Everything you need to know about CRISPR library screening

Below is a general overview of the process of performing a phenotypic screen using a pooled lentiviral sgRNA library. More details are provided in additional sections on this page and in the user manual for the Guide-it CRISPR Genome-Wide sgRNA Library System.

STEP 1: select the phenotypic change you wish to study

The change must provide a basis for enrichment, selection, or depletion of edited cells carrying corresponding gene knockouts in the screened population. In the most straightforward screens, gene knockouts result in the cells having a growth or selection advantage/disadvantage, such as resistance to a drug or increased cell proliferation/viability (positive screens). In other assays, expression of a reporter might enable enrichment by a sorting process (e.g., FACS). More complex screens may be used to identify gene knockouts that are lost from the population under a given treatment (negative screen).

Be sure to include a reference control with your screened sample.

STEP 2: choose the cells you want to use for the screen

The cells need to be a good surrogate for your experimental system but easy to grow and transduce. Study of primary cells might be the goal, but these are often difficult to scale up if they do not proliferate well or require elaborate culture conditions. When using the Guide-it CRISPR Genome-Wide sgRNA Library System, we recommend that you screen using ~76 million cells (see FAQs 2.A and B for more information). To achieve this, you can substitute with a related transformed cell line for the primary screen, followed by use of more relevant primary cells for follow-up confirmation screens/tests.

STEP 3: express Cas9 in your target cells

Transduce your target cells using Cas9-expressing lentivirus and apply selection to enrich for transduced cells. Stable integration of Cas9 lentivirus made using the Guide-it CRISPR Genome-Wide sgRNA Library System can be selected for using puromycin. Isolation of cells expressing Cas9 at an optimal level is critical for performing a successful screen and is addressed in FAQ 2.C.

STEP 4: produce a stock of sgRNA library lentivirus

With our system, this is done by simply adding water to a vial of the Guide-it Genome-Wide sgRNA Library Transfection Mix and then adding the contents to Lenti-X 293T cells in a 10-cm dish; two vials are used for each screen. The virus is collected at 48 and 72 hours post-transfection and pooled. The virus can be titrated easily using Lenti-X GoStix Plus. Either use the virus immediately or freeze while you test your target cells (Step 5).

STEP 5: determine the MOI needed to achieve a 30–40% transduction efficiency for your target cells

Establish the amount of sgRNA library virus that is required to achieve approximately 30–40% transduction efficiency by titrating the virus with your Cas9+ cell line. In our system, this test is done by assaying for expression of the mCherry fluorescent protein that is coexpressed with the guide RNAs. The significance of achieving a 30–40% transduction efficiency is discussed in FAQ 2.D on this page.

STEP 6: express sgRNA library in Cas9+ target cells

Use the amount of virus estimated in Step 5 above to scale up and transduce Cas9+ target cells with the sgRNA library lentivirus to achieve a transduction efficiency of 30–40%. An example of calculations performed to determine appropriate amounts of lentivirus and target cells is provided in Table II of the Guide-it CRISPR Genome-Wide sgRNA Library System User Manual.

STEP 7: perform the screen and harvest genomic DNA

When using the Guide-it CRISPR Genome-Wide sgRNA Library System, we recommend screening with ~76 million cells transduced at an efficiency of 40%. Screens are typically either positive (enrichment) or negative (depletion). Screen regimens can vary widely, but an example of a positive screen could involve culture with and without a drug for 10–14 days. Following the screening, genomic DNA is extracted from 100–200 million cells (~400–1,000 cells for each sgRNA) from both the treated and the untreated populations. It is essential to get the scale for the gDNA isolation correct to maintain sgRNA representation. The genomic DNA isolation cannot be performed using a miniprep, and overloading maxi columns results in reduced sample diversity.

(See other FAQs on this page for additional explanations for DNA purification scale, cell number calculations, required transduction efficiency, and differences in the possible types of screens.)

STEP 8: sequence and analyze

Once a screen is completed, the genomic DNA from each cell population must be isolated, an NGS library prepared and then sequenced. Enrichment or depletion of proviral sgRNAs in screened cells can be analyzed by NGS with the Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Cat. # 632647). If using the library from Takara Bio, it is interpreted as a proxy for enrichment or depletion of cells carrying the corresponding gene knockouts.

