A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture

Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of target site recognition. Cleavage specificity is typically evaluated by low throughput assays (T7 endonuclease I assay, target amplification followed by high-throughput sequencing), which are limited to a subset of potential off-target sites. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites for two guide RNAs. RNA-guided Cas9 binding was highly specific to the target site while off-target binding occurred at much lower intensities. Cas9-bound regions were highly enriched in NGG sites, a sequence required for target site recognition by Streptococcus pyogenes Cas9. To determine the relationship between Cas9 binding and endonuclease activity, we applied targeted sequence capture, which allowed us to survey 1200 genomic loci simultaneously including potential off-target sites identified by ChIP-seq and by computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results confirm the high-specificity of CRISPR endonucleases and demonstrate that sequence capture can be used as a high-throughput genome-wide approach to identify off-target activity.     

Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.     

ggtreeExtra: Compact Visualization of Richly Annotated Phylogenetic Data

We present the ggtreeExtra package for visualizing heterogeneous data with a phylogenetic tree in a circular or rectangular layout (https://www.bioconductor.org/packages/ggtreeExtra). The package supports more data types and visualization methods than other tools. It supports using the grammar of graphics syntax to present data on a tree with richly annotated layers and allows evolutionary statistics inferred by commonly used software to be integrated and visualized with external data. GgtreeExtra is a universal tool for tree data visualization. It extends the applications of the phylogenetic tree in different disciplines by making more domain-specific data to be available to visualize and interpret in the evolutionary context.     

Endophytic bacterial communities in in vitro shoot cultures derived from embryonic tissue of hybrid walnut (<Emphasis Type="Italic">Juglans</Emphasis> × <Emphasis Type="Italic">intermedia</Emphasis>)

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system has emerged as the robust gene editing tool that functions through the double-stranded break repair process leading to targeted mutagenesis in higher genomes. CRISPR/Cas9 has been simplified to a two component system consisting of a single guide RNA (gRNA) that binds Cas9 to target genomic sites in sequence-dependent manner. This RNA-guided nuclease system has mostly been applied for inducing point mutations or short insertion-deletions at one or multiple loci. The present study addressed the utility of this system for excising marker genes from plant genomes, an application highly relevant for developing marker-free transgenic plants. A transgenic rice line expressing β-glucuronidase (GUS) gene was transformed by Agrobacterium or gene gun with a construct expressing Cas9 and two gRNAs to target each end of 1.6 kb GUS gene. Molecular analysis of the transformed lines detected excision at low frequency in the callus lines, but at significantly higher frequency in plant lines, indicating robust efficiency of Cas9:gRNA in regenerated plants. Bi-allelic excisions were observed in plants derived from three independent events, allowing recovery of homozygous excision lines in the first generation (T₀). Notably, the excision in different plant lines was formed by precise cut and ligation of the two blunt ends without mutation at or around the excision site. Since the goal of marker-removal technologies is to precisely excise a defined piece of DNA without introducing mutations in the adjacent sequences, Cas9:gRNA system could be an effective tool for producing marker-free plants.     

CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity

CRISPR-Cas12a (Cpf1) is a bacterial RNA-guided nuclease that cuts double-stranded DNA (dsDNA) at sites specified by a CRISPR RNA (crRNA) guide. Additional activities have been ascribed to this enzyme in vitro: site-specific (cis) single-stranded DNA (ssDNA) cleavage and indiscriminate (trans) degradation of ssDNA, RNA, and dsDNA after activation by a complementary target. The ability of Cas12a to cleave nucleic acids indiscriminately has been harnessed for many applications, including diagnostics, but it remains unknown if it contributes to bacterial immunity. Here, we provide evidence that cleavage of ssDNA in cis or in trans by Cas12a is insufficient to impact immunity. Using LbCas12a expressed in either Pseudomonas aeruginosa or Escherichia coli, we observed that cleavage of dsDNA targets did not elicit cell death or dormancy, suggesting insignificant levels of collateral damage against host RNA or DNA. Canonical immunity against invasive dsDNA also had no impact on the replicative fitness of co-infecting dsDNA phage, ssDNA phage or plasmid in trans. Lastly, crRNAs complementary to invasive ssDNA did not provide protection, suggesting that ssDNA cleavage does not occur in vivo or is insignificant. Overall, these results suggest that CRISPR-Cas12a immunity predominantly occurs via canonical targeting of dsDNA, and that the other activities do not significantly impact infection outcomes.     

CRISPRmap: an automated classification of repeat conservation in prokaryotic adaptive immune systems

Central to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems are repeated RNA sequences that serve as Cas-protein–binding templates. Classification is based on the architectural composition of associated Cas proteins, considering repeat evolution is essential to complete the picture. We compiled the largest data set of CRISPRs to date, performed comprehensive, independent clustering analyses and identified a novel set of 40 conserved sequence families and 33 potential structure motifs for Cas-endoribonucleases with some distinct conservation patterns. Evolutionary relationships are presented as a hierarchical map of sequence and structure similarities for both a quick and detailed insight into the diversity of CRISPR-Cas systems. In a comparison with Cas-subtypes, I-C, I-E, I-F and type II were strongly coupled and the remaining type I and type III subtypes were loosely coupled to repeat and Cas1 evolution, respectively. Subtypes with a strong link to CRISPR evolution were almost exclusive to bacteria; nevertheless, we identified rare examples of potential horizontal transfer of I-C and I-E systems into archaeal organisms. Our easy-to-use web server provides an automated assignment of newly sequenced CRISPRs to our classification system and enables more informed choices on future hypotheses in CRISPR-Cas research: http://rna.informatik.uni-freiburg.de/CRISPRmap.     

Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to ^5′NGG^3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data.     

A cleavage-based surrogate reporter for the evaluation of CRISPR–Cas9 cleavage efficiency

CRISPR–Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR–Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.     

SANTA: Quantifying the Functional Content of Molecular Networks

Linking networks of molecular interactions to cellular functions and phenotypes is a key goal in systems biology. Here, we adapt concepts of spatial statistics to assess the functional content of molecular networks. Based on the guilt-by-association principle, our approach (called SANTA) quantifies the strength of association between a gene set and a network, and functionally annotates molecular networks like other enrichment methods annotate lists of genes. As a general association measure, SANTA can (i) functionally annotate experimentally derived networks using a collection of curated gene sets and (ii) annotate experimentally derived gene sets using a collection of curated networks, as well as (iii) prioritize genes for follow-up analyses. We exemplify the efficacy of SANTA in several case studies using the S. cerevisiae genetic interaction network and genome-wide RNAi screens in cancer cell lines. Our theory, simulations, and applications show that SANTA provides a principled statistical way to quantify the association between molecular networks and cellular functions and phenotypes. SANTA is available from http://bioconductor.org/packages/release/bioc/html/SANTA.html.     

quantro: a data-driven approach to guide the choice of an appropriate normalization method

Normalization is an essential step in the analysis of high-throughput data. Multi-sample global normalization methods, such as quantile normalization, have been successfully used to remove technical variation. However, these methods rely on the assumption that observed global changes across samples are due to unwanted technical variability. Applying global normalization methods has the potential to remove biologically driven variation. Currently, it is up to the subject matter experts to determine if the stated assumptions are appropriate. Here, we propose a data-driven alternative. We demonstrate the utility of our method (quantro) through examples and simulations. A software implementation is available from http://www.bioconductor.org/packages/release/bioc/html/quantro.html.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0679-0) contains supplementary material, which is available to authorized users.