Target-specific precision of CRISPR-mediated genome editing

Abstract

The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms.However, our ability to predict editing accuracy, efficacy and outcome at specific sites is limited by an incomplete understanding of how the bacterial system interacts with eukaryotic genomes and DNA repair machineries. Here, we performed the largest comparison of indel profiles to date, examining over one thousand sites in the genome of human cells, and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing varies considerably among sites, with some targets showing one highly-preferred indel and others displaying a wide range of infrequent indels. Editing precision correlates with editing efficiency, homology-associated end-joining for both insertions and deletions, and a preference for single-nucleotide insertions.Precise targets and the identity of their preferred indel can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the PAM sequence. Regardless of precision, site-specific indel profiles are highly robust and depend on both DNA sequence and chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end-joining that can inform us on DNA repair mechanisms in human cells.

Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair

“…From these data, we trained an L1 regularized logistic regression model that accurately predicts the relative frequency of repair outcomes for any given target sequence. While this manuscript was in preparation, several similar studies appeared as publications (Shen et al 2018) or preprints (Allen et al 2018;Chakrabarti et al 2018) . Chakrabarti et al profiled the mutational outcomes associated with 1,492 target sequences from the human genome, while Allen et al and Shen et al designed and profiled 6,568 and 1,872 synthetic target sequences, respectively (Allen et al 2018;Chakrabarti et al 2018;Shen et al 2018) .…”

“…While this manuscript was in preparation, several similar studies appeared as publications (Shen et al 2018) or preprints (Allen et al 2018;Chakrabarti et al 2018) . Chakrabarti et al profiled the mutational outcomes associated with 1,492 target sequences from the human genome, while Allen et al and Shen et al designed and profiled 6,568 and 1,872 synthetic target sequences, respectively (Allen et al 2018;Chakrabarti et al 2018;Shen et al 2018) . These studies similarly identified sequence context surrounding the DSB, including MH, as a major determinant of repair outcomes, and also trained models to predict the frequency of the most common allele (Chakrabarti et al) or the relative frequencies of all repair outcomes (Allen et al, Shen et al) with comparable performance to that of our model.…”

Sequential Activation of Guide RNAs for Algorithmic Multiplexing of Cas9 Activities

“…However, the efficiency of conversion of different pGuides varied and appeared to depend upon both the spacer 18 sequence and the stem sequence. Nucleotide composition is known to greatly influence DNA repair outcomes at spCas9 generated DSBs (Allen et al, 2018;Chakrabarti et al, 2019;Leenay et al, 2019;Shen et al, 2018;van Overbeek et al, 2016). It is possible that some stem sequences are less amenable to the desired MMEJ repair event if cryptic microhomologies occur because of the nucleotide composition of the spacer region or between the target site in the stem and the ribozyme DNA sequence.…”

Characterization of Cas proteins for CRISPR‐Cas editing in streptomycetes

“…Our study suggests that testing multiple alternative Cas proteins may be more straightforward than calibrating Cas expression to resolve toxicity effects in streptomycetes. Therefore, until we can better understand the mechanisms and rules for successful precise editing by different CRISPR‐Cas systems (Chakrabarti et al, ), having a diverse selection of functional Cas proteins provides us with the versatility to overcome the limitations of individual systems. Beyond genome editing, multiplexing with orthogonal catalytically inactive Cas proteins with different PAM specificities may allow transcriptional control of different gene subsets in actinomycetes using CRISPR interference (Rock et al, ; Tong et al, ; Zhao et al, ).…”