Engineered CAS9 with wider DNA targeting range

A technology of N986R and R991L, applied in the field of engineered CAS9 protein, can solve problems such as limiting the targeting range and application potential

Pending Publication Date: 2022-03-18
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
View PDF62 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although promising for clinical translation due to its compact size, the longer PAM of SaCas9 limits its

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Engineered CAS9 with wider DNA targeting range
  • Engineered CAS9 with wider DNA targeting range
  • Engineered CAS9 with wider DNA targeting range

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] This example demonstrates an all-atom molecular dynamics simulation of a SaCas9 complex.

[0095] The high-resolution SaCas9 complex structure (Nishimasu et al., supra) was examined (although crystal contacts may affect the local structure; see figure 2 ). To establish the dynamic details of the SaCas9 complex in its native state, molecular dynamics (MD) methods were used to model the complex under physiological conditions. MD simulations as a complement to experimental studies have been shown to be effective in understanding protein-DNA interactions (Palermo et al., Proceedings of the National Academy of Sciences, 114:7260-7265 (2017); and Cong et al., Nat. Commun., 3:968 (2012)). All-atom MD simulations were performed to characterize the molecular mechanism of DNA target binding to the SaCas9-sgRNA complex (see figure 1 A).

[0096] In MD analysis, after the binding state with the target DNA substrate reaches equilibrium ( image 3 A), The secondary structures ...

Embodiment 2

[0100] This example describes the use of free energy perturbations and experimental assays to probe Cas9 PAM recognition.

[0101] Completing the system equilibrium and MD analysis reveals previously underestimated dynamics of PAM recognition and addresses one of the fundamental challenges of modeling genome editing tools, namely how to understand in a quantitative manner the contribution of protein residues to target recognition. With this in mind, a combinatorial process was used, in which structural insights guided computational analysis followed by targeted gene editing experiments that further demonstrated computational mapping of Cas9 variant activity, where in silico predictions could correlate with experimental Cas9 editing efficiencies .

[0102] First, the contribution of PI domain residues to SaCas9 PAM recognition was quantified using free energy perturbation (FEP) calculations ( Figure 5 A). Alanine scanning analysis was performed directly on residues near the ...

Embodiment 3

[0104] This example describes the analysis of KKH SaCas9 variants to reveal the molecular mechanism of their expanded PAM.

[0105] The KKH mutant of SaCas9 involves three substitutions: E782K, N968K, and R1015H (Kleinstiver et al., Nature Biotech., 33:1293-1298 (2015). The thermodynamic cycle of R1015H is shown in Figure 5a. In the bound state, R1015 binds to G3 with two hydrogen bonds and is responsible for the PAM-specific NNGRRT. This interaction is further stabilized by the salt bridge between E993 and R1015, which can significantly reduce the conformational fluctuation of R1015. Such as Figure 5 As shown in A, the same salt bridge also exists in the free state of SaCas9. After the R1015H mutation, H1015 in the bound state moves away from G3, thereby releasing the specificity for G3 in the NNGRRT PAM. However, this mutation (in Figure 5 Represented by ΔG1 in A) significantly reduces the binding free energy (or binding affinity). The same mutation process as in th...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention provides a variant Staphylococcus aureus Cas9 (SaCas9) protein having an altered specificity for a prototype spacer adjacent motif (PAM) sequence. The present invention also relates to CRISPR/Cas9 systems and methods for altering the genomic DNA sequence using the variant SaCas9 protein. Also disclosed are methods of producing variant Cas9 proteins with altered PAM specificity.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 838,498, filed April 25, 2019, which is incorporated herein by reference in its entirety. technical field [0003] The present invention relates to engineered CAS9 proteins with a broader DNA targeting range and methods, kits, compositions and systems using them. Background technique [0004] The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, originally discovered in bacteria and archaea, provides adaptive resistance to foreign genetic material, using RNA-guided protein machinery and complex molecular mechanisms to provide microbial immunity (Mojica et al. , J.Mol.Evol., 60:174-182(2005); Bolotin et al., Microbiology, 151:2551-2561(2005); Barrangou et al., Science, 315:1709-1712(2007); Garneau et al., Nature, 468:67(2010); Deltcheva et al., Nature, 471:602(2011); Sapranauskas et al, Nucl. Acids Res., 39:9275-9...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): A61K48/00C12N15/00C12N15/87A61K38/46C07K14/00C12P21/06C07H21/04
CPCC12N9/22C12N15/102C12N15/907C12N2310/20C12Y301/00C12N15/52C12N15/90
Inventor 丛乐
Owner THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap