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CRISPR Researchers Develop Highly Sensitive Method for Identification of Off-Target Effects In Vivo

NEW YORK (GenomeWeb) – An international team of researchers led by Harvard Medical School and Massachusetts General Hospital researcher Keith Joung has developed a highly sensitive method to robustly identify the genome-wide off-target effects of CRISPR–Cas nucleases in vivo.

In a study published in Nature today, the researchers described "verification of in vivo off-targets," or VIVO, and demonstrated using it and a guide RNA deliberately designed to be promiscuous that CRISPR–Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. They also used VIVO to show that appropriately designed guide RNAs can efficiently edit mouse livers in vivo with no detectable off-target mutations.

"VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing," the authors wrote.

The strategy consists of an initial in vitro discovery step where a superset of potential off-target cleavage sites for a nuclease is identified using the circularization for in vitro reporting of cleavage effects by sequencing, or CIRCLE-seq, method. The second step involves in vivo confirmation where the sites identified by CIRCLE-seq are examined for indel mutations in target tissues that have been treated with the nuclease.

To test VIVO, the researchers designed a Streptococcus pyogenes Cas9 guide RNA targeted to the mouse Pcsk9 gene, which they expected would have a high likelihood of inducing multiple off-target mutations in the mouse genome. After infecting two strains of mice with this promiscuous gRNA-CRISPR complex, the researchers determined using VIVO that it caused 3,107 off-target cleavage sites in one mouse strain and 2,663 off-target sites in the other.

To perform the second step of VIVO, the researchers then assessed whether the off-target cleavage sites they identified showed evidence of indels in vivo in the livers of the mice. Of the 45 sites they examined, 19 showed significant evidence of indels. These data suggested to the investigators that Cas9 with a promiscuous gRNA can generate stable, and sometimes high-frequency, off-target mutations in vivo and that VIVO was useful in identifying these off-target effects, even when they occurred with low frequency.

But because potential therapeutic applications would not use a promiscuous gRNA, the team then sought to assess the in vivo off-target profiles of Cas9 gRNAs designed to be more targeted, and so they constructed two additional gRNAs that were targeted to mouse Pcsk9.

They again used VIVO to check for off-target effects, but this time found no significant off-target indels at any of the 181 potential off-target sites identified by CIRCLE-seq in either mouse. To further exclude the possibility that CIRCLE-seq had missed any off-target sites, the researchers also performed targeted amplicon sequencing of the most closely matched sites in the mouse genome that were not identified in the CIRCLE-seq experiments and did not observe significant indels at any of these sites in all treated mice.

"To our knowledge, our report provides the first demonstration that CRISPR–Cas nucleases can robustly induce off-target mutations in vivo. Previous in vivo studies have reported no or very few off-target mutations, but used the cell-based genome-wide unbiased identification of double-strand breaks enabled by sequencing (GUIDE-seq) method or other in silico approaches that have not been validated to effectively identify these sites in vivo," the authors wrote. "By contrast, VIVO enabled the robust and sensitive identification of off-target sites in vivo, even those with mutation frequencies as low as about 0.13 percent. The high sensitivity of CIRCLE-seq is most probably what enabled the identification of a superset of all potential off-target sites, including those actually mutated in vivo."