An Analysis of gRNA Sequence Dependent Cleavage Highlights the Importance of Genomic Context on CRISPR-Cas Activity

Abstract

CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. To better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 39 published data sets. We find strong evidence that the genomic context, which can be defined as the DNA content outside of the gRNA/target sequence itself, greatly contributes to differences in gRNA dependent activity. Context underlies variation in activity often attributed to differences in gRNA sequence. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants.

A Meta-Analysis of gRNA Library Screens Enables an Improved Understanding of the Impact of gRNA Folding and Structural Stability on CRISPR-Cas9 Activity

“…This result, suggests that this observation is due to other context-dependent factors impacted by sequence composition rather than Duplex Stability. 6 This supports a model in which Duplex Stability below the cutoff we established is representative of formation of structure iii, while Duplex Stability above the cutoff does not indicate formation of inhibitory structures but rather may be correlated with other sequence-dependent features (Figure 3c). Finally, we turned to evaluate the impact of these structures across the remaining smaller datasets.…”

“…As mentioned, we have previously compiled 39 datasets from various species. 6 We filtered data from these 39 studies and included only libraries with 10,000 or more gRNA and then compared Spacer MFE and gRNA activity across these datasets, including datasets with different Cas9 variants (Figure 2a-f). 5,[17][18][19][20] Despite differences between datasets, there is a clear relationship between Spacer MFE values and activity when the Spacer MFE is below -5 kcal/mol but not when it is greater.…”

“…Datasets were compiled as described previously. 6 All calculations and generation of figures were performed in Python, using standard libraries. [10][11][12][13][14] Structure and minimum free energy predictions were calculated using ViennaRNA RNAfold package.…”

“…[1][2][3] The mechanisms underlying this sequence dependence have been studied extensively but many unknowns remain. [3][4][5][6][7] One factor reported to negatively impact CRISPR-Cas9 on-target activity is the formation of unwanted secondary structures. 7,8 Unwanted RNA structures and their associated stability can be estimated computationally.…”

“…To do so we expanded upon a meta-analysis we have recently reported, which includes 39 published datasets with gRNA libraries in various organisms. 6 We report two types of secondary structure that can inhibit Cas9 activity, provide estimated free energy cutoffs at which they begin to inhibit activity, and evaluate their prevalence within published datasets.…”