Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

Savić, Nataša; Ringnalda, Femke; Lindsay, Helen; Berk, Christian; Bargsten, Katja; Li, Yizhou; Neri, Dario; Robinson, Mark D.; Ciaudo, Constance; Hall, Jonathan; Jínek, Martin; Schwank, Gerald

doi:10.7554/elife.33761

Cited by 133 publications

(116 citation statements)

References 35 publications

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“…While these studies were underway, reports of genetic fusions of Cas9 to avidin, SNAP tag, or porcine circovirus 2 protein (PCV), which can also bind to donor DNAs, appeared in the literature, [43][44][45][46] and our studies complement these approaches in multiple ways. First, at 17-nt, our Cas9-adaptor constructs are much smaller than the reported constructs, with the possibility of reducing this to as low as 13 nt.…”

Section: Discussionmentioning

confidence: 85%

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Lim

Sreekanth

Cox

et al. 2019

Preprint

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Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, these fusions are polypeptidic, limited to the Cas9 termini and lack multivalent display, and exclude diverse array of molecules. Here, we report a platform for the site-specific and multivalent display of a wide assortment of molecules on both the termini and internal sites on Cas9. Using this platform, we endow Cas9 with the functionality to effect precision genome edits, which involves efficient incorporation of exogenously supplied single-stranded oligonucleotide donor (ssODN) at the break site. We demonstrate that the multivalent display of ssODN on Cas9 significantly increased precision genome edits over those of Cas9 bearing one or no ssODN, and such display platform is compatible with large oligonucleotides and rapid screening of ssODNs. By hijacking the insulin secretion machinery and leveraging the ssODN display platform, we successfully engineer pancreatic β cells to secrete protective immunomodulatory factor interleukin-10. TOC GRAPHIC MAINCRISPR-Cas9 is a DNA endonuclease that can be targeted to a genomic site using a guide RNA (gRNA) bearing sequence complementarity to the target site. 1 Genetic fusion of Cas9 with effector domains (e.g., a transcription activator) has yielded transformative technologies; 2,3 however, this approach is limited to fusions that are generally linear, polypeptidic, and located on the termini of Cas9. A conjugation platform that allows the creation of fusions that are non-polypeptidic (e.g., nucleic acids, small molecules, polyethylene glycol [PEG] chains), unnatural peptides/proteins, internally located on Cas9, and branched with a multivalent display, would provide a greater diversity of technologies and applications. For example, precise sequence alteration at the Cas9 cleavage site requires the efficient incorporation of exogenously supplied single-stranded oligonucleotide donor DNA (ssODN) 4 via the homology-directed repair (HDR) pathway. 5-7 However, most cells instead employ the nonhomologous end-joining (NHEJ) repair, which results in unpredictable insertions and deletions of bases at the cleavage site, some of which are large enough to have pathogenic consequences. 3,8,9 Displaying ssODNs on Cas9 can increase their local concentration around the DNA strand break site to allow enhanced incorporation of the desired sequence. In another application, appending PEG chains to Cas9 may reduce the immunogenicity of this bacterial protein. 10 Multivalent display of DNA repair pathway inhibitors (e.g., NHEJ or p53 pathway inhibitors) or cell-specific ligands could also enhance precision and efficacious genome editing. 11,12 An ideal conjugation platform for Cas9 should have several characteristics. First, the platform should be compatible with a diverse set of cargoes (e.g., small molecules, nucleic acids, nanoparticles, antibodies, PEG chains) and allow their multivalent display. Second, the platform should be robust and implementable by nonspecialists, given the diverse u...

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Section: Discussionmentioning

confidence: 85%

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

Lim

Sreekanth

Cox

et al. 2019

Preprint

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“…3G). We also tested the hypothesis that biotinylating Cas9 and adding a streptavidin tag to the biotinylating ssODN to physically link the CRISPR-Cas9 complex to the repair oligo would increase rates of HDR, as has been previously suggested (50). Despite clear Cas9 biotinylation, we found that this strategy unexpectedly abolished Cas9 activity (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 94%

GenEditID: an open-access platform for the high-throughput identification of CRISPR edited cell clones