To accurately identify sgRNAs present within the purified gDNA isolated from pooled screens, high-quality NGS library production is essential. Sequencing primers used to amplify the sgRNA sequences must contain all the features necessary for amplification and analysis of the sgRNA sequences from the gDNA (including Illumina P5 and P7 flow cell attachment sequences and primer sites for vector amplification and Illumina sequencing). Additional features should include barcodes to help with deconvolution during analysis and primer staggering to maintain library complexity when selected populations become dominated by a smaller number of sgRNAs.

Positive screens are typically followed by sequencing to an NGS read depth of ~1 x 10⁷ reads. Negative screens are more challenging and may require more cells and a greater read depth (more details provided in the FAQ 1.E about positive and negative CRISPR library screens).

An example of a CRISPR pooled sgRNA library screening workflow.

To learn more about CRISPR/Cas9 and the product solutions that we provide, please visit the following pages:

• Gene editing learning center
• Gene editing product catalog

See schematic below explaining CRISPR/Cas9-mediated gene knockout.

Some examples where sgRNA pooled screens have been applied include investigations in:

• Mechanisms associated with ALS and Parkinson's Disease (ALS citation; Parkinson's citation)
• Oncology—looking at growth and metastasis (citation)
• Responses in immune therapy (citation)
• Mechanisms of viral infection (citation)

Phenotypic screens using pooled whole-genome sgRNA libraries have been used for a broad range of applications.

sgRNA vs. RNAi libraries

Unlike RNAi libraries, sgRNA libraries produce a complete knockout (vs. a knockdown) phenotype and have a reduced likelihood of off-target effects. Genes that play a vital role in causing your phenotype of interest but still cause the phenotype at low expression levels may only be detected following the complete knockout resulting from a CRISPR/Cas9 screen.

Pooled vs. targeted arrayed libraries

Arrayed libraries are beneficial if you wish to screen only a known subset of genes for a phenotype or are seeking to assay phenotypes that don’t result in enrichment or depletion; however, these often require automated liquid-handling capabilities, and you almost always need to know what you are looking for. Pooled sgRNA libraries are used more for discovery and allow for an unbiased approach; it is easy to screen the whole genome using a pooled library, and automated liquid handling is not required. The cost of an arrayed targeted library increases with the number of genes assayed.

In simple terms, positive screens are used to determine genes that accumulate in a population because of a treatment and negative screens determine genes that are lost from the population.

Positive screens identify genes that are sensitive to the selection mechanism, such that when these genes are knocked out, the cells survive the selection. In this type of a screen, most cells are lost and only cells that contain sgRNAs knocking out genes which make the cell sensitive to the selection agent will survive. The expected result is that the remaining cells will be enriched for these sgRNAs. In these types of screens, it is essential to culture the cells long enough to allow for the loss of the edited targets and manifestation of the resulting phenotype before sequencing. In our experience, ten days to two weeks is generally sufficient. Because these types of screens can be rather robust, a typical recommended NGS read depth is ~1 x 10⁷ reads.

Negative screens identify genes that are essential for survival under the selective pressure provided by the screening conditions. Cells expressing sgRNAs that trigger null or loss-of-function edits in these genes will be lost from the population upon application of screening conditions, such that the cells expressing other sgRNAs are overrepresented. These types of screens are often more challenging, as most cells survive the screen, and parameters need to be tightly controlled to ensure that statistically significant changes can be detected. For the detection of subtle changes in sgRNA representation in these negative screens, NGS analysis may require read depths of up to ~1 x 10⁸ reads.

Using lentivirus vectors ensures that the researcher can control the presence of only a single sgRNA per cell. Plasmid delivery would result in dozens of different guides per cell which would make it impossible to determine which knockout (or combination) causes the observed phenotype.

To determine which gene has been knocked out in any given cell following a phenotypic screen, you need to determine which sgRNA was delivered to that cell. Therefore, the coding sequence for expressed guide RNA needs to be present after the screen, i.e., stably integrated. Lentivirus delivery under a controlled MOI results in a stable single-copy integration.

