Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats) genome-editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep-sequencing data resulting from genome-editing experiments. However, these tools are typically developed in isolation and oftentimes are not readily translatable into laboratory-based experiments. Here, we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome-editing experiment. This protocol provides instructions for sgRNA design with CRISPOR (computational tool for the design, evaluation, and cloning of sgRNA sequences), experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso (computational tool for analysis of genome-editing outcomes from deep-sequencing data). This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4–5 weeks by non-experts, as well as computational data analysis that can be performed in 1–2 d by both computational and noncomputational biologists alike using web-based and/or command-line versions.

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Figure 1: Schematic of an arrayed genome-editing experiment.
Figure 2: Schematic of a pooled genome-editing experiment.
Figure 3: Design, experimental execution, and data analysis workflows for arrayed and pooled genome-editing experiments.
Figure 4: Locus-specific deep-sequencing analysis of coding and noncoding targeting by CRISPResso.

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Acknowledgements

M.C.C. was supported by a National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Award (F30DK103359). M.H. was funded by National Institutes of Health (NIH)/National Human Genome Research Institute (NHGRI) grant 5U41HG002371-15 and NIH/National Cancer Institute (NCI) grant 5U54HG007990-02 and by a grant from the California Institute of Regenerative Medicine, CIRM GC1R-06673C. D.E.B. was supported by the National Heart, Lung, and Blood Institute (NHLBI) (DP2OD022716, P01HL032262), the Burroughs Wellcome Fund, and a Doris Duke Charitable Foundation Innovations in Clinical Research Award. S.H.O. was supported by an award from the NHLBI (P01HL032262) and an award from the NIDDK (P30DK049216, Center of Excellence in Molecular Hematology). N.E.S. was supported by the NIH through the NHGRI (R00-HG008171). L.P. was supported by an NHGRI Career Development Award (R00HG008399) and the Defense Advanced Research Projects Agency (HR0011-17-2-0042).

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Authors

Contributions

M.C.C., M.H., and L.P. conceived this project. M.H. and J.-P.C. created CRISPOR. L.P., M.C.C., D.E.B., and G.-C.Y. created CRISPResso. M.C.C. and D.E.B. performed the experiments. M.C.C., D.E.B., S.H.O., N.E.S., O.S., G.-C.Y., F.Z., and L.P. analyzed the experimental data. M.C.C., M.H., and L.P. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Luca Pinello.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Screenshot of sharing disk volumes and allocating memory with Docker containers.

a, Screenshot using Docker to ensure that the drive(s) you want to be available to the container is/are checked (under Settings…/ Shared Drives). b, Screenshot using Docker to allocate enough memory to the container (under Settings…/ Advanced).

Supplementary Figure 2 Pooled sgRNA library preparation and analysis.

a, Representative results for lsPCR1. b, Representative results for lsPCR2. c, Gradient PCR for lentiGuide-Puro-specific primers or locus-specific primers for laPCR1. d, Representative results for laPCR2.

Supplementary Figure 3 Example amplicon and regions description files.

a, Example of a properly formatted amplicon description file. This file is a tab delimited text file with up to 5 columns (first 2 columns required). No column heading is required. b, Example of a properly formatted regions description file. This file is a tab delimited text file with up to 7 columns (4 required) and contains the coordinates of the regions to analyze and some additional information. No column heading is required.

Supplementary Figure 4 Visualization of the distribution of identified alleles generated from targeting BCL11A exon 2.

Nucleotides are indicated by unique colors (A = green; C = red; G = yellow; T = purple). Substitutions are shown in bold font. Red rectangles highlight inserted sequences. Horizontal dashed lines indicate deleted sequences. The vertical dashed line indicates the predicted double-strand break position.

Supplementary Figure 5 Direct comparison of BCL11A exon 2 sequence between a BCL11A exon 2 targeted sgRNA sample (“edited”) and a non-edited control sample (“non-edited”).

a, Distribution of editing outcomes (unmodified, NHEJ, HDR, and mixed alleles) for treated (edited) and control (non- edited) samples. b, Comparison of the percent different editing outcomes (unmodified, NHEJ, HDR, and mixed alleles) between the treated (edited) and control (non-edited) samples. c, Combined (substitutions/deletions/insertions) mutation position distribution for treated (edited) and control (non- edited) samples. The vertical dashed line indicates the position of predicted Cas9 cleavage. The position of the sgRNA is shown in gray. d, Comparison of the percent different combined mutations (substitutions/deletions/insertions) between the treated (edited) and control (non-edited) samples. The vertical dashed line indicates the position of predicted Cas9 cleavage. The position of the sgRNA is shown in gray.

Supplementary information

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Supplementary Figures 1–5 and Supplementary Tables 1–8. (PDF 1462 kb)

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Canver, M., Haeussler, M., Bauer, D. et al. Integrated design, execution, and analysis of arrayed and pooled CRISPR genome-editing experiments. Nat Protoc 13, 946–986 (2018). https://doi.org/10.1038/nprot.2018.005

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