The NIH Somatic Cell Genome Editing program

Saha, Krishanu; Sontheimer, Erik J.; Brooks, Philip J.; Dwinell, Melinda R.; Gersbach, Charles A.; Liu, David R.; Murray, Stephen A.; Tsai, Shengdar Q.; Wilson, Ross C.; Anderson, Daniel G.; Asokan, Aravind; Banfield, Jillian F.; Bankiewicz, Krystof S.; Bao, Gang; Bulte, Jeff W.M.; Bursac, Nenad; Campbell, Jarryd M.; Carlson, Daniel F.; Chaikof, Elliot L.; Chen, Zheng Yi; Cheng, Ranran; Clark, Karl J.; Curiel, David T.; Dahlman, James E.; Deverman, Benjamin E.; Dickinson, Mary E.; Doudna, Jennifer A.; Ekker, Stephen C.; Emborg, Marina E.; Feng, Guoping; Freedman, Benjamin; Gamm, David M.; Gao, Guangping; Ghiran, Ionita; Glazer, Peter M.; Gong, Shaoqin; Heaney, Jason D.; Hennebold, Jon D.; Hinson, J. Travis; Khvorova, Anastasia; Kiani, Samira; Lagor, William R.; Lam, Kit S.; Leong, Kam W.; Levine, Jon E.; Lewis, Jennifer A.; Lutz, Cathleen; Ly, Danith H.; Maragh, Samantha; McCray, Paul B.; McDevitt, Todd C.; Mirochnitchenko, Oleg; Morizane, Ryuji; Murthy, Niren; Prather, Randall S.; Ronald, John A.; Roy, Subhojit; Roy, Sushmita; Sabbisetti, Venkata; Saltzman, W. Mark; Santangelo, Philip J.; Segal, David J.; Shimoyama, Mary; Skala, Melissa C.; Tarantal, Alice F.; Tilton, John C.; Truskey, George A.; Vandsburger, Moriel; Watts, Jonathan K.; Wells, Kevin D.; Wolfe, Scot A.; Xu, Qiaobing; Xue, Wen; Yi, Guohua; Zhou, Jiangbing

doi:10.1038/s41586-021-03191-1

Cited by 101 publications

(68 citation statements)

References 155 publications

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“…CasΦ mediate genome editing in eukaryotic cells 2 and conserve the properties of other CRISPR-Cas nucleases in a tiny RNP. Therefore, they could mitigate packing restrictions in the AAV vectors used for delivery 11 . Our study paves the way for the rational redesign of CasΦ endonucleases widening the CRISPR-Cas toolbox.…”

Section: Discussionmentioning

confidence: 99%

“…CRISPR-Cas nucleases have been extensively used as tools for genome editing 6,7 . The redesign of their guide RNA to target specific DNA sites, as well as the manipulation of the protein scaffold 8,9 , has provided a powerful method for genome modification in biomedical and biotechnological applications 10,11 .…”

Section: Figurementioning

confidence: 99%

“…CRISPR-Cas effector complexes are harnessed in vitro and in vivo for genome editing approaches, but specially the latter is limited by delivery problems, which is one of the main unmet needs in the field 11 . Adeno-associated viral vectors (AAV) are commonly used for gene delivery.…”

Section: Figurementioning

confidence: 99%

“…Recently, CasΦ enzymes have been shown to mediate genome editing in mammalian and plant cells 2 expanding our repertoire of genome manipulation tools. The small size CasΦ RNPs can improve our genome editing approaches by alleviating the packing problems in the AAV vectors used for delivery 11 . However, questions regarding the detailed molecular mechanism of target DNA recognition, unzipping and subsequent cleavage by CasΦ nucleases remain unanswered, as no structural information is available.…”

Section: Figurementioning

confidence: 99%

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Structure of the mini-RNA-guided endonuclease CRISPR-CasΦ3