Even when working with well-designed sgRNAs such as those included in the Brunello library, there is a chance for off-target edits. In the context of a genome-wide knockout screen, off-target edits that affect the phenotype of interest can result in false positives, because cells expressing the sgRNA that produced the off-target edit could become enriched or depleted in the screened population, implying that the intended target of the sgRNA is involved in the phenotype. Given that the likelihood of several different guides generating the same off-target edit is negligible, the inclusion of multiple guides targeting the same gene provides a powerful means for avoiding false positives in a screen. When a researcher observes enrichment or depletion of several different guides targeting the same gene, he or she can be reasonably confident that knockout of the intended target of the sgRNA is responsible for the phenotype rather than an off-target edit.

Following positive or negative screens the confidence is far higher when you can confirm that multiple guides targeted to different sites in the same gene show up as positives in your data analysis.

We recommend further investigation for those genes where 3 or 4 of the guides targeting that gene show as positives in the data analysis, as these are unlikely to be artifacts.

NGS provides an efficient method for quantifying and comparing the frequencies of sgRNAs encoded in the screened and control populations, which in turn allows researchers to identify which gene knockouts yielded phenotypes relevant to the screen.

Following the screen, gDNA is purified from the screened and control cell populations, and proviral sgRNA sequences are PCR amplified and incorporated into NGS libraries. Following sequencing, software packages such as MAGeCK or even Excel can be used to compare the frequencies of sequencing reads corresponding to each sgRNA in the respective populations.

The cells need to be a good surrogate for your experimental system but easy to grow and transduce. Primary cells might be the goal but can be difficult to scale up because they often do not proliferate or require elaborate culture conditions. We suggest substituting with a related transformed line for the primary screen, followed by use of more relevant primary cells for follow-up confirmation tests or more targeted screens.

Additionally, because we use lentivirus for delivery of the CRISPR components (Cas9 and sgRNA), it is wise to initially avoid cells that are hard to infect, i.e., those that require high MOI of VSV-g pseudotyped virus for efficient transduction.

We recommend performing each screen using a minimum of 7.65 x 10⁷ cells transduced at 40% efficiency when using the Guide-it CRISPR Genome-Wide sgRNA Library System. See the calculations below:

Total number of guides:
19,114 genes targeted by 4 sgRNAs each = 76,456 sgRNAs (plus 156 control sgRNAs) = 76,612 sgRNAs

Number of cells transduced per guide:
At a low sgRNA representation in the transduced cells (<400 for each sgRNA), the library will have more sgRNAs falling outside 2 standard deviations (SD) of the initial distribution, leading to erroneous interpretation of dropout or enrichment. This noise can be greatly mitigated by achieving fold coverage of 400X or greater (400 cells carrying the same sgRNA).
Total cells = 76,612 x 400 = 3.06 x 10⁷

Transduction efficiency needed:
To ensure that the majority of the cells have only one guide RNA, the cells should be transduced at an efficiency of 20–60% (using Poisson distribution). We choose 30–40%.

Total cell number at the time of transduction:
3.06 x 10⁷/0.4 (40% transduction efficiency) = 7.65 x 10⁷ cells.

When expressed at high levels, Cas9 nuclease can be toxic to mammalian cells, but expression must still be high enough for efficient gene editing. It is, therefore, well worth putting in some effort up front to create the best Cas9+ cell population.

To determine the optimal Cas9 expression for your screening cell line, transduce target cells with different amounts (MOI) of Cas9 lentivirus, select each population on puromycin, and then monitor the cells lines’ growth rates (see figure below). Choose a Cas9 population transduced with the highest MOI that does not exhibit any change in cell growth compared to cells that do not express Cas9.

Expand the population to make a master cell bank with enough vials for all your future screens.

A key goal in producing a Cas9+/sgRNA+ cell population for screening is to balance the need for every guide RNA to be well represented in the screen with having each cell express only a single sgRNA. Transduction efficiencies of 30–40% are expected to yield the highest quantities of cells expressing a single sgRNA while maintaining a reasonable culture size prior to selection in hygromycin (Miles et al. 2016).

This titration is simplified when using the Guide-it Genome-Wide sgRNA Library System due to the presence of the mCherry fluorescent protein on each sgRNA vector. Simply test a small amount of your library virus prep and measure mCherry expression by flow cytometry or microscopy. For your full-scale screen, use the MOI that transduces 30–40% of the cells.

Reference:
Miles, L. A. et al. Design, execution, and analysis of pooled in vitro CRISPR/Cas9 screens. The FEBS J. 283, 3170–3180 (2016).

