200

1.52 × 107

5.1 × 107

26 plates × 100 mm

400

3.04 × 107

1.02 × 108

52 plates × 100 mm

H. Calculation of volume of lentiviral particles needed for transduction
Before proceeding with the Edit-R Lentiviral sgRNA pooled library screen, confirm that you have sufficient lentiviralparticles to perform the screen at the desired fold coverage, given the relative transduction efficiency of your cell line andthe number of biological replicates you wish to perform. Follow the steps below to calculate the volume of lentiviralparticles required for each pool. Additional lentiviral particles can be ordered separately.
First, identify the number of lentiviral particles (or transducing units, TU) necessary for each biological replicate basedon the required number of cells at the time of transduction by applying the following formula:

MOI

10

×

Number of cells required at
the time of transduction=

Required number oflentiviral particles (TU)Next, determine the volume of lentiviral particles required based on the following formula:

Required number oflentiviral particles (TU)

Functional titer in yourcell line (TU/mL)

÷

Volume of lentiviralparticles per biologicalreplicate (mL)=

To determine the total volume of lentiviral particles required for the screen, use the following formula:

Volume of lentiviral particles
per biological replicate (mL)×

Number of biologicalreplicates=Total volume oflentiviral particles (mL)

Calculation example
For example, at an MOI of 0.3 and a 200-fold average sgRNA representation at transduction, you will have
prepared 5.1 × 107 cells at the time of transduction. Based on the above formula, calculate the number oflentiviral particles needed:
0.3 (MOI) × 5.1 × 107 cells = 1.52 × 107 TU of lentiviral particles are required for transduction
If you have determined that you need 1.52 × 107 TU and you have a functional titer of 2.35 × 108TU/mL in yourcell line, you can calculate the volume of lentiviral particles needed as follows:
1.52 × 107 TU ÷ 2.35 × 108 TU/mL = 0.065 mL of lentiviral particles
If you are performing two biological replicates, you can determine the total volume of lentiviral particles
required as follows:
0.65 mL of lentiviral particles × 2 biological replicates = 0.13 mL of lentiviral particles

6 Primary screen
The following sections describe the primary screening workflow (Figure 3). Please use the Pooled-sgRNA-screeningprotocol-tracking-worksheet as a guide.

A. Cell transductions and selection screen
The experimental conditions described here serve as a guide for performing lentiviral transductions. However, the precise
cell number and volume of lentiviral particles necessary to achieve the desired MOI and average sgRNA foldrepresentation should be determined specifically for each cell line of interest and each intended screening experiment, as
outlined in Section 5. Similarly, conditions should be clearly defined prior to starting the screen for application of selective
pressure and/or sorting of cells exhibiting the phenotype of interest.

Figure 4. Timeline of primary screen.

11

i. Transduction of cells with Edit-R Lentiviral Cas9 Nuclease particles
Prior to performing the screen, a population of stable Cas9-expressing cells is first generated using Edit-R Lentiviral Cas9
particles (with the most active cellular promoter). If a clonal Cas9-expressing cell line is preferred, isolate and characterizeclonal cell lines using protocols appropriate for your cells of interest.
Day 1:
1. Seed cells in Growth Medium. The number of cells seeded should be determined by extrapolating from the number ofcells desired at the time of transduction and the doubling time of your cell type. Incubate overnight.
Day 2:
1. The next day, remove the medium and add optimized Transduction Medium (see Section 5.B.) with the appropriate
amount of Edit-R Lentiviral Cas9 Nuclease particles so that the cells are just covered.
2. After the appropriate transduction time (see Section 5.B.), add additional Growth Medium to your cells such that thecells can be incubated for 48-72 hours.
Days 4-15:
1. At 48-72 hours post-transduction begin blasticidin selection to remove non-transduced cells.
2. Every 48-72 hours, replace with fresh Selection Medium containing blasticidin and passage cells as needed.
Use the appropriate concentration of puromycin and the minimum number of days required to kill non-transduced
cells as determined by a puromycin kill curve (see Section 5.D.).
Once a pure population of transduced cells has been obtained (7-15 days), expand the cell population to have enough cells
for your screen with the pooled lentiviral sgRNA library as determined in Section 5.G. Calculation of number of cells
needed for transduction.
We recommend evaluating the generated Cas9-expressing cell line by estimating gene editing events with sgRNA
positive control lentiviral particles (see Section 4.B.) before performing the screen.

