Effect of Different Caenorhabditis elegans U6 Promoters on the Efficiency of CRISPR/Cas9-Mediated Gene Editing

FENG Lixiang, HUANG Ying, ZHAO Rongqian, ZHANG Kui, YANG Wenxing

Abstract


Objective 

To investigate the effects of Caenorhabditis elegans (C. elegans) endogenous U6 promoters on dpy-10 gene editing efficiency.

Methods 

We screened endogenous U6 small nuclear RNA (snRNA) genes of C. elegans from the WormBase database and constructed 14 editing plasmids targeting dpy-10 by replacing the U6r07e5.16 promoter in the pSX524 plasmid (Peft-3::cas9::tbb-2 terminator::U6 r07e5.16::dpy-10 sgRNA) through molecular cloning. Gene editing was performed in wild-type C. elegans using a standardized microinjection protocol. Gene editing efficiency and the high-efficiency gene editing index were quantified based on the screening of dpy-10 mutant phenotypes in the F1 progeny.

Results 

A total of 15 U6 snRNA genes (r07e5.16, f35c11.9, t20d3.13, k09b11.15, k09b11.16, w05b2.8, c28a5.7, f54c8.9, k09b11.11, k09b11.12, k09b11.14, t20d3.12, f54c8.8, f54c8.10, and k09b11.13) were identified from the WormBase database. Based on the editing efficiency and high-efficiency gene editing index, the activity of these promoters was evaluated, and 4 U6 promoters (w05b2.8, c28a5.7, f54c8.9, and k09b11.11) were found to have significantly enhanced gene editing success rates, outperforming other promoters, including U6r07e5.16 and U6k09b11.12, which are commonly used in the C. elegans research community. Notably, the gRNAF+E scaffold did not show superior editing efficiency over the gRNA scaffold when paired with the optimal U6w05b2.8 promoter.

Conclusion 

In this study, U6 promoters that significantly improve gene editing efficiency in C. elegans are identified and the critical role of promoter optimization in CRISPR-Cas9 systems is highlighted. These findings provide a valuable foundation for improving genome editing strategies and offer new ideas for optimizing the CRISPR technology applied in nematode research.

 

Keywords: Caenorhabditis elegans, U6 promoter, CRISPR/Cas9, Gene editing, Efficiency

 

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References


DICKINSON D J, WARD J D, REINER D J, et al. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods, 2013, 10(10): 1028-1034. doi: 10.1038/nmeth.2641.

FRIEDLAND A E, TZUR Y B, ESVELT K M, et al. Heritable genome editing in C.elegans via a CRISPR-Cas9 system. Nat Methods, 2013, 10(8): 741-743. doi: 10.1038/nmeth.2532.

CHIU H, SCHWARTZ H T, ANTOSHECHKIN I, et al. Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas. Genetics, 2013, 195(3): 1167-1171. doi: 10.1534/genetics.113.155879.

LO T W, PICKLE C S, LIN S, et al. Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics, 2013, 195(2): 331-348. doi: 10. 1534/genetics.113.155382.

SHEN B, ZHANG J, WU H, et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res, 2013, 23(5): 720-723. doi: 10.1038/cr.2013.46.

NIU Y, SHEN B, CUI Y, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell, 2014, 156(4): 836-843. doi: 10.1016/j.cell.2014.01.027.

BASSETT A R, TIBBIT C, PONTING C P, et al. Highly efficient targeted mutagenesis of drosophila with the CRISPR/Cas9 system. Cell Rep, 2013, 4(1): 220-228. doi: 10.1016/j.celrep.2013.06.020.

CHO S W, LEE J, CARROLL D, et al. Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics, 2013, 195(3): 1177-1180. doi: 10.1534/genetics.113.155853.

PAIX A, FOLKMANN A, RASOLOSON D, et al. High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes. Genetics, 2015, 201(1): 47- 54. doi: 10.1534/genetics.115.179382.

CHEN B, GILBERT L A, CIMINI B A, et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell, 2013, 155(7): 1479-1491. doi: 10.1016/j.cell.2013.12.001.

YANG W, WU T, TU S, et al. Redundant neural circuits regulate olfactory integration. PLoS Genet, 2022, 18(1): e1010029. doi: 10.1371/journal.pgen.1010029.

WANG Y, SUN X, FENG L, et al. Nervous system guides behavioral immunity in Caenorhabditis elegans. PeerJ, 2024, 12: e18289. doi: 10.7717/peerj.18289.

KERN C C, SRIVASTAVA S, EZCURRA M, et al. C.elegans ageing is accelerated by a self-destructive reproductive programme. Nat Commun, 2023, 14(1): 4381. doi: 10.1038/s41467-023-40088-1.

ZHANG J, YANG W, LI Z, et al. Multigenerational exposure of cadmium trans-generationally impairs locomotive and chemotactic behaviors in Caenorhabditis elegans. Chemosphere, 2023, 325: 138432. doi: 10.1016/j. chemosphere.2023.138432.

FARBOUD B, MEYER B J. Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design. Genetics, 2015, 199(4): 959-971. doi: 10.1534/genetics.115.175166.

KATIC I, XU L, CIOSK R. CRISPR/Cas9 genome editing in Caenorhabditis elegans: evaluation of templates for homology-mediated repair and knock-ins by homology-independent dna repair. G3 (Bethesda), 2015, 5(8): 1649-1656. doi: 10.1534/g3.115.019273.

WARD J D. Rapid and precise engineering of the Caenorhabditis elegans genome with lethal mutation co-conversion and inactivation of NHEJ repair. Genetics, 2015, 199(2): 363-377. doi: 10.1534/genetics.114.172361.

ARRIBERE J A, BELL R T, FU B X, et al. Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics, 2014, 198(3): 837-846. doi: 10.1534/genetics.114. 169730.

KIM H, ISHIDATE T, GHANTA K S, et al. A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans. Genetics, 2014, 197(4): 1069-1080. doi: 10.1534/genetics.114.166389.

SMITH A, BERGWELL M, SMITH E, et al. CRISPR/Cas9 editing of the C. elegans rbm-3. 2 gene using the dpy-10 Co-CRISPR screening marker and assembled ribonucleoprotein complexes. J Vis Exp, 2020, 166: e62001. doi: 10.3791/62001.

TOKER I A, HOBERT O. The Cbr-DPY-10(Arg92Cys) modification is a reliable co-conversion marker for CRISPR/Cas9 genome editing in Caenorhabditis briggsae. microPubl Biol, 2022, 2022: 10.17912/micropub. biology.000554. doi: 10.17912/micropub.biology.000554.

LEVY A D, YANG J, KRAMER J M. Molecular and genetic analyses of the Caenorhabditis elegans dpy-2 and dpy-10 collagen genes: a variety of molecular alterations affect organismal morphology. Mol Biol Cell, 1993, 4(8): 803-817. doi: 10.1091/mbc.4.8.803.

LI J, QIN Y, SHEN C, et al. A new miniMOS tool kit capable of visualizing single copy insertion in C.elegans. PeerJ, 2023, 11: e15433. doi: 10.7717/peerj.15433.

JINEK M, CHYLINSKI K, FONFARA I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012, 337(6096): 816-821. doi: 10.1126/science.1225829.

FROEHLICH J J, UYAR B, HERZOG M, et al. Parallel genetics of regulatory sequences using scalable genome editing in vivo. Cell Rep, 2021, 35(2): 108988. doi: 10.1016/j.celrep.2021.108988.


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