In a perfect whole genome library, each guide RNA is present in an equal copy number (equal representation). In practice, no library is perfect, due to variability in the processes used to make it (oligo synthesis, cloning, transformation, library amplification, DNA extraction, etc.). Our experience of creating, amplifying, and validating libraries ensures that our library is produced to high standards and that representation of guides included in our system is as equal as possible.

For the Guide-it CRISPR Genome-Wide sgRNA Library System, the sgRNA content of the library plasmids is verified in every lot by NGS to contain >90% of the sgRNAs within a 10-fold distribution range (see image below). We have additionally measured representation in a transduced cell population and confirmed an extremely high correlation between the starting plasmid material and the transduced cell population (Spearman and Pearson correlations >0.95). This high correlation shows that it is possible to maintain representation through all the steps from transfection, harvest of the virus, and transduction of target cells using our format and protocols. The cells produced using this system are then the starting point from which you begin your screening process.

Although you only need a small amount of DNA for sequencing, we recommend that you isolate DNA from 100–200 million cells for pooled screens. This typically amounts to 300–500 µg of genomic DNA. The reason for this is that it is essential to generate an NGS library that is representative of the diversity of the edited cells (and hence sgRNAs) still in the population after treatment. Purifying the DNA at this scale ensures that you can generate sequencing data for ~400–1,000 cells per individual sgRNA included in the library.

False positives from a screen can result from a guide RNA targeting an off-target sequence that is unrelated to the sequence for which it was designed. We have significantly negated the risk of this happening with the design of the library and the optimization of the screening process. This library contains sgRNAs from the Brunello library which were chosen using algorithms that maximize on-target specificity and activity while minimizing off-target activity (Doench et al. 2016).

Additionally, each gene is targeted by four highly active sgRNAs. We recommend choosing positives from your screen based on the criteria that at least three guides for a given gene must be over/underrepresented after screening compared to their numbers before screening. The probability that three guides for the same gene also target the same unrelated off-target gene is close to zero.

Lastly, false positives can arise from co-transduction events where one sgRNA not related to the knockout phenotype is present in the same cell as an sgRNA that is responsible for that cell's enrichment. Statistically, the ineffective sgRNA will be represented by only one guide, and therefore we can regard it as not being a qualified hit. We can easily limit this phenomenon by adjusting the MOI for transduction to achieve ~30–40% transduction efficiency (see FAQ 2.D for more details.)

References:
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR Cas9. Nat. Biotechnol. 34, 184–191 (2016).

Yes. Click on the link below to see data generated in a screen for genes involved in resistance to the purine analog 6-thioguanine (6-TG).

Technical note: Performing a phenotypic screen using the Guide-it CRISPR Genome-Wide sgRNA Library System

Genome-wide CRISPR screens using pooled lentiviral sgRNA libraries typically take several months to complete. Below is a timeline for various aspects of the workflow based on the experiences of our R&D team. NOTE: The workflow below does not include the time required to produce the sgRNA library. Our kit provides the researcher with a high-quality, validated library for expediting the process.

There are five components to the library system as described below:

632646 Guide-it CRISPR Genome-Wide sgRNA Library System, 5 Screens

• Guide-it Genome-Wide sgRNA Library Transfection Mix, 2 x 5 vials
• Guide-it Cas9 Lentiviral Transfection Mix, 5 vials
• pLVXS-sgRNA-mCherry-hyg Vector, 5 μg
• Lenti-X 293T Cell Line, 2 x 10⁶ cells
• Lenti-X GoStix Plus (Sample), 3 tests