ii. Transduction of cells with Edit-R Lentiviral sgRNA Pooled Library
Day 1:
1. Seed the Cas9-expressing cells in Growth Medium in the number of plates determined in Section 5.G. The number ofcells seeded should be determined by extrapolating from the number of cells needed at the time of transduction and the
doubling time of your cell type. Incubate overnight.
Day 2:
1. The next day, remove the medium and add optimized Transduction Medium (see Section 5.B.) with the appropriate
volume of lentiviral particles (Section 5.H.) so that the cells are just covered. If a single lentiviral pool will be added tomultiple plates, as determined in Section 5, divide the volume of lentiviral particles evenly among plates.
2. After the appropriate transduction time (see Section 5.B.), add additional Growth Medium to your cells such that thecells can be incubated for 48-72 hours.
Days 4-18:
1. At 48-72 hours post-transduction, begin puromycin selection to remove non-transduced cells.
2. Every 48-72 hours, replace with fresh Selection Medium containing puromycin and passage cells as needed.
Use the appropriate concentration of puromycin and the minimum number of days required to kill non-transduced
cells as determined by a puromycin kill curve (see Section 5.D.).

12

Once a pure population of puromycin-resistant cells has been obtained (2-6 days), begin screening. Split cells into at least
two populations: one as a reference and another (experimental) for application of selective pressure and/or sorting ofcells exhibiting the phenotype of interest. To maintain your desired sgRNA fold representation at each cell passage, always
retain at least the number of cells that corresponds to the desired number of lentiviral integrants.

B. Genomic DNA isolation
Following selection, gDNA should be isolated from control and experimental cell populations. Isolation of gDNA from cells
transduced with lentiviral pooled libraries has been optimized in the protocol below using a Qiagen Blood and Cell Culture
DNA Maxi Kit; however, kits from other manufacturers may also be suitable.
1. Collect cells by trypsinization for counting and gDNA isolation. To maintain your desired sgRNA fold representation
during gDNA isolation, use at least the number of cells that corresponds to the desired number of lentiviral integrants. The
most accurate results can be obtained by counting cell number prior to gDNA isolation. Follow the manufacturer’s
protocol for purification of gDNA from cell cultures.
It is important that you elute gDNA samples in EDTA-free buffer to prevent inhibition of subsequent PCR reactions.
2. Quantify the isolated gDNA using a spectrophotometer and assess the DNA purity by measuring the ratio of theabsorbance at 260 and 280 nm (A260/280) and at 230 nm (A260/230). High-quality gDNA samples should have an A260/280 ratioof 1.8 to 2.0, indicating the absence of contaminating proteins, and an A 260/230 ratio of > 2.0, indicating the absence ofother organic contaminants.

C. PCR amplification of constructs from genomic DNA
The PCR amplification step described here has been designed to amplify sgRNA constructs from gDNA without bias, suchthat differences in sgRNA representation after sequencing are due to enrichment or depletion that occurs during the
primary screen. It is important to use adequate template copies per construct in the PCR amplification step such that thedesired fold representation at transduction is maintained. This ensures assay reproducibility and facilitates hit
identification6. Note that the NGS Hit Identification Primers have been designed to add partial TruSeq® adapters designed
for use with Illumina® sequencing platforms. They are not compatible with other high-throughput sequencing systems.
Libraries targeting mouse genes and human libraries ordered prior to 2024 must be amplified with Edit-R Mouse PooledsgRNA Indexing PCR and Sequencing Primer Kits.

i. Number of PCR reactions
Calculate the minimum amount of gDNA required to maintain sgRNA fold representation, assuming a single lentiviral
integration event per cell (genome), using the following formula:
Number of cells withlentiviral integrations

×

6.6x10-3 ng/ genome (seenote below)=

Mass of gDNA required tomaintain representation ofeach sgRNA (ng)The calculation above assumes a diploid human genome. For cells that are not diploid, calculations should be
adjusted accordingly.

