Although great progress has been made in the characterization of off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification–mediated modification of a previously published high-throughput, genome-wide translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN-nucleases revealed off-target hotspots for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases by more than 10-fold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.

SUMMARY RAG initiates antibody V(D)J recombination in developing lymphocytes by generating “on-target” DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically-inserted RSS pairs to identify huge numbers of RAG “off-target” breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage-site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation-dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them.

Unbiased, high-throughput assays to detect and quantify DNA double-stranded breaks (DSBs) genome-wide in mammalian cells will facilitate basic studies of mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as evaluating on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for detecting genome-wide “prey” DSBs via their translocation in cultured mammalian cells to a fixed “bait” DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina paired-end Miseq sequencing. A custom bioinformatic pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis are necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable, and straightforward to implement with a turnaround time of less than one week.

Summary Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single stranded DNA targets. While largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-Seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting “convergent” transcription arises from antisense transcription that emanates from Super-Enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells.

During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons1,2. In mice, six additional sets of constant region exons (CHs) lie 100-200 kb downstream in the same transcriptional orientation as V(D)J and Cμ exons2. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH2. Activation-Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region2,3; these lesions are converted into DNA double-strand breaks (DSBs) by general DNA repair factors3. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB (Fig. 1a). However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high-throughput genome-wide translocation sequencing (HTGTS)4 into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor.

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ∼10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.

Efficient and precise genome editing is essential for clinical applications and generating animal models, which requires engineered nucleases with high editing ability while low off-target activity. Here we present a high-throughput sequencing method, primer-extension-mediated sequencing (PEM-seq), to comprehensively assess both editing ability and specificity of engineered nucleases. We showed CRISPR/Cas9-generated breaks could lead to chromosomal translocations and large deletions by PEM-seq. We also found that Cas9 nickase possessed lower off-target activity while with some loss of target cleavage ability. However, high-fidelity Cas9 variants, including both eCas9 and the new FeCas9, could significantly reduce the Cas9 off-target activity with no obvious editing retardation. Moreover, we found AcrIIA4 inhibitor could greatly reduce the activities of Cas9, but off-target loci were not so effectively suppressed as the on-target sites. Therefore, PEM-seq fully evaluating engineered nucleases could help choose better genome editing strategy at given loci than other methods detecting only off-target activity.

BackgroundThe CRISPR/Cas9 system has become an efficient gene editing method for generating cells carrying precise gene mutations, including the rearrangement and deletion of chromosomal segments. However, whether an entire chromosome could be eliminated by this technology is still unknown.ResultsHere we demonstrate the use of the CRISPR/Cas9 system to eliminate targeted chromosomes. Using either multiple cleavages induced by a single-guide RNA (sgRNA) that targets multiple chromosome-specific sites or a cocktail of multiple sgRNAs, each targeting one specific site, we found that a sex chromosome could be selectively eliminated in cultured cells, embryos, and tissues in vivo. Furthermore, this approach was able to produce a targeted autosome loss in aneuploid mouse embryonic stem cells with an extra human chromosome and human induced pluripotent stem cells with trisomy 21, as well as cancer cells.ConclusionsCRISPR/Cas9-mediated targeted chromosome elimination offers a new approach to develop animal models with chromosome deletions, and a potential therapeutic strategy for human aneuploidy diseases involving additional chromosomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1354-4) contains supplementary material, which is available to authorized users.