1. The Guide-it Genome-Wide sgRNA Library Transfection Mix is the library itself in our easy-to-use lyophilized Lenti-X Single Shots format and is used for performing phenotypic knockout screens in human cells. There is no need to amplify the library, as each vial contains all the necessary plasmids (library and lentiviral packaging plasmids) and transfection reagents to generate self-inactivating lentiviral particles containing a complete sgRNA library targeting the entire human genome (>19,000 genes). Simply resuspend the lyophilized transfection mix in water and add the mixture to Lenti -X 293T cells to produce lentiviral particles. The sgRNA library is expressed from the U6 promoter and includes four highly active guides per gene, with guide design based on the Brunello library (Doench et al. 2016). The library backbone is the pLVXS-sgRNA-mCherry-hyg Vector. In addition to expressing sgRNA, the sgRNA lentiviral particles also code for the mCherry fluorescent protein to facilitate simple, functional titer assays, as well as hygromycin resistance to enable selection for stably transduced cells. Representation of the guide RNAs has been verified by NGS in both the plasmid library as well as in transduced target cell populations to be >90% within a 10-fold range to improve the power of your screens. Use two vials per library screen (one vial for the treatment population, and the other for the untreated control population).
2. The Guide-it Cas9 Lentiviral Transfection Mix contains the pLVXS-EF1a-Cas9-PGK-Puro Vector in our easy-to-use Lenti-X Single Shots format. This Cas9 transfection mix is used to create your Cas9-expressing target cell line. Each vial includes all the necessary plasmids and transfection reagents to generate self-inactivating lentiviral particles that express Cas9 under the EF1-alpha promoter in transduced target cells. Simply add water to the vial, mix well, and then add the transfection mix to Lenti-X 293T cells to produce lentiviral particles ready to transduce your target cells. The vector contains a puromycin resistance gene to enable positive selection for stably transduced cells. Cell lines expressing Cas9 produced using this system can be used for gene editing experiments, including phenotypic knockout screens.
3. The pLVXS-sgRNA-mCherry-hyg Vector is the empty version of the vector used for manufacturing the library; it can be used to clone specific sgRNAs and confirm hits after performing screens. The cloning site features a BsmBI restriction site to enable insertion of candidate single-guide RNAs (sgRNAs) for downstream assays. Once the sgRNAs are re-cloned, the researcher can produce virus using the Lenti-X Packaging Single Shots (not included).
4. Our Lenti-X 293T Cell Line was clonally selected because it is easily transfected and supports a high-level expression of viral proteins, allowing you to produce the highest possible lentiviral titers (up to 10⁸ IFU/ml) required for library screens.
5. We provide a sample kit of Lenti-X GoStix Plus, which is the simplest method ever to measure lentiviral titer. The test involves applying 20 µl of supernatant to a GoStix cassette and waiting 10 minutes for the appearance of test and control bands that indicate the presence of lentiviral p24. The results on the cassette can then be analyzed using a free smartphone app, which quantifies lentivirus titer by comparing the intensities of the test and control bands to an internal control curve. Learn more about Lenti-X GoStix Plus.

The Lenti-X packaging single shots format provides a straightforward and consistent method to package high-titer lentivirus. The lyophilized pellet (see image below) contains our fourth-generation lentiviral packaging system, a premixed formulation of five Lenti-X packaging plasmids and the library (>76,000 sgRNA lentiviral plasmids) all combined in a single tube at preoptimized ratios.

No additional transfection reagent is needed because the single shots vials for the library also contain prealiquoted, lyophilized Xfect Transfection Reagent.

A high-titer virus is produced by simply reconstituting this mixture in sterile water and adding it to Lenti-X 293T Cells (Cat. # 632180), in a 10-cm dish. Typically, titers of 10⁷–10⁸ IFU per ml can be expected, and such high titers are critical for providing sufficient virus for a whole genome library screen.

Researchers conducting pooled, lentiviral CRISPR knockout screens often start either with an sgRNA library in plasmid form or premade lentiviral particles. Starting from plasmid involves a much lower upfront cost, but the process of amplifying the library, verifying sgRNA representation, and producing lentivirus require familiarity with a wide array of techniques and often proves to be time-consuming and overly challenging for some researchers. Starting from lentiviral particles allows researchers to circumvent the challenges of library amplification and validation and production of lentivirus but is exceedingly expensive.

Both our sgRNA library and the Cas9 sequence are cloned into a lentiviral vector backbone consisting of a 3' self-inactivating LTR and sequence elements (e.g., cPPT/CTS and WPRE) to enhance titer and transduction efficiency (see schematic below).

Cas9 (from S.pyogenes) is expressed from the human EF1-alpha promoter, and stable integrants can be selected using puromycin.

The sgRNA library is expressed from a human U6 promoter, and hygromycin B can be used to select for stable integration of the lentivirus. The red fluorescent protein mCherry is also expressed, which significantly simplifies the process to determine which MOI gives a transduction efficiency of 30–40% in your target cells (see FAQ 3.N). It also allows visualization of a fully selected population (i.e., one in which 100% of the cells are red).

Guide RNAs in our library target the entire human genome of 19,114 genes (including essential genes). There are four guides per gene, which, including controls amounts to 76,612 different sgRNA vector constructs.

