Using the CRISPR-Cas9 System Analogous to Restriction Enzymes Whose Recognition Site is 23 Nucleotides to Alter Pooled CRISPR Libraries

Abstract

Pooled CRISPR libraries in recent years have been widely used to perform genetic screens. Such a library contains tens of thousands of single guide RNA (sgRNA) constructs that target a set of genes of interest. A perfect library is a collection of all intended constructs, and all library members are uniformly distributed. However, in reality, that is never the case. During library production, unwanted sgRNA constructs might be introduced and some constructs are more abundant than other. These issues can cause issues with screen setup and data analysis. For example, a library that has high proportion of unwanted constructs, or a library that have lopsided abundance distribution may force us to scale up the screen setup, in order to have large portion of the library members screened. Currently there is no good way to remove sgRNA constructs from plasmid pooled CRISPR libraries, other than to remake the library. Knowing that the CRISPR-Cas9 system can cleave a 20-nucleotide region, this study aimed to show that sgRNA constructs present within a CRISPR Library could be removed by using sgRNA- Cas9 complexes in similar manner to restriction enzymes by using a 23-nucleotide recognition sequence. First, cleaving a plasmid DNA containing a single sgRNA construct was demonstrated, by designing a sgRNA oligo targeting the 20-nucleotide sgRNA construct present within a plasmid DNA sample. After the initial successful test, an additional seven sgRNA oligos were designed to target the complimentary sgRNA constructs present within a genome-wide CRISPR library, a library of 70,000 members. The results from the first attempt came out inconclusive, due to shallow sequencing that resulted in non-targeted sgRNA constructs appearing to be depleted after analyzing the sequencing data. The second attempt however, with some optimizations was able to prove that sgRNA constructs could be removed up to 8-fold from a pooled CRISPR library. An additional experiment showed that after digestion of plasmid DNA with a sgRNA-Cas9 complex, the cleaved 20-nucleotide sequence has the ability to be ligated back into the opened plasmid with blunt-end ligation. Just like restriction enzymes, in our sgRNA- Cas9 system, the cleavage was not 100% in either the single plasmid DNA or the pooled CRISPR library, further optimizations past this study will need to be completed to help obtain higher cleavage efficiency.