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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
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.

1932-6203
Dillon, James
f406e30a-3ad4-4a53-80db-6694bab5e3ed
Rawsthorne-Manning, Helena
17228f7b-6d7b-4f73-9dbc-6849c3f1f496
Dillon, James
f406e30a-3ad4-4a53-80db-6694bab5e3ed
Rawsthorne-Manning, Helena
17228f7b-6d7b-4f73-9dbc-6849c3f1f496

Dillon, James and Rawsthorne-Manning, Helena (2022) Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes. PLoS ONE, 17 (1 January), [e0253351]. (doi:10.1371/journal.pone.0253351).

Record type: Article

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.

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Accepted/In Press date: 29 October 2021
Published date: 18 January 2022

Identifiers

Local EPrints ID: 456266
URI: http://eprints.soton.ac.uk/id/eprint/456266
ISSN: 1932-6203
PURE UUID: b12a2008-4b4e-4cd0-a591-ff8d36f8d8da
ORCID for James Dillon: ORCID iD orcid.org/0000-0003-3244-7483

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Date deposited: 27 Apr 2022 00:57
Last modified: 28 Apr 2022 01:52

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Contributors

Author: James Dillon ORCID iD
Author: Helena Rawsthorne-Manning

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