A Rapid Combinatorial Approach to Assembling Synthetic Prokaryotic and Eukaryotic Protein Expression Vectors.

Vector construction and gene cloning are ubiquitous techniques essential to all fields of biological and medical research. They are the first steps in many endeavors leading to expressing proteins to understand gene function and regulation. However, they can often be rate-limiting, particularly in multi-gene studies, due to the time and effort required to assemble gene constructs and to identify the optimal constructs for protein expression.

Construction of CRISPR Libraries for Functional Screening.

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.