We have optimized the PCR conditions to remain in the linear phase of log amplification using Phusion Hot Start II DNA Polymerase.
We recommend a maximum of 825 ng of gDNA per PCR; using more gDNA per PCR will inhibit the efficiency of the reaction
and will result in either failure of the PCR or biased amplification.

13

Calculate the number of PCRs required for each sample using the following formula:

Mass of gDNA required tomaintain representation ofeach sgRNA (ng)÷825 ng per reaction=

Number of PCR reactions required
to maintain representation ofeach sgRNA (ng)** Round up the value obtained to the next whole number

The calculations above are used to determine the number of PCR reactions required per sample. To determine the total
number of PCR reactions required for your screen use the following formula:
Number of PCR reactions per
sample×

Number of samples=

Total number of PCR reactions

Calculation example
If you anticipate 1.52 × 107 cells with lentiviral integrations, the mass of gDNA required would be calculated asfollows:
1.52 × 107 lentiviral integrations × 6.6 × 10-3 ng/genome = 1× 105 ng
The maximum amount of gDNA per PCR is 825 ng, therefore the number of PCR reactions necessary can becalculated as follows:
1 × 105 ng gDNA ÷ 825 ng/PCR = 122 PCR reactions
If you have four samples (reference and experimental samples in biological duplicates), then you need thefollowing total number of PCR reactions:
122 PCR reactions × 4 samples = 488 PCR reactions

ii. PCR from genomic DNA
Once you have determined the number of PCR reactions you need per sample, you can proceed with the PCR amplificationof the gDNA samples. Sequence amplification from gDNA has been optimized using Phusion Hot Start II DNA Polymerase.
The amplification conditions recommended below have been determined to provide maximum yields of PCR product whilestaying within the linear phase of log amplification.
1. Perform PCR in 50 µL reactions using 96-well PCR plates. PCR conditions have been optimized to amplify 825 ng of gDNA
per 50 µL reaction. As described above, calculate the number of reactions needed for the amplification of total gDNA
necessary to maintain the fold representation used at transduction. Table 5 provides the components and volumes
needed for one 50 µL PCR amplification reaction. Libraries targeting mouse genes and human libraries ordered prior to2024 must be amplified with Edit-R Mouse Pooled sgRNA Indexing PCR and Sequencing Primer Kits.

14

Table 5. PCR components for amplification of genomic DNA.

Component

Volume per reaction (μL)

Final concentration

5x Phusion HF Buffer

101x

10 mM dNTPs

1

200 μMNGS Hit Identification Primers mCMV* (50 μM)

1

1 μM

gDNA (825 ng) + PCR grade H2O

36

16.5 ng/μL

Phusion Hot Start II DNA Polymerase (2 U/μL)

2

0.08 U/μL

TOTAL

50 μL

*For libraries targeting mouse genes and human libraries ordered prior to 2024, use the Edit-R Mouse Pooled sgRNA Forward PCR Primer and Edit-R

Mouse Pooled sgRNA Reverse Index PCR Primers included with the Edit-R Mouse Pooled sgRNA Indexing PCR and Sequencing Primer Kit in place of NGS
Hit Identification Primers with Forward and Reverse primers each at a final concentration of 0.5 μM.

2. To PCR amplify the Edit-R sgRNA constructs, use the cycling conditions outlined in Table 6. The conditions indicated inthis protocol ensure that amplification is in the exponential phase when using Phusion Hot Start II DNA Polymerase; ifusing another polymerase, the optimal cycle conditions should be determined empirically.