Xue

Tung

Siersbæk

et al. 2019

Preprint

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25CRISPR-Cas9-based gene editing is a powerful tool to reveal genotype-phenotype 26 relationships, but identifying cell clones carrying desired edits remains challenging. To 27 address this issue we developed GenEditID, a flexible, open-access platform for sample 28 tracking, analysis and integration of multiplexed deep sequencing and proteomic data, and 29 intuitive plate-based data visualisation to facilitate gene edited clone identification. To 30 demonstrate the scalability and sensitivity of this method, we identified KO clones in parallel 31 from multiplexed targeting experiments, and optimised conditions for single base editing 32 using homology directed repair. GenEditID enables non-specialist groups to expand their 33 gene targeting efforts, facilitating the study of genetically complex human disease. 34 35 KEYWORDS 36 CRISPR-Cas9; gene editing; GWAS; Illumina sequencing; multiplexed; pluripotent stem cell, 37 LIMS, In-Cell Western 38 39 BACKGROUND 40In the last decade, there has been an explosion of data from the sequencing of human 41 populations, including genome-wide association studies (GWAS) based on DNA microarrays 42 and increasingly also whole exomes and whole genomes (1). These studies have revealed 43 thousands of replicable genetic associations for complex diseases such as diabetes, obesity, 44Alzheimer's Disease and breast cancer (2-4). However, mechanistically determining how 45 these genetic associations contribute to disease remains challenging. Causal evidence 46 requires careful functional follow-up experiments in model cellular systems, organisms and 47 eventually in humans, but the traditional approach of characterising one gene at a time 48 cannot keep pace with the rate of genetic discovery. Furthermore, many associated variants 49 are non-coding, so the genetic elements responsible for conferring disease risk are often 50 unclear (5, 6). This issue is exemplified by the fat mass and obesity associated (FTO) locus, 51 3 in which intronic SNPs are strongly associated with obesity, largely due to increased food 52 intake (7-10) irrespective of gender, age or ethnicity (11, 12). Despite intense study, the 53 identify of the genetic elements that mediate SNP-associated phenotypes remains 54 controversial. Some studies suggest that effect on appetite might not be driven by the FTO 55 gene itself as initially thought, but instead by the nearby genes retinitis pigmentosa GTPase 56 regulator-interacting protein-1 like (RPGRIP1L) (13-15), or by iroquois homeobox 3 (IRX3) 57 and iroquois homeobox 5 (IRX5) (16, 17). Two powerful tools have recently emerged to help 58 meet the challenge of uncovering disease mechanisms from the translating the growing 59 wealth of genetic data: human pluripotent stem cells (hPSCs) and the CRISPR-Cas9 system 60 (18, 19). 62hPSCs facilitate human disease modelling since they can be indefinitely maintained in a 63 pluripotent state and can theoretically be differentiated into any cell type in the body, 64including disease-relevant cell populations (20, 21). For ex...

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“…Indeed, it has been reported that covalently linking an ssDNA repair template to Cas9 (Fig. 3G) increased HDR frequency by up to 30 times in multiple cell lines (Aird et al, 2018;Savic et al, 2018). An alternative attachment method for dsDNA could include adding a site-specific DNA-binding domain to Cas9 and including its binding site at the end of the dsDNA repair template (Fig.…”

Section: Thousands Of Birdsmentioning

confidence: 89%

Genome editing approaches to augment livestock breeding programs

Bishop

Eenennaam

2020

Journal of Experimental Biology

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The prospect of genome editing offers a number of promising opportunities for livestock breeders. Firstly, these tools can be used in functional genomics to elucidate gene function, and identify causal variants underlying monogenic traits. Secondly, they can be used to precisely introduce useful genetic variation into structured livestock breeding programs. Such variation may include repair of genetic defects, the inactivation of undesired genes, and the moving of useful alleles and haplotypes between breeds in the absence of linkage drag. Editing could also be used to accelerate the rate of genetic progress by enabling the replacement of the germ cell lineage of commercial breeding animals with cells derived from genetically elite lines. In the future, editing may also provide a useful complement to evolving approaches to decrease the length of the generation interval through in vitro generation of gametes. For editing to be adopted, it will need to seamlessly integrate with livestock breeding schemes. This will likely involve introducing edits into multiple elite animals to avoid genetic bottlenecks. It will also require editing of different breeds and lines to maintain genetic diversity, and enable structured crossbreeding. This requirement is at odds with the process-based trigger and event-based regulatory approach that has been proposed for the products of genome editing by several countries. In the absence of regulatory harmony, researchers in some countries will have the ability to use genome editing in food animals, while others will not, resulting in disparate access to these tools, and ultimately the potential for global trade disruptions.

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Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

Cited by 133 publications

References 35 publications

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

A conjugation platform for CRISPR-Cas9 allows efficient β-cell engineering

GenEditID: an open-access platform for the high-throughput identification of CRISPR edited cell clones

Genome editing approaches to augment livestock breeding programs

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