Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Abstract

Spotting off-targets from gene editing Unintended genomic modifications limit the potential therapeutic use of gene-editing tools. Available methods to find off-targets generally do not work in vivo or detect single-nucleotide changes. Three papers in this issue report new methods for monitoring gene-editing tools in vivo (see the Perspective by Kempton and Qi). Wienert et al. followed the recruitment of a DNA repair protein to DNA breaks induced by CRISPR-Cas9, enabling unbiased detection of off-target editing in cellular and animal models. Zuo et al. identified off-targets without the interference of natural genetic heterogeneity by injecting base editors into one blastomere of a two-cell mouse embryo and leaving the other genetically identical blastomere unedited. Jin et al. performed whole-genome sequencing on individual, genome-edited rice plants to identify unintended mutations. Cytosine, but not adenine, base editors induced numerous single-nucleotide variants in both mouse and rice. Science , this issue p. 286 , p. 289 , p. 292 ; see also p. 234

RGEN-seq for highly sensitive amplification-free screen of off-target sites of gene editors

“…In the resulting alignment, sites cleaved by the RGENs yield sequence read pileups that terminate at the cut site, ~ 3 nucleotides proximal to the PAM, producing a distinct signature that can be detected computationally. This signature is similar to those observed with DIGENOME-Seq 23 , SITE-Seq 28 , and DISCOVER-Seq 22 , which allowed us to use previously developed software for the analysis of RGEN-seq experimental data 22 , 25 , 28 .…”

“…1 b), and one targeting an integrated vector sequence. Cas9 cut sites were identified with BLENDER 22 pipeline. Figure 1 b shows an IGV snapshot of the read coverage within the aforementioned genomic region—note the absence of reads between target cut sites.…”

“…This concern drives developing strategies for improving targeted nuclease specificities 14 – 17 as well as methods for genome-wide off-target sites detection. Cell-based and in vivo genome-wide methods 18 – 22 rely on cellular events for the integration of donor sequences or chromosomal translocations. These methods are assumed to be precise, with few or no false positives as they operate in the context of intracellular chromatin state and modifications.…”

The Off-Targets of Clustered Regularly Interspaced Short Palindromic Repeats Gene Editing

“…ChIP-seq using a catalytically inactive Cas9 revealed that the potential off-targets are enriched in the open chromatin regions, and the number of off-targets varied widely from around 10 to over 1,000 depending on the sgRNA used (Kuscu et al, 2014). DISCOVER-Seq is another ChIP-seq approach, but based on the tracking of the MRE11, a repair factor that is recruited to the Cas9 cut site and allows the identification of the nuclease cutting site with a single-base resolution (Wienert et al, 2019).…”

Will Plant Genome Editing Play a Decisive Role in “Quantum-Leap” Improvements in Crop Yield to Feed an Increasing Global Human Population?

“…For example, cytosine and adenine base editors converting C to T and A to G, respectively, fuse a nickase-type Cas9 with a deaminase domain and, thus, do not induce DSBs (Figure 2). The two single-base editors (cytidine deaminase and adenosine deaminase) were fused to produce simultaneous double-base conversions (C → T and G → A) [15,[154][155][156][157][158][159].…”