Temperature

Time

98 ⁰C

3 minutes

98 ⁰C

10 seconds

60 ⁰C

15 seconds

72 ⁰C

15 seconds

72 ⁰C

5 minutes

23 Cycles

Table 6. PCR cycle parameters

3. Confirm that amplicons of the appropriate size are obtained from each sample by running 10 µL of PCR product on a 2%
agarose gel. The amplicons will be 330-339 bp. For libraries targeting mouse genes and human libraries ordered prior to2024, gDNA amplification with Edit-R Mouse Pooled sgRNA Reverse Index PCR Primers will result in a 357 bp amplicon.
Due to the low number of PCR cycles, the product will be a faint band on a gel before purification. Note too that the
NGS Hit Identification FWD primers are staggered to introduce diversity and will generate ten roughly equimolar
amplicons varying by 1 bp in length.
4. Combine reactions amplifying the same gDNA sample into a single 1.5 mL tube. Purify the combined PCR amplicons foreach gDNA sample using a PCR purification kit.
Please be aware that gDNA often co-purifies with the PCR products. Although, contaminating gDNA does notinterfere with the next-generation sequencing, it does affect the quantification of the amount of PCR amplicons byspectrophotometric or fluorometric methods. Therefore, it is recommended to evaluate the concentration of each
amplicon using quantitative PCR or chromatographic methods such as analysis on a Revvity LabChip Nucleic Acid
Analyzer. Alternatively, the amplicons can be purified and quantified by a fluorometric DNA quantification method,such as QuBit (Thermo Fisher).

15

7 Library prep and Illumina platform sequencing
Illumina P5 and P7 adapters and TruSeq indexes can be added to the amplified library generated in Section 6.C.ii through asecond low amplification PCR step prior to sequencing. Please follow the manufacturer’s instructions for Illumina platform
sequencing.
Libraries targeting mouse genes and human libraries ordered prior to 2024 must be amplified with Edit-R Mouse PooledsgRNA Indexing PCR and Sequencing Primer Kits. Following gDNA amplification, please refer to Appendix A for information
regarding multiplexing and sequencing.
For accurate hit identification, we recommend obtaining a minimum of 1000 1 × 150 bp reads per sgRNA. If the output ofyour sequencing platform is higher than the number of reads required per sample, it may be possible to multiplex samples
in the same run. To calculate the maximum number of samples that can be loaded per lane, it is advisable to assume alower number of sequence reads than the maximum obtained on an optimum sequencing run. We suggest estimating thenumber of reads by adjusting the manufacturer's specifications by a factor of 0.7.

8 Hit identification and follow up
Alignment of sequence files and hit identification can be performed using open-source programs that require somebioinformatics knowledge. We recommend that you consult with a bioinformatician with proficiency in using commandline tools for data analysis and hit identification. A detailed protocol utilizing freely available software can be downloaded
from the Edit-R Lentiviral sgRNA Pooled Library webpage under the Resources tab. Samples should be de-multiplexed on
the sequencer to bin each index tag with the appropriate sample. Sequences should be trimmed so that the first 19 basesof each read correspond to the sgRNA target sequence. The remaining bases in the read include the sgRNA vector
backbone and can be ignored in the subsequent analysis. Using an alignment tool, such as Bowtie 2 7, align the first 19
bases of each read to the reference file provided with your lentiviral pooled library. Bowtie 2 has an option to ignore baseson the 3' end of the read; use this option to align only the first 19 bases of the sgRNA strand. Using the output of thealigner, count the number of alignments for each construct. Next, use a differential expression tool built for discrete countdata, such as DESeq28, to determine primary hits. Hits can be confirmed and studied further using individual Edit-R
Lentiviral sgRNAs.

