Why do I need to use next-generation sequencing (NGS) to analyze the results of the screen? How can I perform this analysis?

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 [...]

Why do we need multiple guides targeting the same gene?

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, [...]

Why are guide RNA libraries supplied on lentivirus vectors but not on regular plasmids?

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 [...]

What are positive and negative CRISPR library screens?

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 [...]

Pooled sgRNA libraries vs. pooled RNAi libraries vs. targeted arrayed sgRNA libraries

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. [...]