A complete list of all sgRNAs in our library and their representation data can be requested at takarabio.com/genomewidelibrary.

19,114 genes targeted by four sgRNAs each = 76,456 sgRNAs (plus 156 control sgRNAs) = 76,612 sgRNAs

Smaller libraries such as the Guide-it CRISPR Genome-Wide sgRNA Library System have a higher percentage of active and specific sgRNAs, which streamlines the screening process and makes screening far more cost-effective. The four guides per gene in our library are designed to be highly active since they are chosen on the basis of an algorithm that ensures high on-target activity (Doench et al. 2016) and improved sgRNA scaffold design (Chen et al. 2013; see FAQ 3.J).

Libraries that use less well-designed guide RNAs may provide eight guides per gene to counteract the lower percentage of active and specific sgRNAs. Users of libraries with eight guides need to screen double the number of cells, which represents a significant amount of extra work (more virus production, more cells to transduce) and additional sequencing, analysis, and cost.

References
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR Cas9. Nat. Biotechnol. 34, 184–191 (2016).

Chen, B. et al. Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System. Cell 155, 1479–1491 (2013).

The target sequences cloned into the Guide-it Genome-Wide sgRNA Library are the same as described by Doench et al. in 2016. In this paper, the authors created and modified their algorithm based on real screening and activity data from positive and negative screens. They named their human library "Brunello."

Reference
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR Cas9. Nat. Biotechnol. 34, 184–191 (2016).

Two parts of every guide RNA are essential for its activity: the 20-nucleotide target sequence which determines the specificity to the target gene and the looped-structured scaffold that determines how well the guide RNA binds to the Cas9 protein. The sgRNA scaffold encoded by the Guide-it library vector pLVXS-sgRNA-mCherry-hyg uses an optimized sequence for improved editing efficiency (see image below). This modified scaffold maximizes the binding affinity between the sgRNA and the Cas9 endonuclease (Chen et al. 2013), which increases editing efficiency, and compared to some traditional scaffolds used in other libraries, provides a greater chance that each guide is active.

Reference
Chen, B. et al. Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System. Cell 155, 1479–1491 (2013).

It is impossible to test all 76,456 sgRNAs functionally. However, the target sequences cloned into the Guide-it Genome-Wide sgRNA Library are the same as those described by Doench et al. in 2016. In this paper, the authors created and modified their Brunello algorithm based on real screening and activity data from positive and negative screens. At Takara Bio we verified the editing activity of a selected number of clones chosen at random and confirmed that all worked well (see data below).

Reference
Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR Cas9. Nat. Biotechnol. 34, 184–191 (2016). 

Our library is supplied lyophilized in our Single Shots format with our fourth-generation lentiviral packaging system, designed to produce high titers (up to 10⁸ IFU/ml) without the need for optimization or post-production processes, such as concentration. A high-titer virus is essential for library screening and is produced by simply reconstituting this mixture with sterile water and adding it to the Lenti-X 293T Cells supplied with the system. It is that simple.

Watch our fourth-generation lentiviral packaging overview video for more information »

Below are links to the sequence and map files for the vectors used in the Guide-it CRISPR Genome-Wide sgRNA Library System.

Cas9 vector - map file
Cas9 vector - genbank sequence file

sgRNA Library vector - map file
Library vector - genbank sequence file

The red fluorescent protein mCherry is expressed from the same vector that expresses each sgRNA and is included to simplify the process of determining the optimal MOI for the screen. The aim is to obtain a transduction efficiency of 30–40% in your target cells, which ensures that most transduced cells contain only a single sgRNA construct. For the calculation of transduction efficiency, a small-scale titration is performed and cells are analyzed for mCherry expression by flow cytometry or microscopy.

The Guide-it CRISPR Genome-Wide sgRNA Library NGS Analysis Kit (Cat. # 632647) enables preparation of Illumina-ready NGS libraries from cell populations screened using the Guide-it CRISPR Genome-Wide sgRNA Library System (Cat. # 632646), allowing for identification of sgRNA sequences and corresponding gene knockouts that have increased or decreased in frequency under the conditions of the screen. The kit provides all components necessary to prepare 10 sequencing libraries (20 PCR amplifications), including a genomic DNA purification kit, primers, PCR enzyme and buffers, and a PCR cleanup kit.