9 Appendix
A. Multiplexing and Illumina platform sequencing of gDNA amplified with Edit-R
Mouse Pooled sgRNA Indexing PCR and Sequencing Primer Kits (for mouse and pre2024 libraries)
The workflow described here (Figure 5) has been designed to amplify and sequence sgRNA constructs from gDNA withoutbias, such that differences in sgRNA representation after sequencing are due to enrichment or depletion that occurs
during the primary screen. For accurate hit identification, we recommend obtaining a minimum of 1000 reads per sgRNA.
Most high-throughput sequencing platforms provide a sufficiently high number of sequence reads to allow multiplexing ofsamples in sequencing runs (running multiple samples in one lane). If the output of your sequencing platform is higher
than the number of reads required per sample, it may be possible to multiplex samples in the same run. To calculate themaximum number of samples that can be loaded per lane, it is advisable to assume a lower number of sequence reads
than the maximum obtained on an optimum sequencing run. We suggest estimating the number of reads by adjusting the
manufacturer's specifications by a factor of 0.7.
From this information the number of samples you can run per lane can be determined using the following calculations:16

Number of sgRNA constructs
in lentiviral pool×

Expected numberof reads per
sequencing lane

0.7 (numberof reads
adjustment)

×1000 read coverage perconstruct÷=

Output reads
required per
sampleOutput reads required per
sample=Number of sample
indices per laneCalculation example
If your pool contains 1000 sgRNAs, then the number of output reads required per sample can be calculated asfollows:
1000 sgRNAs × 1000 reads/sgRNA = 1 million output reads required per sample
If you are using a sequencing platform with a projected output of 15 million reads per lane then the number ofsamples per lane can be calculated as:
1.5 × 107 reads per lane × 0.7 (number of reads adjustment) ÷ 1 × 10 6 output reads required per sample =10 samples per lane

Edit-R Mouse Pooled sgRNA Indexing PCR primers are provided as 50 µM forward and reverse indexed primers. The index
tags are shown in Table 7.

Figure 5. PCR amplification and Illumina high-throughput sequencing workflow with Edit-R Mouse Pooled sgRNA Indexing PCR and Sequencing Primer Kits.

17

Table 7. The index tags on Edit-R Mouse Pooled sgRNA Indexing PCR primers are compatible with Illumina TruSeq LT indices.

Kit A
Item

Index

Index Sequence

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 2

2

CGATGT

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 4

4

TGACCA

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 5

5

ACAGTG

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 6

6

GCCAAT

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 7

7

CAGATC

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 1212

CTTGTA

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 13

13

AGTCAA

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 14

14

AGTTCC

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 15

15

ATGTCA

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 16

16

CCGTCC

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 18

18

GTCCGC

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 19

19

GTGAAA

Item

Index

Index Sequence

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 1

1

ATCACG

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 3

3

TTAGGC

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 88

ACTTGA

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 9

9

GATCAG

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 10

10

TAGCTT

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 11

11

GGCTAC

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 20

20

GTGGCC

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 21

21

GTTTCG

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 22

22

CGTACG

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 23

23

GAGTGG

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 25

25

ACTGAT

Edit-R Mouse Pooled sgRNA Reverse Index PCR Primer 27

27

ATTCCT

Kit B

Please follow the manufacturer’s instructions for Illumina platform sequencing. Quantify the library using the quality
standards that are recommended for your Illumina sequencing platform and then follow Illumina’s instructions for dilution
and denaturation of the library.
For the Illumina MiSeq Desktop Sequencer, we recommend spiking the library with 10% PhiX Control to serve as aninternal control and loading the library onto the flow cell at 7 to 10 pM using standard loading volumes. After loading yoursample into the Illumina MiSeq Reagent Cartridge, add 5 µL of the 100 µM Edit-R Mouse Pooled sgRNA Read 1 Sequencing
Primer stock to the "Read 1 Primer mix" reservoir and 5 µL of the 100 µM Edit-R Mouse Pooled sgRNA Index Read
Sequencing Primer stock to the "Index Primer Mix" reservoir on the Illumina MiSeq reagent cartridge (please check the
MiSeq System User Guide to confirm the inlet positions).
For the Illumina HiSeq 2500 system, we recommend spiking the library with 10% PhiX Control to serve as an internal
control and loading the library onto the flow cell at 6 pM for Rapid Run with onboard clustering or 7.5 pM for High Output
Run with c-bot clustering using standard loading volumes. Dilute the Edit-R Mouse Pooled sgRNA Read 1 Sequencing
Primer and Edit-R Mouse Pooled sgRNA Index Read Sequencing Primer into the respective Illumina primer reagents to a
final concentration of 0.5 µM.
18

At minimum, 19 base single-end reads are required to identify the sgRNA target sequence, as outlined in Figure 6.

