Target-Specific Precision of CRISPR-Mediated Genome Editing

Abstract

Summary The CRISPR-Cas9 system has successfully been adapted to edit the genome of various organisms. However, our ability to predict the editing outcome at specific sites is limited. Here, we examined indel profiles at over 1,000 genomic sites in human cells and uncovered general principles guiding CRISPR-mediated DNA editing. We find that precision of DNA editing (i.e., recurrence of a specific indel) varies considerably among sites, with some targets showing one highly preferred indel and others displaying numerous infrequent indels. Editing precision correlates with editing efficiency and a preference for single-nucleotide homologous insertions. Precise targets and editing outcome can be predicted based on simple rules that mainly depend on the fourth nucleotide upstream of the protospacer adjacent motif (PAM). Indel profiles are robust, but they can be influenced by chromatin features. Our findings have important implications for clinical applications of CRISPR technology and reveal general patterns of broken end joining that can provide insights into DNA repair mechanisms.

A Universal Approach to Target Various HBB Gene Mutations in Hematopoietic Stem/Progenitor Cells for Beta-Thalassemia Gene Therapy by CRISPR/Cas9 and the rAAV6 Vector

“…We next compared the different electroporation parameters, and the results illustrated that 1,600 V was the most optimized electroporation parameter for the high indel frequency and cell survival rate ( Figure 2C). It has previously been shown that 2'-O-methyl 3'phosphorothioate (MS) modi cations to the three terminal nucleotides at the 5' and 3' ends of the sgRNA signi cantly increase the ability of the Cas9/sgRNA system to induce DSBs in HSPCs [16,20]. Based on previous work, we determined whether modi ed sgRNA was more effective than in vitro transcription sgRNA (IVT sgRNA).…”

Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses

“…Protein-based delivery of Cas9 circumvents potentially detrimental integration of commonly used DNA-based delivery vectors. Further, several studies have recently revealed more precise, predictable outcomes, following targeting via a single CRISPR/Cas9 nuclease, at many loci [88,89]. As these models are primarily based upon the sequence context of the PAM site, they enable rapid and efficient development of precise CRISPR/Cas9-based strategies resulting in predictably homogenous DSB repair outcomes for reframing and knockout approaches.…”

One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation

“…[ 6,7 ] To fabricate these new edited cell lines, evaluating the properties of single clones (i.e., a single edited cell) is especially important, as biallelic editing differences can occur and non‐homologous end‐joining DSB repair mechanisms generate indels that differ between individual clones. [ 8–10 ] Isolating single clones provides a method for enriching correctly edited cells, of which one can correlate the phenotypic changes to a specific clonal genotype and facilitate downstream characterization.…”