Construction of CRISPR Libraries for Functional Screening

Abstract

Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides. Here, we describe a method for constructing complex plasmid libraries based on pooled designed oligomers with high representation and tight distributions. The procedure allows construction of plasmid libraries of >60,000 members with a 95th/5th percentile ratio of less than 3.5.

CRISPR screens guide the way for PARP and ATR inhibitor biomarker discovery

“…Frequently, a frameshift mutation will occur, resulting in loss of function of the target gene. CRISPR‐Cas9 technology allows for delivery of pooled genome‐wide gRNA libraries to generate mutant cell populations [18]. In these pooled genetic screens, each cell receives a different gRNA, causing each individual cell to have loss of function of a single gene.…”

Molecular Switch Engineering for Precise Genome Editing

“…With CRISPR-Cas9, only 20-nt sequences need be identified as targets for gRNA design, which also simplifies construction of library plasmids. 19,20 Nonetheless, there are still some problems to be solved in order to use genome editing tools safely and efficiently. "Offtarget effects", unexpected cleavages at sequences similar to the target, 21−24 are among them.…”

“…With ZFN or TALEN, each DNA-binding domain for the target sequence must be constructed, which requires laborious subcloning steps. With CRISPR-Cas9, only 20-nt sequences need be identified as targets for gRNA design, which also simplifies construction of library plasmids. , …”