Figure 6. Position of Edit-R Mouse Pooled sgRNA Read 1 and Index Read Sequencing primers and contents of 50 base sequencing read, including the sgRNAtarget sequence.

B. Stability and storage
Edit-R Lentiviral sgRNA Pooled Libraries are shipped on dry ice and must be stored at -80 °C. Under these conditions,lentiviral particles are stable for at least 3 years. Repeated freeze-thaw cycles should be avoided, as this is expected tonegatively affect titer. When setting up a pooled lentiviral screening experiment, lentiviral particles should be thawed onice, divided into smaller aliquots (if necessary) and immediately returned to -80 °C.

C. Quality assurance and control
Edit-R Pooled Lentiviral sgRNA Screening Libraries are subject to stringent quality control at multiple steps during the
manufacturing process, including:
1. Evaluation of sgRNA representation by high-throughput sequencing on Illumina sequencing platform:
Lentiviral pooled libraries are sequenced using 2 × 250 base reads, filtered by PHRED score > 30, and > 1000-foldread coverage. Pools have greater than 95% of the constituent constructs recovered. The library has highstandards for uniform construct distribution with 70% and 90% of the population within 10-fold and 25-fold ofeach other, respectively. A data file with gene annotation, sgRNA target sequences information and counts per
million mapped reads is provided with each screening library.
2. Lentiviral titering using quantitative PCR (qPCR) after transduction in HEK293T cells for functional titers.
3. Examination of each batch to ensure preparations are free from mold and bacterial contamination.
4. Specified lentiviral titers are included in data file with each shipment.

10 Frequently asked questions
How many lentiviral particles do I need for my screen?
The number of lentiviral particles (reported as transducing units (TU)) required for a screen will depend on:1) The relative transduction efficiency of your specific cell line/cell type2) The fold representation you choose to maintain throughout the screen3) The number of biological replicates you intend to include
Once these parameters have been determined, the Pooled-sgRNA-screening-protocol-tracking-worksheet can be used to
calculate the number of TU needed for your screen.

19How do I choose between the various Edit-R Lentiviral Cas9 Nuclease promoter options?
Choose the promoter option that has been demonstrated by your own experimental observations or shown to actively
express a transgene in your cells of choice through references in the published literature. For optimal experimental
confidence or if such information is not available, consider testing multiple lentiviral promoter-Cas9 constructs.

I am using human cell lines in my research; should I always choose a human promoter for optimal
Cas9 activity?
In some instances, promoter activity correlates with the species from which it is derived. However, promoter activity does
not always follow a species-specific expression pattern. For example, we have observed mouse promoters to be the mostactive in some human cell lines, whereas both human and mouse promoters were most active in some rat cells. Choosing
the most effective promoter in a particular cell line is not always predictable, and therefore should be determined
empirically.

Do the Edit-R Lentiviral sgRNA pooled libraries target lncRNAs or regulatory regions?
No. The libraries only contain sgRNAs targeting protein-coding genes.

Are Dharmacon™ lentiviral particle products safe to use in the laboratory? What precautions should
be taken when handling lentiviral particles?
Lentiviral delivery systems have been employed in many research laboratories worldwide without incident. Handling oflentiviral products requires extensive experience with cell culture techniques. It is vital that the protocols provided by
Revvity and the safety guidelines described for appropriate handling and storage are fully understood and followed
precisely (see Section 12, Lentiviral particle product safety level information).

Can Dharmacon™ lentiviral particles be further propagated in the lab?
No. Edit-R lentiviral products are engineered for biosafety and are provided as replication-incompetent lentiviral particles.

