Massively targeted evaluation of therapeutic CRISPR off-targets in cells

Abstract

Methods for sensitive and high-throughput evaluation of CRISPR RNA-guided nucleases (RGNs) off-targets (OTs) are essential for advancing RGN-based gene therapies. Here we report SURRO-seq for simultaneously evaluating thousands of therapeutic RGN OTs in cells. SURRO-seq captures RGN-induced indels in cells by pooled lentiviral OTs libraries and deep sequencing, an approach comparable and complementary to OTs detection by T7 endonuclease 1, GUIDE-seq, and CIRCLE-seq. Application of SURRO-seq to 8150 OTs from 110 therapeutic RGNs identifies significantly detectable indels in 783 OTs, of which 37 OTs are found in cancer genes and 23 OTs are further validated in five human cell lines by targeted amplicon sequencing. Finally, SURRO-seq reveals that thermodynamically stable wobble base pair (rG•dT) and free binding energy strongly affect RGN specificity. Our study emphasizes the necessity of thoroughly evaluating therapeutic RGN OTs to minimize inevitable off-target effects.

Off-target effects in CRISPR/Cas9 gene editing

“…In this method, genomic DNA is first sheared and circularized by intramolecular ligation. With the presence of Cas9/sgRNA complexes, the circular DNA fragments were selectively linearized upon Cas9 nuclease cleavage before they become available for library construction and high-throughput sequencing ( Tsai et al, 2017 ; Lv et al, 2022 ; Pan et al, 2022 ). In this method, non-specific linear DNA and undigested circular DNA can be efficiently removed, greatly reducing the background in off-target detection.…”

“…GUIDE-seq is more sensitive than the IDLV method because dsODNs integrate more efficiently and precisely into DSBs, while the integration events of IDLV is low in number and can distribute as far as 500bp away from the actual DSB sites ( Tsai et al, 2015 ; Cromer et al, 2023 ). A primary limitation of GUIDE-seq is relevant to the low delivery efficiency of dsODNs into cells, which results in detection of only 30%–50% of all the DSBs ( Tsai et al, 2015 ; Pan et al, 2022 ).…”

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

“…Besides targeted deep sequencing, other validation methods such as CUT-PCR 33 and TIDE/TIDER 34 are greatly limited by their validation scales, as well as limited by the detection sensitivity in the case of sanger sequencing-based TIDE/TIDER 34 . Nevertheless, integration of candidate off-target site libraries by SURRO-seq 35 , 36 and an improved dual-target system 37 allow large-scale validation of CRISPR-Cas nuclease off-targets identified from other methods in a genomic and cellular context, although these methods may still not fully mimic the editor delivery and chromatin state expected in target cells for the gene editing therapy.…”

Chemically Modified Platforms for Better RNA Therapeutics

“…However, the widespread application of RNA-based drugs as a versatile therapeutic modality is impeded by significant challenges, including susceptibility to nucleases, large size and fragile nature of RNA molecules, pronounced negative charge, immunogenicity, off-target effects, and the absence of sophisticated delivery carriers. The inherent instability of RNA molecules can lead to shortened half-life, immune recognition, and subsequent elimination, thus limiting their sustained therapeutic benefits. − Additionally, off-target effects may result in serious side effects, while the strong negative charge of RNA impedes cellular membrane penetration and cytoplasmic targeting of disease-related genes. − Moreover, the lack of effective delivery technologies or carriers further hampers the clinical translation of RNA-based drugs. Overcoming these barriers necessitates additional platform technologies.…”