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Researchers Publish Two New Methods for Analysis of CRISPR Off-Target Effects

NEW YORK (GenomeWeb) – Two studies published simultaneously in Nature Methods yesterday describe new methods for analyzing the off-target effects of CRISPR/Cas-9 genome editing. One method identifies off-target mutations associated with cell-type-specific SNPs, while the other detects potential off-target cleavage sites.

In the first study, researchers at Massachusetts General Hospital and Harvard described circularization for in vitro reporting of cleavage effects by sequencing, or CIRCLE-seq — calling it an in vitro screening method that is highly sensitive and performs better than existing cell-based or biochemical approaches for identifying CRISPR/Cas9 genome-wide off-target mutations.

"In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences," the authors wrote. "Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR/Cas9."

The team designed restriction-enzyme-independent strategies to generate and enzymatically select for the conversion of randomly sheared DNA into one of two different types of covalently closed DNA structures: attachment of stem-loops to linear DNA ends or circularization of linear fragments. They found that circularization was at least an order of magnitude more effective when enriching for Cas9-nuclease cleaved genomic DNA fragments than attaching stem loops to linear DNA.

The researchers then tested the sensitivity of their new method by comparing off-target results of a single-guide RNA (sgRNA) targeting the HBB gene with off-target profiles of the same sgRNA generated by another off-target identification method called Digenome-seq. They found that CIRCLE-seq identified 26 of 29 off-target sites previously seen with Digenome-seq, and further identified 156 new sites the other method did not catch.

"These results demonstrate that CIRCLE-seq possesses higher signal-to-noise ratios relative to those of Digenome-seq and using approximately 100-fold fewer sequencing reads; this likely accounts for its greater sensitivity for identifying genome-wide off-target sites," they wrote.

In the second study, researchers from Caribou Biosciences and DuPont Pioneer described a biochemical method called SITE-Seq, which uses Cas9 programmed with sgRNAs to identify the sequence of cut sites within genomic DNA.

"We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells," the team said. "The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type, and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity."

The SITE-Seq procedure involves selectively tagging, enriching, sequencing, and mapping cleaved genomic DNA to a corresponding reference genome. In the resulting alignment, sites cleaved by the sgRNP produce a distinct signature that can be detected computationally, the researchers noted, adding that this signature is similar to that observed with Digenome-seq.

They tested their method on human embryonic kidney genomic DNA digested with Cas9, and found 771 biochemical cleavage sites for SITE-Seq to target. They then transfected AAVS1 sgRNA into HEK293 cells stably expressing Cas9–GFP and measured off-target editing three days later. They selected for evaluation a subset of 68 sites from the original 771 containing a wide range of nucleotide substitutions, and found 29 cellular off-targets with mutation frequencies ranging from 66.6 percent to 0.1 percent.

"Taken together, these data suggest that SITE-Seq enables us to uncover the full landscape of sgRNP biochemical cleavage sites and then pinpoint the subset of those targets that accumulate off-target mutations in a cellular context," the team wrote.

"This research marks a significant step towards the confirmation of CRISPR-Cas9 as a safe and effective gene-editing tool. By providing researchers with a way to accurately map the genomic landscape of CRISPR–Cas9 activity, we can reduce the occurrence of off-target effects by selecting for targets with the highest possible activity and specificity," Caribou President and CEO Rachel Haurwitz added in a statement.

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