GuideScan software for improved single and paired CRISPR guide RNA design

Perez, Almudena Chaves; Pritykin, Yuri; Vidigal, Joana A.; Chhangawala, Sagar; Zamparo, Lee; Leslie, Christina; Ventura, Andrea

doi:10.1038/nbt.3804

Cited by 218 publications

(203 citation statements)

References 25 publications

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“…Even though the number of reads on target was sufficient for analyzing the repeats in this experimental setup, there was a high background of reads derived from off‐target Cas9 activity or by unspecific pulldown of SMRTbell molecules not cleaved by Cas9. Computational gRNA design tools that allows for selection of gRNAs with minimal off‐targets sites (Heigwer, Kerr, & Boutros, ; Naito, Hino, Bono, & Ui‐Tei, ; Perez et al., ) could result in a more sensitive assay. Also, it would be interesting to evaluate high‐fidelity Cas9 enzymes that have proven to decrease off‐target effects while retaining on‐target activity (Kleinstiver et al., ).…”

Section: Discussionmentioning

confidence: 99%

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing

et al. 2018

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Amplification of DNA is required as a mandatory step during library preparation in most targeted sequencing protocols. This can be a critical limitation when targeting regions that are highly repetitive or with extreme guanine–cytosine (GC) content, including repeat expansions associated with human disease. Here, we used an amplification‐free protocol for targeted enrichment utilizing the CRISPR/Cas9 system (No‐Amp Targeted sequencing) in combination with single molecule, real‐time (SMRT) sequencing for studying repeat elements in the huntingtin (HTT) gene, where an expanded CAG repeat is causative for Huntington disease. We also developed a robust data analysis pipeline for repeat element analysis that is independent of alignment of reads to a reference genome. The method was applied to 11 diagnostic blood samples, and for all 22 alleles the resulting CAG repeat count agreed with previous results based on fragment analysis. The amplification‐free protocol also allowed for studying somatic variability of repeat elements in our samples, without the interference of PCR stutter. In summary, with No‐Amp Targeted sequencing in combination with our analysis pipeline, we could accurately study repeat elements that are difficult to investigate using PCR‐based methods.

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Section: Discussionmentioning

confidence: 99%

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing

et al. 2018

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“…For this purpose we used one of the available bioinformatics tool (see section 2.3.1) that provides a list of potential target sites with a score indicating the faithfulness (i.e., on-target activity) and the predicted number of off-target sites. Other software, which uses different algorithms, e.g., Cas-OFFinder [39] and GuideScan [40], is also available for the design of gRNAs. (For a more complete list, see [41]).…”

Section: Discussionmentioning

confidence: 99%

Generation of chromosomal translocations that lead to conditional fusion protein expression using CRISPR-Cas9 and homology-directed repair

Vanoli

Jasin

2017

Methods

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Recurrent chromosomal translocations often lead to expression of fusion proteins associated with oncogenic transformation. To study translocations and downstream events, genome editing techniques have been developed to generate chromosomal translocations through non-homologous end joining of DNA double-strand breaks introduced at the two participating endogenous loci. However, the frequencies at which these events occur is usually too low to efficiently clone cells carrying the translocation. This article provides a detailed method using CRISPR-Cas9 technology and homology- directed repair to efficiently isolate cells harboring a chromosomal translocation. For an additional level of control, the resulting fusion protein is conditionally expressed to allow early events in oncogenic transformation to be studied. We focus on the generation of the EWSR1-WT1 fusion using human mesenchymal cells, which is associated with the translocation found in desmoplastic small round cell tumors.

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“…Overall, off-targets can be minimized by iterating a process of: 1) selecting specific sgRNAs (Doench et al, 2016; Perez et al, 2017; Scott and Zhang, 2017), 2) employing optimized Cas9 variants and delivery methods, 3) detecting mutations with unbiased genome-wide methods, and 4) validating off-target edits with sensitive targeted sequencing.…”

Section: Figurementioning

confidence: 99%

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

et al. 2017

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The past several years have seen an explosion in development of applications for the CRISPR-Cas9 system, from efficient genome editing, to high-throughput screening, to recruitment of a range of DNA and chromatin-modifying enzymes. While homology-directed repair (HDR) coupled with Cas9 nuclease cleavage has been used with great success to repair and re-write genomes, recently developed base editing systems present a useful orthogonal strategy to engineer nucleotide substitutions. Base editing relies on recruitment of cytidine deaminases to introduce changes (rather than double stranded breaks and donor templates), and offers potential improvements in efficiency while limiting damage and simplifying the delivery of editing machinery. At the same time, these systems enable novel mutagenesis strategies to introduce sequence diversity for engineering and discovery. Here, we review the different base editing platforms, including their deaminase recruitment strategies and editing outcomes, and compare them to other CRISPR genome editing technologies. Additionally, we discuss how these systems have been applied in therapeutic, engineering, and research settings. Lastly, we explore future directions of this emerging technology.

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GuideScan software for improved single and paired CRISPR guide RNA design

Cited by 218 publications

References 25 publications

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing

Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing

Generation of chromosomal translocations that lead to conditional fusion protein expression using CRISPR-Cas9 and homology-directed repair

Methods and Applications of CRISPR-Mediated Base Editing in Eukaryotic Genomes

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