Abstract
This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR–Cas9 (clustered regularly interspaced short palindromic repeats–CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.
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Acknowledgements
We thank A. Fossum (BRIC), C.K. Beier Holden, C. Andersen, and S. Narimatsu (Copenhagen Center for Glycomics), and A. Dürr (the Danish Stem Cell Center) for excellent technical assistance. We thank S. Miller, Washington University, for critical reading of the manuscript. This work was supported by the Danish Cancer Society (R124-A7632-15-S2 to M.F.), the Danish Council for Independent Research (0602-02368B to M.F.), the University of Copenhagen Excellence Programme for Interdisciplinary Research—CDO2016 (to M.F., E.P.B. and H.H.W.), the Novo Nordisk Foundation (to M.F., Y.N. and Z.Y., and NNF15CC0018344 to E.A.O.), the Lundbeck Foundation (R165-2013-15743 to F.N.), the National Institutes of Health (R21CA184656 to S.H.H.), the Medical Research Council UK (U117581329 to E.A.O.) and the Danish National Research Foundation (DNRF107 to E.P.B., H.H.W., Y.N., Z.Y. and H.C.).
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All authors were involved in designing and/or performing experiments underlying the manuscript or the development of the methods described. L.A.L., E.P.B. and M.F. wrote the manuscript with comments from the other authors.
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E.P.B. declares competing financial interests (see the HTML version of this article for details). E.P.B. possesses the rights to a patent application describing the IDAA methodology.
Integrated supplementary information
Supplementary Figure 1 Sensitivity and quantitative performance of IDAA performed in an ABI 3500 instrument is comparable to Next-Generation Sequencing (MiSeq).
Indel analysis by (a) IDAA or (b) MiSeq of PCR amplicons from the Cosmc locus edited by CRISPR/Cas9 in a CHO cell pool. On the x-axis, indel sizes (bp) are indicated for some of the amplicons. In the zoom-in panels, a stippled line indicates the background signal level, defined based on the signals from 45-55 bp insertions that were considered background, since CRISPR/Cas9 rarely elicits insertions of such sizes. (c) Graphs of frequencies of indels a-m from the IDAA and MiSeq analyses. Note the high degree of agreement between the two analyses.
Supplementary Figure 2 IDAA can generate useful indel profiles from minute amounts of IDAA PCR amplicons.
The Trp53 locus was targeted with CRISPR/Cas9 in a mouse Neuro2A cell pool and the target site was amplified by IDAA PCR. The indicated amounts of the IDAA PCR were analyzed by (a) agarose gel electrophoresis (an arrow indicates the IDAA PCR amplicon) or (b) IDAA capillary electrophoresis. The size and frequency of selected indels are indicated. IDAA was performed in an ABI 3500 instrument.
Note that the indel profiles are almost identical across the range of IDAA amplicons analyzed and that a high-quality profile can be generated from amplicon amounts barely visible or undetectable by agarose gel analysis.
Supplementary Figure 3 Upper and lower detection levels for IDAA amplicons in ABI 3130 and 3500 instruments.
(a) Various amounts of a wt IDAA amplicon from a non-edited control sample were analyzed in ABI 3130 and 3500 instruments. When the loaded amplicons exceed the dynamic range, a smaller peak artifact often appears 20-30 bp ahead of the true signal (indicated by asterisk in the upper 3130 panel). (b) Zoom in on the peaks boxed in (a). The lower 3130 panel illustrates that also below the dynamic range, a specific signal can be discriminated from background signals.
Supplementary Figure 4 IDAA reveals the indel signature of a given gRNA, TALEN or ZFN.
IDAA profiles for two independent experiments using the same construct of FP-linked CRISPR/Cas9 (a,b), TALENs (c) or ZFNs (d), targeting various loci in K562 or HEK293 cells, as indicated. Prior to analysis, the nuclease-transfected cells were subjected to bulk FACS for the top 30% most fluorescent cells. The size and frequency of selected indels are indicated. IDAA profiles were generated in an ABI 3130 instrument.
Supplementary Figure 5 Using IDAA to estimate probability of germline transmission of indels from F0 mosaic fish and to identify F1 mutant fish in Danio rerio genome editing.
(a) In zebrafish genome editing, a major challenge is determination of the nature and degree of indel mosaicism and hence, probability of germline transmission of indels from F0 fish derived from one-cell embryos injected with Cas9 and gRNA. IDAA enables easy and rapid evaluation of these variables through analysis of F0 fin DNA, allowing optimal set-up of F0 breeding pairs. IDAA also enables easy identification of indels in subsequent generations through fin DNA analysis. (b) Somatic IDAA profiles of the bambi locus in fin DNA of F0 fish targeted at the one-cell embryo stage via injection of Cas9 and gRNA. F0 male #1 and F0 female #4 were chosen for mating due to the presence of predominant indels indicated by open or closed circles, suggesting high probability of germline transmission of these mutations. (c) IDAA profiles of 200 downstream F1 fish, showing that 4 out of 5 fish harbored the predominant indels identified in the F0 breeding pair, of which 3 were in the biallelic state (F1 #1, #3, #4) and one homozygous for an indel (F1 #4). In this example, IDAA was performed on embryos, but IDAA could also have been performed on fin DNA. The size and frequency of selected indels are indicated. IDAA profiles were generated in an ABI 3130 instrument.
Supplementary Figure 6 QCgRNA amplicon expression cassettes.
(a) Schematic showing the various elements of the QCgRNA amplicon primers QCgFwd, QCgX and QCgRev. Sequences annealing to U6 promotor template are shown in orange; gRNA design in red; tracr elements in blue; restriction enzyme sites for sub-cloning to pEPB104 in italics. Note that the gRNA design is incorporated into QCgX as the complementary sequence to the target, which is shown in green. The nucleotide (c) is only included in the QCgX primer, if the gRNA (=target) does not contain a G as the first (5´) nucleotide, which is the case in the present example. (b) Agarose gel (2%) electrophoresis check for the formation of full-length (f) products (arrow) by QCgRNA amplicon tri-primer PCRs, as compared to control (c) PCRs containing only QCgFwd and QCgX primers (Step 3 in Procedure). Various amounts of MassRuler Low Range DNA ladder (M) are run alongside to enable quantitation of the QCgRNA amplicons. (c) The indel profiles elicited by a gRNA design in a QCgRNA amplicon and a plasmid vector are identical, as illustrated by targeting GALNT10 in HEK293 cells. The GALNT10 QCgRNA amplicon was subcloned into pEPB104 plasmid (Addgene #68369; Supplementary Sequence 1) using EcoRI and KpnI restriction endonuclease sites present in both constructs. (d) A QCgRNA design found non-functional in one cell type typically remains non-functional when tested in other cell types, as illustrated with a POMT2 QCgRNA. IDAA profiles were generated in an (c) ABI 3500 or and (d) ABI 3130 instrument.
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Lonowski, L., Narimatsu, Y., Riaz, A. et al. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis. Nat Protoc 12, 581–603 (2017). https://doi.org/10.1038/nprot.2016.165
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DOI: https://doi.org/10.1038/nprot.2016.165
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