Arrayed gene knockout (KO) libraries represent a valuable resource for performing functional genomics screening. Current generation arrayed KO libraries for the whole human genome rely on either single CRISPR sgRNAs to generate frameshift-causing indels or a mixture of several sgRNAs pooled together using only top ranked on-target predicted sgRNAs with no geographical consideration. However, there are considerable drawbacks to both of these approaches, since many indels do not cause frameshifts and current on-target prediction is unreliable. We designed a next generation library for generating gene KOs in an arrayed library format by multiplexing sgRNAs to localized, early exon regions of genes. We found that these multiplexed sgRNAs work cooperatively to generate larger sequence deletions thank indels at an efficient rate, allowing researchers to de-risk an arrayed approach to CRISPR library screening and assuring that each well within a library can produce a strong KO phenotype. 

Learning Objectives: 

1. Understand how optimizing sgRNA design and CRISPR protocols can yield the most effective methods for generating gene knockouts.
2. Learn how these methods have been adapted to generating next-generation whole human genome gene knockout libraries, that can be purposed for functional genomics screening.