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. 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.

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.The SureVector system was developed to address this by enabling the rapid and reliable assembly of multiple DNA modules into a recombinant plasmid containing a gene-of-interest (GOI). It harnesses the power of synthetic biology to combine DNA modules from standard parts into a customized vector that expresses proteins in bacterial, mammalian, or yeast cells. The key advantages of the innovative SureVector system include rapid custom vector generation, enhanced flexibility to assemble new vectors quickly as experimental requirements change, and the reliable and precise assembly of fully interchangeable standard DNA modules that retain their functionality. The SureVector system is the only next-generation plasmid assembly technology to guarantee assembly of multiple functional DNA modules.