Materials and Methods

Confounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes

C. elegans culturing and strains used

All C. elegans strains were maintained using standard conditions (Brenner, 1974). Strains used: Bristol N2 (wild-type), VC228 nlg-1(ok259) X (x6 outcrossed), CB665 unc-58(e665) X, TN64 dpy-10(cn64) II, provided by Caenorhabditis Genetics Center (CGC). XA3780 nlg-1(qa3780) X (x2 outcrossed), XA3773 unc-58(qa3788) X; nlg-1(qa3780) X (x1 outcrossed), XA3788 unc-58(qa3788) X (x2 outcrossed), generated in this study. JIP1154 unc-58(bln223) X, provided by Thomas Boulin.

CRISPR/Cas9 genome editing

The previously reported method by El Mouridi et al was followed for CRSIPR/Cas9 editing (El Mouridi et al., 2017). L4+1 day old hermaphrodites were microinjected with expression vectors for Cas9 and sgRNAs targeting nlg-1 and unc-58 or dpy-10 for the first and second round of CRISPR, respectively. The design of the sgRNAs and repair templates to edit unc-58, dpy-10 and nlg-1 genes were as previously reported (Rawsthorne et al., 2020; Arribere et al., 2014). sgRNAs and repair templates were purchased from Integrated DNA Technologies (IDT™ – Integrated DNA Technologies). All injection reagents were diluted in molecular grade water to a final concentration of 50ng/µl in the CRISPR mix.

Molecular Screening and sequencing

nlg-1 primers, forward 5’-ATGAGTATACAGATTGGGAAAATCCC-3’ and reverse 5’-ACTGTTTGGTTGCTCTTGGCTCCAAG-3’, were used to amplify the CRISPR targeted region of nlg-1 using a single worm PCR protocol (He, 2011). A BanI site was used to screen PCR amplicons from individual worms for the incorporation or loss of the d10 sequence in nlg-1. The primers used for sequencing of the target regions of unc-58 and dpy-10 were: forward 5’-GACTCGGAGATATCGTTGTGACTG-3’, reverse 5’-CGCGGAGTTCGTTATCCAGGAAG-3’ and forward 5’-ACTAATTCAGAGTCATCATCTCGCC-3’, reverse 5’-CATCAATTCCCTTAAGTCCTGGTGG-3’ respectively.

Removal of unc-58 background mutation

unc-58(qa3788); nlg-1(qa3780) double CRISPR mutant strain was backcrossed with N2 males once to generate heterozygous F1 progeny. These progeny were cloned by picking a single F1 onto individual plates before being left for 2-3 days to self-fertilise and F2 progeny were screened using a single worm PCR protocol (He, 2011) and restriction digest. Restriction digest of unc-58 and nlg-1 PCR amplicons were performed using restriction enzymes BsiWI and BstBI respectively to screen for the presence or absence of CRISPR mediated edits. Restriction enzymes were supplied by New England BioLabs (NEB) and used according to manufacturer instructions.

Food leaving assay

Food leaving assays were carried out as previously described (Rawsthorne et al., 2020). Briefly, 50µl of OP50 E. coli at OD600 of 0.8 was gently spotted on to the middle of an unseeded plate the day prior to the assay. Seven age synchronised L4+1 day old hermaphrodites were gently picked onto the centre of the bacterial lawn on the assay plate. At 2 and 24 hours food leaving events were counted visually using a Nikon SMZ800 microscope (X10 magnification) during 30 minute observations. A food leaving event was defined as when the whole of the worm’s body exited the bacterial lawn. N2 animals were used as a paired control, run in parallel with the strain under investigation and the investigator was blind to the genotypes being observed.

Thrashing assay

Using a 24 well plate, a single worm was picked per well containing 500µl of M9 with 0.1% bovine serum albumin and left for 5 minutes before thrashing was measured. For each worm, thrashing events were visually counted under a Nikon SMZ800 microscope (X30 magnification) for a period of 30 seconds. This was repeated three consecutive times and the mean was calculated. Each thrash was defined as a complete movement through the midpoint of the worms body and back. N2 animals were used as a paired control, run in parallel with the strain under investigation and the investigator was blind to the genotypes.

unc-58 protein sequence analysis

UNC-58, isoform b protein sequence was downloaded from WormBase version WS278. The wild-type or CRISPR mutant protein sequence was entered into the membrane topology prediction tool TMHMM (v.2.0) (Sonnhammer, von Heijne and Krogh, 1998; Krogh et al., 2001) to determine the predicted transmembrane topology.

Article TitleConfounds of using the unc-58 selection marker highlights the importance of genotyping co-CRISPR genes

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, outcrossing can be avoided by selecting worms of wild type appearance 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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