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.
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DOI：
10.1371/journal.pone.0253351
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
Dillon J
中科院分区：
文献类型：
--
作者：
Rawsthorne-Manning H;Calahorro F;G Izquierdo P;Tardy P;Boulin T;Holden-Dye L;O'Connor V;Dillon J
利用模式遗传生物秀丽隐杆线虫（C. elegans），在提高CRISPR/Cas9编辑效率方面取得了多项进展。在这里，我们报告了co-CRISPR“标记”基因的使用：发生co-CRISPR事件的蠕虫具有明显的可见表型，这有助于选择在靶基因中含有CRISPR事件的蠕虫。然后通过与野生型的异交去除co-CRISPR基因的突变，但如果CRISPR和co-CRISPR基因难以分离，这可能是具有挑战性的。然而，如果选择的蠕虫是野生型的，并且是从一窝中奖的幼崽中挑选出来的，那么分离出co-CRISPR修饰基因就不那么困难了。在这些卵群中，单个注射的蠕虫的后代中有很高比例显示出co-CRISPR表型，这表明CRISPR效率很高。这可以在没有共crispr突变的情况下，在目标基因位点上携带所需突变的蠕虫。我们已经成功地在秀丽隐杆线虫nlg-1基因使用这种方法产生离散突变。然而，在测序以验证nlg-1基因编辑的过程中，我们发现在co-CRISPR基因unc-58上出现的基因组重排，通过视觉观察，这种重排在表型上是沉默的，但仍然导致通过拍打行为评分的运动性显着降低。这突出表明，在对基因功能进行下游分析之前，应仔细考虑co-CRISPR介导的遗传变化的潜在后果。鉴于此，我们建议在利用表型选择作为管道一部分的CRISPR程序之后对co-CRISPR基因进行测序。
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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DOI：
10.1534/g3.117.040824
发表时间：
2017-05-05
期刊：
G3 (Bethesda, Md.)
影响因子：
--
作者：
El Mouridi S;Lecroisey C;Tardy P;Mercier M;Leclercq-Blondel A;Zariohi N;Boulin T
通讯作者：
Boulin T
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
16.6
作者：
Ben Soussia, Ismail;El Mouridi, Sonia;Boulin, Thomas
通讯作者：
Boulin, Thomas
影响因子：
3.9
作者：
Mojica, FJM;Díez-Villaseñor, C;Soria, E
通讯作者：
Soria, E