Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes
Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes
Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR 'marker' genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, segregating away the co-CRISPR modified gene can be less challenging if the worms selected appear wild type and are selected from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co- CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.
Rawsthorne-Manning, Helena
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Calahorro, Fernando
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Izquierdo, Patricia G.
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Tardy, Philippe
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Boulin, Thomas
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Holden-Dye, Lindy
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O'Connor, Vincent
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Dillon, James
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18 January 2022
Rawsthorne-Manning, Helena
17228f7b-6d7b-4f73-9dbc-6849c3f1f496
Calahorro, Fernando
dddfa373-d3cc-433f-8851-9ca37f2f3950
Izquierdo, Patricia G.
bc166241-fb24-44c1-bf25-c25c384dedbe
Tardy, Philippe
90f5d3bc-9f20-4cc5-94b3-107e2e86e71f
Boulin, Thomas
23be2237-568d-44e6-bbed-c5369de84132
Holden-Dye, Lindy
8032bf60-5db6-40cb-b71c-ddda9d212c8e
O'Connor, Vincent
8021b06c-01a0-4925-9dde-a61c8fe278ca
Dillon, James
f406e30a-3ad4-4a53-80db-6694bab5e3ed
Rawsthorne-Manning, Helena, Calahorro, Fernando, Izquierdo, Patricia G., Tardy, Philippe, Boulin, Thomas, Holden-Dye, Lindy, O'Connor, Vincent and Dillon, James
(2022)
Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes.
PLoS ONE, 17 (1), [e0253351].
(doi:10.1371/journal.pone.0253351).
Abstract
Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR 'marker' genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, segregating away the co-CRISPR modified gene can be less challenging if the worms selected appear wild type and are selected from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co- CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.
Text
Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes
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Accepted/In Press date: 29 October 2021
Published date: 18 January 2022
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© 2022 Rawsthorne-Manning et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Local EPrints ID: 456266
URI: http://eprints.soton.ac.uk/id/eprint/456266
ISSN: 1932-6203
PURE UUID: b12a2008-4b4e-4cd0-a591-ff8d36f8d8da
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Date deposited: 27 Apr 2022 00:57
Last modified: 19 Jun 2025 01:49
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Author:
Helena Rawsthorne-Manning
Author:
Patricia G. Izquierdo
Author:
Philippe Tardy
Author:
Thomas Boulin
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