2
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      CHANGE-seq reveals genetic and epigenetic effects on CRISPR-Cas9 genome-wide activity

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Current methods can illuminate the genome-wide activity of CRISPR-Cas9 nucleases, but are not easily scalable to the throughput needed to fully understand the principles that govern Cas9 specificity. Here we describe ‘circularization for high-throughput analysis of nuclease genome-wide effects by sequencing’ (CHANGE-seq), a scalable, automatable tagmentation-based method for measuring the genome-wide activity of Cas9 in vitro. We applied CHANGE-seq to 110 sgRNA targets across 13 therapeutically relevant loci in human primary T-cells and identified 201,934 off-target sites, enabling the training of a machine learning model to predict off-target activity. Comparing matched genome-wide off-target, chromatin modification and accessibility, and transcriptional data, we found that cellular off-target activity was two to four times more likely to occur near active promoters, enhancers, and transcribed regions. Finally, CHANGE-seq analysis of 6 targets across 8 individual genomes revealed that human single-nucleotide variation had significant effects on activity at ~15.2% of off-target sites analyzed. CHANGE-seq is a simplified, sensitive, and scalable approach to understanding the specificity of genome editors.

          Related collections

          Most cited references48

          • Record: found
          • Abstract: found
          • Article: found
          Is Open Access

          Trimmomatic: a flexible trimmer for Illumina sequence data

          Motivation: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. Results: The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. Availability and implementation: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic Contact: usadel@bio1.rwth-aachen.de Supplementary information: Supplementary data are available at Bioinformatics online.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: found
            Is Open Access

            Fast and accurate short read alignment with Burrows–Wheeler transform

            Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals. Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ∼10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package. Availability: http://maq.sourceforge.net Contact: rd@sanger.ac.uk
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              FLASH: fast length adjustment of short reads to improve genome assemblies.

              Next-generation sequencing technologies generate very large numbers of short reads. Even with very deep genome coverage, short read lengths cause problems in de novo assemblies. The use of paired-end libraries with a fragment size shorter than twice the read length provides an opportunity to generate much longer reads by overlapping and merging read pairs before assembling a genome. We present FLASH, a fast computational tool to extend the length of short reads by overlapping paired-end reads from fragment libraries that are sufficiently short. We tested the correctness of the tool on one million simulated read pairs, and we then applied it as a pre-processor for genome assemblies of Illumina reads from the bacterium Staphylococcus aureus and human chromosome 14. FLASH correctly extended and merged reads >99% of the time on simulated reads with an error rate of <1%. With adequately set parameters, FLASH correctly merged reads over 90% of the time even when the reads contained up to 5% errors. When FLASH was used to extend reads prior to assembly, the resulting assemblies had substantially greater N50 lengths for both contigs and scaffolds. The FLASH system is implemented in C and is freely available as open-source code at http://www.cbcb.umd.edu/software/flash. t.magoc@gmail.com.
                Bookmark

                Author and article information

                Journal
                9604648
                20305
                Nat Biotechnol
                Nat Biotechnol
                Nature biotechnology
                1087-0156
                1546-1696
                8 June 2020
                15 June 2020
                November 2020
                15 December 2020
                : 38
                : 11
                : 1317-1327
                Affiliations
                [1 ]Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USA
                [2 ]Computational Biology Department, School of Computer Science, Carnegie Mellon University, Pittsburgh, PA, USA
                [3 ]Roche Sequencing & Life Science, Roche Diagnostics, Indianapolis, IN, USA
                [4 ]Present address: Department of Human Nutrition, Food and Animal Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI, USA
                [5 ]National Institute of Standards and Technology, Gaithersburg, MD, USA
                [6 ]Department of Bone Marrow Transplantation & Cellular Therapy, St. Jude Children’s Research Hospital, Memphis, TN, USA
                [7 ]Physical Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA
                [8 ]Maryland NanoCenter, University of Maryland, College Park, MD, USA
                Author notes

                Author Contributions

                C.R.L. and S.Q.T. conceived of and designed the study. C.R.L., N.L.M, G.L., E.C., Y.H., X.L., K.J., V.K., N.G.K., E.S., and C.T.P. performed experiments. Y.L., Y.Y., R.Z., Y.H., performed computational analyses. S.M., G.K., J.M., Y.C., and S.Q.T. supervised the project. C.R.L. and S.Q.T. wrote the paper with input from all authors.

                Article
                NIHMS1591991
                10.1038/s41587-020-0555-7
                7652380
                32541958
                3c78591a-8988-480a-9bf2-a540f5b0e3f2

                Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                Categories
                Article

                Biotechnology
                Biotechnology

                Comments

                Comment on this article