How do I perform a genome-wide CRISPR knockout screen?
How do I perform a genome-wide CRISPR knockout screen?
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. 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 (see vector map). Isolation of cells expressing Cas9 at an optimal level is critical for performing a successful screen.
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.
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.