Following phenotypic selection, genomic DNA is extracted from the selected clones, alongside a control cell population. In the most common protocols for genome-wide knockouts, a Next-generation sequencing (NGS) library is created by a two step polymerase chain reaction (PCR). The first step amplifies the sgRNA region, using primers specific to the lentiviral integration sequence, and the second step adds Illumina i5 and i7 sequences. NGS of the PCR products allows the recovered sgRNAs to be identified, and a quantification step can be used to determine the relative abundance of each sgRNA.
The final step in the screen is to computationally evaluate the significantly enriched or depleted sgRNAs, trace them back to their corresponding genes, and in turn determine which genes and pathways could be responsible for the observed phenotype. Several algorithms are currently available for this purpose, with the most popular being the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) method. Developed specifically for CRISPR/Cas9 knockout screens in 2014, MAGeCK demonstrated better performance compared with alternative algorithms at the time, and has since demonstrated robust results and high sensitivity across different experimental conditions. As of 2015, the MAGeCK algorithm has been extended to introduce quality control measurements, and account for the previously overlooked sgRNA knockout efficiency. A web-based visualisation tool (VISPR) was also integrated, allowing users to interactively explore the results, analysis, and quality controls.