Project description:The Detect-seq provides an unbiased method for genome-wide identification of CBE induced off-targets inside of cells. Purpose: Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, its genome-wide specificity remains poorly understood, precluding its therapeutic applications. Unbiased tools are urgently needed to comprehensively evaluate CBE off-targets at the genome-wide level. Methods: The genomic DNA from CBE transfected cells are processed with fragmentation, endogenous d5fC protection, damage repair, biotin-dUTP and 5fdCTP labeling, and pull-down steps. After the sequencing library preparation, the FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates. Results: Through Detect-seq, we comprehensively profiled the genome-wide off-targets of CBE for several representative sgRNAs, and observed both weak, “random-like” off-targets as well as strong Cas9-dependent off-targets in different human cell lines. Cas9-dependent off-targets can be prevalent for several sgRNAs, and demonstrate a divergent degrees of sequence similarity to the sgRNA. These CBE-induced off-target sites differ greatly from those caused by Cas9 nuclease alone, suggesting inherently different properties among these genome editing tools. Targeted amplicon sequencing further corroborated the novel off-target sites by Detect-seq; quite a few sites showed editing ratio that is on the same order of magnitude or even comparable to the on-target sites. Detect-seq also revealed two unexpected types of Cas9-dependent off-targets, which are out-of-protospacer editing and target-strand editing. These novel off-targets were likely caused by an unstable structure at the PAM-distal side of the Cas9-sgRNA-DNA complex. Based upon such understanding to the off-target editing, we engineered several CBE variants bearing mutations in APOBEC1 and obtained improved CBEs with significantly reduced off-target editing activities. Detect-seq utilized a dU-mapping strategy to profile on-target and off-target editing events by CBE at the whole-genome level. We applied Detect-seq to off-target evaluation in HEK293T and MCF7 cells for BE4max with several frequently used sgRNAs: “VEGFA site 2”, “HEK293 site 4”, “EMX1”, and “RNF2”.

Project description:BackgroundADAR (adenosine deaminase acting on RNA) proteins convert adenosine into inosine in double-stranded RNAs and have been shown to increase gene product diversity in a number of bilaterians, particularly mammals and flies. This enzyme family appears to have evolved from an ADAT (adenosine deaminase acting on tRNA) ancestor, via the addition of a double-stranded RNA binding domain. The modern vertebrate ADAR family is comprised of ADAD, ADAR2 and ADAR1, each of which has a conserved domain architecture. To reconstruct the origin of this protein family, we identified and categorised ADAR family members encoded in the genomes and/or transcriptomes of early-branching metazoan and closely related non-metazoan taxa, including thirteen sponge and ten ctenophore species.ResultsWe demonstrate that the ADAR protein family is a metazoan innovation, with the three ADAR subtypes being present in representatives of the earliest phyletic lineages of animals - sponges and ctenophores - but not in other closely related choanoflagellate and filasterean holozoans. ADAR1 is missing from all ctenophore genomes and transcriptomes surveyed. Depending on the relationship of sponges and ctenophores to the rest of the Metazoa, this is consistent with either ADAR1 being lost in ctenophores, as it has been in multiple metazoan lineages, or being an innovation that evolved after ctenophores diverged from the rest of the animal kingdom. The presence of Z-DNA binding domains in some sponge ADARs indicates an ancestral ADAR included this domain and it has been lost in multiple animal lineages.ConclusionsThe ADAR family appears to be a metazoan innovation, with all family members in place in the earliest phyletic branches of the crown Metazoa. The presence of ADARs in sponges and ctenophores is consistent with A-to-I editing being a post-transcriptional regulatory mechanism that was used by the last common ancestor to all living animals and subsequently has been preserved in most modern lineages.

Project description:The Detect-seq provides an unbiased method for genome-wide identification of CBE induced off-targets inside of cells. Purpose: Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, its genome-wide specificity remains poorly understood, precluding its therapeutic applications. Unbiased tools are urgently needed to comprehensively evaluate CBE off-targets at the genome-wide level. Methods: The genomic DNA from CBE transfected cells are processed with fragmentation, endogenous d5fC protection, damage repair, biotin-dUTP and 5fdCTP labeling, and pull-down steps. After the sequencing library preparation, the FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates. Results: Through Detect-seq, we comprehensively profiled the genome-wide off-targets of CBE for several representative sgRNAs, and observed both weak, “random-like” off-targets as well as strong Cas9-dependent off-targets in different human cell lines. Cas9-dependent off-targets can be prevalent for several sgRNAs, and demonstrate a divergent degrees of sequence similarity to the sgRNA. These CBE-induced off-target sites differ greatly from those caused by Cas9 nuclease alone, suggesting inherently different properties among these genome editing tools. Targeted amplicon sequencing further corroborated the novel off-target sites by Detect-seq; quite a few sites showed editing ratio that is on the same order of magnitude or even comparable to the on-target sites. Detect-seq also revealed two unexpected types of Cas9-dependent off-targets, which are out-of-protospacer editing and target-strand editing. These novel off-targets were likely caused by an unstable structure at the PAM-distal side of the Cas9-sgRNA-DNA complex. Based upon such understanding to the off-target editing, we engineered several CBE variants bearing mutations in APOBEC1 and obtained improved CBEs with significantly reduced off-target editing activities.

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The origin of unwanted editing byproducts in gene editing.

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The origin of unwanted editing byproducts in gene editing.

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