Rey

Fuglsang

Temperini

et al. 2021

Preprint

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CasΦ is a novel family of miniaturized RNA-guided endonucleases from phages 1,2. These novel ribonucleoproteins (RNPs) provide a compact scaffold gathering all key activities of a genome editing tool2. Here, we provide the first structural insight into CasΦ singular DNA targeting and cleavage mechanism by determining the cryoEM structure of CasΦ3 with the triple strand R-loop generated after DNA cleavage. The structure reveals the unique machinery for target unwinding to form the crRNA-DNA hybrid and cleaving the target DNA. The protospacer adjacent motif (PAM) is recognised by the target strand (T-strand) and non-target strand (NT-strand) PAM interacting domains (TPID and NPID). Unwinding occurs after insertion of the conserved α1 helix disrupting the dsDNA, thus facilitating the crRNA-DNA hybrid formation. The NT-strand is funnelled towards the RuvC catalytic site, while a long helix of TPID separates the displaced NT-strand and the crRNA-DNA hybrid avoiding DNA re-annealing. The crRNA-DNA hybrid is directed to the stop (STP) domain that splits the hybrid guiding the T-strand towards the RuvC active site. The conserved RuvC insertion of the CasΦ family is extended along the hybrid, interacting with the phosphate backbone of the crRNA. A cluster of hydrophobic residues anchors the RuvC insertion in a cavity of the STP domain. The assembly of the hybrid promotes the shortening of the RuvC insertion, thus pulling the STP towards the RuvC active site to activate catalysis. These findings illustrate why CasΦ unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. Site-directed mutagenesis in key residues support CasΦ3 target DNA and non-specific ssDNA cutting mechanism. Our analysis provides new avenues to redesign the compact CRISPR-CasΦ nucleases for genome editing.

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Section: Discussionmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

See 2 more Smart Citations

Structure of the mini-RNA-guided endonuclease CRISPR-CasΦ3

Rey

Fuglsang

Temperini

et al. 2021

Preprint

View full text Add to dashboard Cite

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“…Diverse Cas9 nuclease variants are involved in clinical trials to develop new therapies against human diseases 16 . The redesign of the CRISPR-Cas guide RNA, as well as the modification of the protein 17 , 18 , has provided a powerful method for genome editing in biomedical and biotechnological applications 19 , 20 . In some of these applications Adeno-associated viral (AAV) vectors are commonly used for gene delivery.…”

Section: Introductionmentioning

confidence: 99%

Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

et al. 2021

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CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.

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Moving away from one disease at a time: Screening, trial design, and regulatory implications of novel platform technologies

Lekstrom-Himes

Brooks

Koeberl

et al. 2023

American J of Med Genetics Pt C

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Most rare diseases are caused by single‐gene mutations, and as such, lend themselves to a host of new gene‐targeted therapies and technologies including antisense oligonucleotides, phosphomorpholinos, small interfering RNAs, and a variety of gene delivery and gene editing systems. Early successes are encouraging, however, given the substantial number of distinct rare diseases, the ability to scale these successes will be unsustainable without new development efficiencies. Herein, we discuss the need for genomic newborn screening to match pace with the growing development of targeted therapeutics and ability to rapidly develop individualized therapies for rare variants. We offer approaches to move beyond conventional “one disease at a time” preclinical and clinical drug development and discuss planned regulatory innovations that are necessary to speed therapy delivery to individuals in need. These proposals leverage the shared properties of platform classes of therapeutics and innovative trial designs including master and platform protocols to better serve patients and accelerate drug development. Ultimately, there are risks to these novel approaches; however, we believe that close partnership and transparency between health authorities, patients, researchers, and drug developers present the path forward to overcome these challenges and deliver on the promise of gene‐targeted therapies for rare diseases.

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The NIH Somatic Cell Genome Editing program

Cited by 101 publications

References 155 publications

Structure of the mini-RNA-guided endonuclease CRISPR-CasΦ3

Structure of the mini-RNA-guided endonuclease CRISPR-CasΦ3

Structure of the mini-RNA-guided endonuclease CRISPR-Cas12j3

Moving away from one disease at a time: Screening, trial design, and regulatory implications of novel platform technologies

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