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Compositions and methods of improving specificity in genomic engineering using rna-guided endonucleases

A nucleotide and polynucleotide technology, applied in the field of compositions and methods for improving the specificity of genome engineering using RNA-guided endonucleases, which can solve problems affecting potential uses and the like

Active Publication Date: 2018-07-31
DUKE UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been several reports of off-target binding and cleavage by Cas9, which can adversely affect its potential use in practice

Method used

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  • Compositions and methods of improving specificity in genomic engineering using rna-guided endonucleases
  • Compositions and methods of improving specificity in genomic engineering using rna-guided endonucleases
  • Compositions and methods of improving specificity in genomic engineering using rna-guided endonucleases

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example 1

[0294] Materials and Methods

[0295] Material. Tris-HCl (pH 7.6) buffer was obtained from Corning LifeSciences. L-glutamic acid monopotassium salt monohydrate, dithiothreitol (DTT) and magnesium chloride were obtained from Sigma Aldrich Co., LLC.

[0296] Cloning of Cas9, dCas9 and sgRNA expression plasmids ; Plasmids encoding Cas9, dCas9 and sgRNA targeting the AAVS1 locus of human chromosome 19 were cloned, expressed and purified using standard techniques. DNA substrates for imaging were also generated using standard techniques - (i) a 1198 bp substrate derived from a segment of the AAVS1 locus of human chromosome 19; (ii) containing a series of six complete, partial or mismatched and (iii) a 1078 bp "nonsense" substrate with no homology (>3 bp) to the protospacer. Plasmids encoding wild-type Cas9 and dCas9 were obtained from Addgene (plasmid 39312 and plasmid 47106). Plasmids for expression of Cas9 and dCas9 in bacteria were cloned using Gateway cloning (Life Technol...

example 2

[0315] Atomic force microscopy captures specifically and nonspecifically bound Cas9 / dCas9 along engineered DNA substrates at high resolution

[0316] Analysis of crystallographic and biochemical experiments revealed that specificity for protospacer binding and cleavage is conferred by first recognizing the PAM site by Cas9 itself, followed by strand invasion and spacer binding to the protospacer by the bound RNA complex Watson-Crick base pairing directly (Fig. 1A), but the full mechanistic picture has not yet emerged. To directly probe the relative propensity to bind protospacers and off-target sites at single-molecule resolution, 50 nM Cas9-sgRNA or dCas9-sgRNA complexes targeting the AAVS1 locus of human chromosome 19 were synthesized in combination with three DNA substrates ( 2.5nM) and imaged by AFM in air after incubation with:

[0317] (i) 1198 bp segment of the AAVS1 locus containing the complete target site followed by a PAM (hre "TGG") (Fig. 1C);

[0318] (ii) A 989...

example 3

[0347] An sgRNA (tru-gRNA) with two nucleotide truncations at the 5' end does not increase the binding specificity of dCas9 in vitro

[0348] Cas9 was found to exhibit cleavage activity even when the guide (protospacer-targeting) segment of the sgRNA or crRNA was truncated by up to four nucleotides from its 5' end, and Fu et al. (21) recently showed that using a These 5'-truncated (optimally truncated by 2-3 nucleotides) sgRNAs can actually lead to an order-of-magnitude increase in the fidelity of Cas9 cleavage in vivo. It has been suggested that the increased sensitivity to mismatch sites (MM) using these truncated sgRNAs (termed "tru-gRNAs", Figure 2A) is due to their binding between the guide RNA and the in situ spacer site Can reduce the result. This implies that the binding energy conferred by the extra 5'-nucleotides on the sgRNA can compensate for any mismatched nucleotides and stabilize Cas9 at the incorrect site, whereas the tru-gRNA will bind on the DNA if there is ...

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Abstract

Disclosed herein are optimized guide RNAs (gRNAs) and methods of designing and using said optimized gRNAs that have increased target binding specificity and reduced off-target binding.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Application No. 62 / 209,466, filed August 25, 2015, which is hereby incorporated by reference in its entirety. [0003] Statement of Government Interest [0004] This invention was made with government support under Federal Grant Nos. MCB1244297 and CBET1151035 awarded by the National Science Foundation and F32GM11250201, R01DA036865 and DP2OD008586 awarded by the National Institutes of Health. The government has certain rights in this invention. technical field [0005] The present disclosure relates to optimization of guide RNAs (gRNAs) and methods of designing and using said gRNAs with increased target binding specificity and reduced off-target binding. Background technique [0006] RNA-guiding endonucleases, especially the protein Cas9, have been hailed as potentially "perfect genome engineering tools" because they can be guided by a single 'guide RNA' molecule to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N33/543G01N33/553G16B25/00
CPCC12N15/113C12N9/22C12N15/09C12N2310/20G16B25/00C12N15/111C12N15/11A61P43/00C12N15/907G16B15/10G16B15/30G16B30/10C12N15/102C12N15/86C12N2740/15041
Inventor E.约瑟夫斯D.科卡克P.马萨莱克C.A.格斯巴赫
Owner DUKE UNIV
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