Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools

Hu, Chunyi; Ni, D.R.; Nam, Ki Hyun; Majumdar, Sonali; McLean, Justin; Stahlberg, Henning; Terns, Michael P.; Ke, Ailong

doi:10.1016/j.molcel.2022.06.007

Cited by 44 publications

(54 citation statements)

References 70 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In our CryoEM reconstruction, we did not observe discernible density near the 3’ end of the RNA corresponding to the Csb2 subunit. As Csb2 makes up a stoichiometric component of the Cascade protein complex used for CryoEM analysis (Figure 3), we interpret the failure to observe this subunit to be a consequence of inherent flexibility of this part of the complex – as observed previously for the type I-A, I-C and I-F complexes (7,8,49).…”

Section: Resultssupporting

confidence: 65%

“…High resolution crystal or cryo-EM structures are available for type I-A (7), I-C (8,9), I-D (10), I-E (11)(12)(13)(14), I-F (15), I-Fv (16) and type I-F-TniQ (17) complexes. However, within this broad family there are many variations.…”

Section: Introductionmentioning

confidence: 99%

“…In type I-C systems, the Cas5 subunit possesses crRNA processing activity and Cas6 is absent (21). Although more complex than the streamlined class 2 systems such as Cas9 and Cas12, type I effectors have been utilised for a growing range of genome editing approaches and are particularly useful for long range genome edits (7,(22)(23)(24)(25).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Structure and mechanism of the type I-G CRISPR effector

Shangguan

Graham

Sundaramoorthy

et al. 2022

Preprint

View full text Add to dashboard Cite

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3-prime end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. Type I-G Cascade is one of the most streamlined Class 1 CRISPR effectors. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

show abstract

Section: Resultssupporting

confidence: 65%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Structure and mechanism of the type I-G CRISPR effector

Shangguan

Graham

Sundaramoorthy

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…High resolution crystal or cryo-EM structures are available for type I-A ( 7 ), I-C ( 8 , 9 ), I-D ( 10 ), I-E ( 11–14 ), I-F ( 15 ), I-Fv ( 16 ) and type I-F-TniQ ( 17 ) complexes. However, within this broad family there are many variations.…”

Section: Introductionmentioning

confidence: 99%

“…In type I-C systems, the Cas5 subunit possesses crRNA processing activity and Cas6 is absent ( 21 ). Although more complex than the streamlined class 2 systems such as Cas9 and Cas12, type I effectors have been utilised for a growing range of genome editing approaches and are particularly useful for long range genome edits ( 7 , 22–25 ).…”

Section: Introductionmentioning

confidence: 99%

Structure and mechanism of the type I-G CRISPR effector

Shangguan

Graham

Sundaramoorthy

et al. 2022

Nucleic Acids Research

View full text Add to dashboard Cite

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3′ end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

show abstract

Brain‐Targeted Cas12a Ribonucleoprotein Nanocapsules Enable Synergetic Gene Co‐Editing Leading to Potent Inhibition of Orthotopic Glioblastoma

Ruan,

Xu,

et al. 2024

Advanced Science

View full text Add to dashboard Cite

Gene‐editing technology shows great potential in glioblastoma (GBM) therapy. Due to the complexity of GBM pathogenesis, a single gene‐editing‐based therapy is unlikely to be successful; therefore, a multi‐gene knockout strategy is preferred for effective GBM inhibition. Here, a non‐invasive, biodegradable brain‐targeted CRISPR/Cas12a nanocapsule is used that simultaneously targeted dual oncogenes, EGFR and PLK1, for effective GBM therapy. This cargo nanoencapsulation technology enables the CRISPR/Cas12a system to achieve extended blood half‐life, efficient blood‐brain barrier (BBB) penetration, active tumor targeting, and selective release. In U87MG cells, the combinatorial gene editing system resulted in 61% and 33% knockout of EGFR and PLK1, respectively. Following systemic administration, the CRISPR/Cas12a system demonstrated promising brain tumor accumulation that led to extensive EGFR and PLK1 gene editing in both U87MG and patient‐derived GSC xenograft mouse models with negligible off‐target gene editing detected through NGS. Additionally, CRISPR/Cas12a nanocapsules that concurrently targeted the EGFR and PLK1 oncogenes showed superior tumor growth suppression and significantly improved the median survival time relative to nanocapsules containing single oncogene knockouts, signifying the potency of the multi‐oncogene targeting strategy. The findings indicate that utilization of the CRISPR/Cas12a combinatorial gene editing technique presents a practical option for gene therapy in GBM.

show abstract

Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools

Cited by 44 publications

References 70 publications

Structure and mechanism of the type I-G CRISPR effector

Structure and mechanism of the type I-G CRISPR effector

Structure and mechanism of the type I-G CRISPR effector

Brain‐Targeted Cas12a Ribonucleoprotein Nanocapsules Enable Synergetic Gene Co‐Editing Leading to Potent Inhibition of Orthotopic Glioblastoma

Contact Info

Product

Resources

About