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Variants of cpf1 (cas12a) with altered pam specificity

A technology of S170R, cpf1, applied in DNA/RNA fragments, stable introduction of foreign DNA into chromosomes, recombinant DNA technology, etc.

Pending Publication Date: 2020-02-14
THE GENERAL HOSPITAL CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One possible reason is the need for a longer PAM, which limits targeting to approximately once every 43 bp of random DNA sequence compared to every 8 bp of SpCas9

Method used

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  • Variants of cpf1 (cas12a) with altered pam specificity
  • Variants of cpf1 (cas12a) with altered pam specificity
  • Variants of cpf1 (cas12a) with altered pam specificity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0372] Example 1. Variants of AsCpf1 with altered PAM specificity

[0373] In an attempt to alter the targeting range of Cpf1 nucleases, we first examined the existing crystal structures of AsCpf1 and LbCpf1 (Dong, Nature 2016; Yamano, Cell 2016). Among other observations, these structures demonstrate that PAM specificity is mediated by a combination of electrostatic interactions and indirect base reading. We therefore hypothesized that combinations of certain amino acid substitutions at residues of DNA bases that are sterically close to PAM bases may produce variants with altered or relaxed PAM recognition preferences. To test this, we examined the region of AsCpf1 spanning residues G131-L137, S161-S181, N534-I555, Y595-T616, L628-F632 and S685-I693 near the PAM (Table 1). We focused on amino acids in the reference AsCpf1 sequence whose three-dimensional positions meet at least one of the following criteria: 1) spatially close to PAM DNA bases (on the target or non-target st...

Embodiment 1B

[0393] Example 1B. Further characterization of AsCas12a variants with altered PAM specificity and improved on-target activity

[0394] Previous characterization of Cas12a orthologs in human cells revealed that As and LbCas12a are consistently more efficient nucleases for sites with TTTV PAMs (Kim et al., Nat Biotechnol., 2016, 34:863-8) , Fn and MbCas12a may possess the relaxed PAM preference of NTTN (Zetsche et al., Cell, 2015, 163:759-71). To more thoroughly assess the activity and PAM preference of each ortholog, their genome editing activity was examined in human cells using two sets of twelve crRNAs targeting sites with TTTN or VTTNPAM ( Figure 19A ). We observed similar gene disruption among the four orthologs for the TTTN PAM site, however target-specific differences were observed. Furthermore, Fn and Mb can target VTTN PAMs more efficiently when compared with As and LbCas12a, but consistent with previous reports, their average activity towards VTTN sites is too low ...

Embodiment 4

[0408] Example 4 provides additional evidence to support the observation that the E174R substitution enhances on-target activity.

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Abstract

Engineered CRISPR from Prevotella and Francisella 1 (Cpf1) nucleases with improved targeting range and enhanced on-target activity, and their use in genomic engineering, epigenomic engineering, base editing, genome targeting, genome editing, and in vitro diagnostics.

Description

[0001] priority statement [0002] This application claims the benefit of U.S. Patent Application Serial No. 62 / 488,426, filed April 21, 2017, and U.S. Patent Application Serial No. 62 / 616,066, filed January 11, 2018. The entire contents of the above are incorporated herein by reference. [0003] Federally Sponsored Research or Development [0004] This invention was made with Government support under Grant Nos. GM105378 and GM118158 awarded by the National Institutes of Health. The US Government has certain rights in this invention. technical field [0005] The present invention relates at least in part to engineered Prevotella and Francisella CRISPR 1 (CRISPR from Prevotella and Francisella 1, Cpf1 ) nucleases with altered and improved target specificity, and their Uses in genome engineering, epigenome engineering, genome targeting, genome editing, and in vitro diagnostics. Background technique [0006] CRISPR-Cas Cpf1 nuclease (also known as Cas12a nuclease) was recen...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N9/16C12N9/22C12N15/11C12N15/62C12N15/63C12N15/79C12N15/90
CPCC12Q1/6813C12N9/88C12N9/22C12Y401/99013C12N9/52C12N15/62Y02A50/30C12Q2521/301C12N2310/20
Inventor J·K·乔昂格B·克莱因斯蒂弗A·苏萨
Owner THE GENERAL HOSPITAL CORP
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