Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans

Paix, Alexandre; Folkmann, Andrew W.; Seydoux, Géraldine

doi:10.1016/j.ymeth.2017.03.023

Cited by 228 publications

(226 citation statements)

References 24 publications

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“…2A). To test this further, using genome editing (Paix et al, 2017), we 106 regenerated the meg-4 deletion in a line carrying the meg-3 deletion to generate three new meg-3 meg-107 4 lines (meg-3 meg-4 #2 , meg-3 meg-4 #3 , and meg-3 meg-4 #4 ). Strikingly, we found that the newly 108 generated meg-3 meg-4 lines remained competent for RNAi for at least five generations before 109 beginning to exhibit resistance.…”

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confidence: 99%

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P granules protect RNA interference genes from silencing by piRNAs

Ouyang

Folkmann

Bernard

et al. 2019

Preprint

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15P granules are perinuclear condensates in C. elegans germ cells proposed to serve as hubs for 16 self/non-self RNA discrimination by Argonautes. We report that a mutant (meg-3 meg-4) that does not 17 assemble P granules in primordial germ cells loses competence for RNA-interference over several 18 generations and accumulates silencing small RNAs against hundreds of endogenous genes, including the 19 RNA-interference genes rde-11 and sid-1. In wild-type, rde-11 and sid-1 transcripts are heavily targeted 20 by piRNAs, accumulate in P granules, but maintain expression. In the primordial germ cells of meg-3 21 meg-4 mutants, rde-11 and sid-1 transcripts disperse in the cytoplasm with the small RNA biogenesis 22 machinery, become hyper-targeted by secondary sRNAs, and are eventually silenced. Silencing requires 23 the PIWI-class Argonaute PRG-1 and the nuclear Argonaute HRDE-1 that maintains trans-generational 24 silencing of piRNA targets. These observations support a "safe harbor" model for P granules in 25 protecting germline transcripts from piRNA-initiated silencing. 26 27In the germ cells of animals, dense RNA-protein condensates accumulate on the cytoplasmic 29 face of the nuclear envelope. These condensates, collectively referred to as nuage, contain components 30 of the small RNA (sRNA) machinery that scan germline transcripts for foreign sequences. For example, in 31Drosophila, components of the piRNA machinery in nuage amplify small RNAs that target transcripts 32 from transposable elements for destruction (Huang et al., 2017). In C. elegans, the PIWI-class Argonaute 33 PRG-1 associates with ~15,000 piRNAs encoded in the genome that scan most, if not all, germline 34 1). Segregation of Argonautes and proteins required for 22G-RNA production into distinct nuage 60 compartments (P granules versus Z granules and mutator foci) could also play a role in sorting 22G-RNAs 61 or limiting their production (Wan et al., 2018). A direct test of these hypotheses, however, has been 62 difficult to obtain as complete loss of P granules causes sterility. 63We previously identified a mutant that affects P granule coalescence only during embryogenesis 64 (Wang et al., 2014). MEG-3 and MEG-4 are intrinsically-disordered proteins present in the germ plasm, a 65 specialized cytoplasm that is partitioned with the germ lineage during early embryonic cleavages (Wang 66 3 and Seydoux, 2013). MEG-3 and MEG-4 form gel-like scaffolds that recruit and stimulate the coalescence 67 of P granule proteins in germ plasm to ensure their partitioning to the embryonic germline and the 68 primordial germ cells Z2 and Z3 (Fig. 1;Putnam et al., 2019). In meg-3 meg-4 embryos, P granules do not 69 coalesce in germ plasm, causing granule components to be partitioned equally to all cells and turned 70 over ( Fig. 1; Wang et al., 2014). Despite lacking P granules during embryogenesis, meg-3 meg-4 71 assemble P granules de novo when the primordial germ cells resume divisions in the first larval stage to 72 generate the ~ 2000 germ cells that constitu...

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confidence: 99%

“…Results 86 meg-3 meg-4 mutants are defective in exogenous RNA-mediated interference 87 JH3475 is a strain in which both the meg-3 and meg-4 open reading frames have been deleted 88 by genome editing (Smith et al, 2016;Paix et al, 2017). This strain (meg-3 meg-4 #1 ) has been passaged 89 over 100 times.…”

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confidence: 99%

P granules protect RNA interference genes from silencing by piRNAs

Ouyang

Folkmann

Bernard

et al. 2019

Preprint

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“…Null, tagged, and mutated versions of gcna-1 were created by CRISPR/Cas9 genome engineering. For null alleles ea43 and linked double mutant lines, 5' gcna-1 crRNA CTC GGG TGA AGC TAT CTA TAC TCA TGT AAT TTT TAT TAT GTA CTA CAC T. Injection mixes containing crRNA, tracrRNA, ssDNA ultramers, and Cas9 protein were prepared as described (Paix et al, 2017). crRNA and ssDNA repair template for dpy-10(cn64) co-injection marker were also included (Arribere et al, 2014).…”

Section: Generation Of C Elegans Crispr Allelesmentioning

confidence: 99%

GCNA preserves genome integrity and fertility across species

Bhargava

Goldstein

Russell

et al. 2019

Preprint

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SUMMARYThe propagation of species depends on the ability of germ cells to protect their genome in the face of numerous exogenous and endogenous threats. While these cells employ a number of known repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. Here, we identify Germ Cell Nuclear Acidic Peptidase (GCNA) as a highly conserved regulator of genome stability in flies, worms, zebrafish, and humans. GCNA contains a long acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, GCNA mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with a second SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit meiotic and replicative damage, most of GCNA’s function maps to its IDR. This work shows GCNA protects germ cells from various sources of damage, providing novel insights into conserved mechanisms that promote genome integrity across generations.HighlightsGCNA ensures genomic stability in germ cells and early embryos across speciesGCNA limits replication stress and DNA double stranded breaksGCNA restricts DNA-Protein Crosslinks within germ cells and early embryosThe IDR and SprT domains of GCNA govern distinct aspects of genome integrityGraphic Abstract

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“…This is in contrast to the HMEJ experiments described above [Yao et al 2017] where long homology arms perform better than short homology arms. A detailed protocol on performing edits of either DNA insertions or deletions using short homology arms has recently been published and contains useful tips [Paix et al 2017b]. …”

Section: Recent Advances For Site-specific Insertion Of Relatively Lamentioning

confidence: 99%

Making ends meet: targeted integration of DNA fragments by genome editing

Yamamoto

Gerbi

2018

Chromosoma

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Targeted insertion of large pieces of DNA is an important goal of genetic engineering. However, this goal has been elusive since classical methods for homology-directed repair are inefficient and often not feasible in many systems. Recent advances are described here that enable site-specific genomic insertion of relatively large DNA with much improved efficiency. Using the preferred repair pathway in the cell of nonhomologous end-joining, DNA of up to several kb could be introduced with remarkably good precision by the methods of HITI and ObLiGaRe with an efficiency up to 30-40%. Recent advances utilizing homology-directed repair (methods of PITCh; short homology arms including ssODN; 2H2OP) have significantly increased the efficiency for DNA insertion, often to 40-50% or even more depending on the method and length of DNA. The remaining challenges of integration precision and off-target site insertions are summarized. Overall, current advances provide major steps forward for site-specific insertion of large DNA into genomes from a broad range of cells and organisms.

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Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans

Cited by 228 publications

References 24 publications

P granules protect RNA interference genes from silencing by piRNAs

P granules protect RNA interference genes from silencing by piRNAs

GCNA preserves genome integrity and fertility across species

Making ends meet: targeted integration of DNA fragments by genome editing

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