User:Maria Kuzmina/sandbox

CRISPR screening
The clustered regularly interspaced short palindrome repeats (CRISPR)/Cas9 system is a new gene-editing technology that can induce double-strand breaks (DSBs), single-strand nicks, or anywhere guide ribonucleic acids (RNAs) can bind with the protospacer adjacent motif sequence. By simple changing sequence of gRNA, Cas9-endonuclease can be delivered to a gene of interest and induce DSBs. The efficiency of Cas9-endonuclease and the ease by which genes can be targeted led to the development of CRISPR-knockout (KO) libraries both for mouse and human cells, which can cover either specific gene sets of interest or the whole-genome. Knock-out libraries are created in a way to achieve equal representation and performance across all expressed gRNAs and carry an antibiotic or fluorescent selection marker that can be used to recover transduced cells. There are two plasmid systems in CRISPR/Cas9 libraries. First, is all in one plasmid, where sgRNA and Cas9 are produced simultaneously in a transfected cell. Second, is a two-vector system: sgRNA and Cas9 plasmids are delivered separetly. It's important to deliver thousands of unique sgRNAs-containing vectors to a single vessel of cells by viral transduction at low multiplicity of infection (MOI, typically at 0.1-0.6), it prevents the probability that an individual cell clone will get more than one type of sgRNA otherwise it can lead to incorrect assignment of genotype to phenotype.

Once pooled library is prepared it is necessary to carry out a deep sequencing (NGS, next generation sequencing) of PCR-amplifed plasmid DNA in order to reveal abundance of sgRNAs. Cells of interest can be consequentially infected by the library and then selected according to the phenotype. There are 2 types of selection: negative and positive. By negative selection dead or slow growing cells are efficiently detected. It can identify survival-essential genes, which can be further serve as candidates for molecularly targeted drugs. On the other hand, positive selection gives a collection of growth-advantage acquired populations by random mutagenesis. After selection genomic DNA is collected and sequenced by NGS. Depletion or enrichment of sgRNAs is detected and compared to the original sgRNA library, annotated with the target gene that sgRNA corresponds to. Statistical analysis then identify genes that are significantly likely to be relevant to the phenotype of interest.