![]() 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%. STEP 6: express sgRNA library in Cas9+ target cells The significance of achieving a 30–40% transduction efficiency is discussed in FAQ 2.D on this page. In our system, this test is done by assaying for expression of the mCherry fluorescent protein that is coexpressed with the guide RNAs. STEP 5: determine the MOI needed to achieve a 30–40% transduction efficiency for your target cellsĮstablish 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. Either use the virus immediately or freeze while you test your target cells (Step 5). ![]() The virus can be titrated easily using Lenti-X GoStix Plus. The virus is collected at 48 and 72 hours post-transfection and pooled. 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. STEP 4: produce a stock of sgRNA library lentivirus Isolation of cells expressing Cas9 at an optimal level is critical for performing a successful screen and is addressed in FAQ 2.C. 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). Transduce your target cells using Cas9-expressing lentivirus and apply selection to enrich for transduced cells. STEP 3: express Cas9 in your target 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. 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). 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. The cells need to be a good surrogate for your experimental system but easy to grow and transduce. STEP 2: choose the cells you want to use for the screen More complex screens may be used to identify gene knockouts that are lost from the population under a given treatment (negative screen).īe sure to include a reference control with your screened sample. In other assays, expression of a reporter might enable enrichment by a sorting process (e.g., FACS). 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). The change must provide a basis for enrichment, selection, or depletion of edited cells carrying corresponding gene knockouts in the screened population. STEP 1: select the phenotypic change you wish to study 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. What you need to know about Takara Bio's Guide-it CRISPR Genome-Wide sgRNA Library System-discusses the solutions offered by Takara Bio and details the features and benefits of the Guide-it CRISPR Genome-Wide sgRNA Library Systemīelow is a general overview of the process of performing a phenotypic screen using a pooled lentiviral sgRNA library.What you need to know about the sgRNA library screening protocol-provides a step-by-step walkthrough of what to do and what to avoid.General information about phenotypic library screening-provides a background and general information about the technologies used for phenotypic screens.We have arranged the content in the following three sections click on the tabs to expand the FAQs and tips in each section. We have compiled a comprehensive list of questions to walk the reader through the screening workflow and provide useful tips on the process of conducting a phenotypic screen using a whole-genome sgRNA library. CRISPR/Cas9 gene editing technology combined with next-generation sequencing (NGS) is making LoF screens easier to do and more broadly accessible to researchers than ever before, but it involves a lot of optimization at each step and careful planning to avoid the pitfalls. Genome-wide loss-of-function (LoF) phenotypic screens using a single guide RNA (sgRNA) library provide extremely powerful ways to identify novel protein functions by knocking out genes across a population of cells, applying selective pressure, and then identifying genes that are either enriched or depleted in the selected cell population relative to a control population.
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