Can I order the Edit-R Lentiviral sgRNA Pooled libraries as plasmid DNA or glycerol stocks?
No. Our Edit-R Lentiviral sgRNA Pooled Libraries are sold exclusively as lentiviral particles.

How should lentiviral particle products be stored?
All lentiviral particle products must be stored at -80 °C. If necessary, the particles can be aliquoted upon the first thaw toconvenient volumes and the aliquots stored at -80 °C to minimize the number of future freeze-thaws. However, werecommend avoiding multiple freeze-thaw cycles as much as possible.

Can lentiviral particle products be kept at 4 °C?
No. Once thawed, lentiviral particle preparations begin to decrease in titer. We advise against storing any lentiviralparticles at 4 °C. All lentiviral particle products must be kept frozen and stored at -80 °C until transductions are performed.

How are lentiviral particle products shipped?
Lentiviral particle products are shipped on dry ice for overnight domestic delivery or priority international for delivery
outside of the U.S.

Where can I find titer information for my pooled library?
The titer of your lentiviral pools will be indicated in the data file sent with your library shipment and can also be requested
from Scientific Support.

Where can I find the targeting sequences for sgRNAs included in my pooled library?
The sgRNA target sequences are in the data files sent with your library shipment. Please contact Scientific Support withyour Sales Order number (SO-XXXXXXXG) if you did not receive the data files in your shipment.

How is gDNA input calculated to maintain desired construct fold representation?
The quantity of gDNA required for maintaining a desired fold representation can be calculated using the formulas andvalues (Table 8) below.

20

Table 8. Constants needed for calculating gDNA input.

Constants
Mass of a base pairBase pairs per diploid human genome
Avogadro constantValue
660 g/mol/bp
6 × 109 bp
6.02 × 1023 mol-1

Example:Calculate the mass of human genome:(6 × 109 bp/genome) × (660 g/mol/bp) = 4 × 1012
g/mol/genome(4 × 1012 g/mol/genome) ÷ (6.02 × 1023 mol -1) = 6.6 × 10 12
g/genome

The calculation above assumes a diploid human genome. For cells that are not diploid, calculations should be
adjusted accordingly.

How can I index and sequence the gDNA from my screen?
gDNA can be amplified, indexed, and prepared for NGS sequencing with a two-step PCR protocol. We provide vectorspecific NGS hit identification primers for the first round of amplification of your gDNA. The forward and reverse primers
contain the following Illumina® compatible TruSeq® partial adapters:
Forward primer extension: 5’ ACACTCTTTCCCTACACGACGCTCTTCCGATCT 3’
Reverse primer extension: 5’ GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT 3’
Illumina P5 and P7 adapters and TruSeq indexes can then be added to the amplified library through a second low
amplification PCR step prior to sequencing. Note that the forward primers contain a stagger site between the 5’ Illumina
extension and the priming site to introduce nucleotide diversity during sequencing, which will need to be adjusted forwhen the sequences are trimmed. For more information, please see our Bioinformatic Analysis of Dharmacon™ LentiviralsgRNA Pooled Library Screens Protocol.

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

Bhaya, et al. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu.
Rev. Genet. 45, 273-297 (2011).
M. Jinek, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821(2012).
Deltcheva, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor Nuclease III. Nature 471, 602-607(2011).
P. Mali, et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013).
T.R. Sampson, and D.S. Weiss. Exploiting CRISPR/Cas systems for biotechnology. Bioessays 36, 34-38 (2014)
Ž. Strezoska, et al. Optimized PCR conditions and increased shRNA fold representation improve reproducibility of pooled
shRNA screens. PLoS One 7, e42341 (2012).
Langmead and S. Salzberg. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357-359 (2012).
S. Anders and W. Huber. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

12 Lentiviral particle product safety level information
This Lentiviral Particle Product Safety Level Information constitutes Product Documentation according to clause 1 of the Product Terms
and Conditions. It is applicable to all Dharmacon lentiviral particle products.
Any investigator who purchases Dharmacon lentiviral particle products is responsible for consulting with their institution’s healthand biosafety personnel for specific guidelines on the handling of lentiviral vector particles. Furthermore, each investigator is fully
responsible for obtaining the required permissions for research use and the acceptance of replication -incompetent SIN lentiviral vectors
and replication-defective lentiviral particles into their local jurisdiction and institution.
The Products are solely for internal research use (as set forth in the Product Terms and Conditions) in laboratories where thecontainment measures stated below and in applicable laws and regulations are met. Products may not be used for diagnostic,
therapeutic or other commercial purposes and may not to be administered to humans for any purpose or to animals for therapeutic
purposes. The Products are replication-incompetent, self-inactivating (SIN) and non-pathogenic (do not cause infectious humandisease).
21

For questions concerning the design or production of the products, please contact our Scientific Support team.
Revvity2650 Crescent Drive
Lafayette, CO 80026 USA
Tel: +1 800 235 9880; 303 604 9499 (United States)
Fax: +1 800 292 6088; 303 604 9680 (United States)
Email: technical@horizondiscovery.com (North America and Europe)
In the US:
For US guidance on containment for lentiviral vectors, please refer to:
1.
2.
3.

The Recombinant DNA Advisory Committee (RAC) guidelines for research with lentiviral vectors(https://osp.od.nih.gov/wp-content/uploads/Lenti_Containment_Guidance.pdf);
The U.S. Department of Health and Human Services Centers for Disease Control and Prevention and National Institutes of
Health, Biosafety in Microbiological and Biomedical Laboratories (BMBL);

The NIH Guidelines For Research Involving Recombinant DNA Molecules (NIH Guidelines)(https://osp.od.nih.gov/wp-content/uploads/NIH_Guidelines.pdf)

In the EU:
For the EU directives, please consult the following:
1.

2.

Council Directive 2009/41/EC of the European Parliament and of the Council of 6 May 2009 on the contained use ofgenetically modified micro-organisms. (revised version of Directive 90/219/EEC of the European Parliament and of theCouncil of 23 April 1990 on the contained use of genetically modified micro-organisms, amended by Council
Directive 98/81/EC of 26 October 1998); andCouncil Directive 2001/18/EC of the European Parliament and of the Council of 12 March on the deliberate release into theenvironment of genetically modified organisms and repealing Council Directive 90/220/EEC.

In Germany:
Required Containment Measures: The containment requirements as stated in the German Genetic Safety Ordinance (GentechnikSicherheitsverordnung) of Safety Level 2* or higher have been assigned to the handling of the above- mentioned lentiviral vector
particles. Please note a higher Security Level might be required if the lentiviral vector particles are used for genetic engineering
operations with other products which require a higher Security Level.
*Safety Level 2: activities of low risk for human health and the environment by the state of scientific knowledge (Stand derWissenschaft).
For the German regulations, please consult the following:
1.
2.

German Genetic Engineering Act (Gentechnikgesetz - GenTG); andGenetic Engineering Safety Ordinance (Gentechnik-Sicherheitsverordnung - GenTSV).

13 Limited use licenses
The Products, use and applications, are covered by pending and issued patents. Certain Label licenses govern the use of
the Products, these can be found at Dharmacon Licensing Statements. 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 Products.
If you have any questions contact
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 https://horizondiscovery.com/en/contact-us
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Illumina, HiSeq, MiSeq, and TruSeq are trademarks or registered trademarks of Illumina, Inc. Limited permission to copy or distributesequence information. Oligonucleotide sequences 2007-2011 Illumina, Inc. All rights reserved. Derivative works created by Illumina customers
are authorized for use with Illumina Instruments and products only. All other uses are strictly prohibited. Phusion is a trademark of Thermo Fisher Scientific,
Inc. TurboGFP and TurboRFP are trademarks of Evrogen Inc.

22

Revvity
940 Winter Street
Waltham, MA 02451 USA

23

www.revvity.com

For a complete listing of our global offices, visit www.revvity.com
Copyright ©2023, Revvity. All rights reserved.