Crispr/cpf1 systems and methods

A technology for ascpf1, cell lines, applied in biochemical equipment and methods, other methods of inserting foreign genetic material, DNA/RNA fragments, etc.

Pending Publication Date: 2019-11-01
INTEGRATED DNA TECHNOLOGIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the FnCpf1 nuclease does not function in mammalian cells for genome editing

Method used

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  • Crispr/cpf1 systems and methods
  • Crispr/cpf1 systems and methods
  • Crispr/cpf1 systems and methods

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] DNA and amino acid sequences of wild-type As Cpf1 polypeptides as encoded in isolated nucleic acid vectors

[0089] The table below shows wild-type (WT) As Cpf1 nucleases expressed as polypeptide fusion proteins described in the present invention. Those skilled in the art will appreciate that many different DNA sequences can encode / express the same amino acid (AA) sequence, as in many cases more than one codon can encode the same amino acid. The DNA sequences shown below are used as examples only and other DNA sequences encoding the same protein (eg same amino acid sequence) are contemplated. It is also understood that additional features, elements or tags, such as NLS domains etc., may be added to the sequence. The examples show WT AsCpf1 showing the amino acid and DNA sequences of those proteins that are Cpf1 alone and Cpf1 fused to both the C-terminal and N-terminal SV40NLS domains and the HIS tag.

[0090] Amino acid sequences representing NLS sequences, domain li...

Embodiment 2

[0125] An isolated vector expressing a nucleic acid encoding a human codon-optimized AsCpf1 polypeptide fusion protein and a human cell line stably expressing the AsCpf1 polypeptide fusion protein were prepared.

[0126] The reference amino acid for AsCpf1 has been published. See Zetsche, B., Gootenberg, J.S., Abudayyeh, O.O., Slaymaker, I.M., Makarova, K.S., Essletzbichler, P., Volz, S.E., Joung, J., van der Oost, J., Regev, A., Koonin, E.V. and Zhang, F. (2015) Cpf1 is a single RNA-guidedendonuclease of a class 2 CRISPR-Cas system. Cell 163:1-13. Plasmids encoding human codon-optimized AsCpf1, flanked by a nuclear localization signal (NLS) and a 5'V5 epitope tag were generated by the Synthetic Biology division of Integrated DNATechnologies. The expression cassette is flanked by 5'XhoI and 3'EcoRI restriction enzyme sites ( figure 2 ). The Cpf1 plasmid was digested with XhoI and EcoRI (NEB), gel purified using a column-based purification system (Qiagen) and ligated into a...

Embodiment 3

[0145] crRNA length optimization: testing for truncation of the 5'20 base universal loop domain.

[0146] A set of 6 loci in the human HPRT1 gene was selected to study the length optimization of AsCpf1 crRNA. Target-specific protospacer domains each with 3' 24 bases and 5' loop structures with 20, 19, 18 and 17 bases representing a set of consecutive 1 base deletions from the 5' end were synthesized domain of a series of crRNAs. A second set of crRNAs were synthesized, each having a 3' 21 base target-specific protospacer domain and likewise a 5' loop domain of 20, 19, 18 and 17 bases at the same position.

[0147] A HEK cell line stably expressing the AsCpf1 endonuclease was used in these studies (Example 2). Anti-HPRT1 crRNA was separately mixed with Lipofectamine RNAiMAX (Life Technologies) in reverse transfection format and transfected into HEK-Cpf1 cell line. Transfections were performed at 40,000 cells per well in a 96-well plate format. RNA was introduced at a final ...

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Abstract

This invention pertains to recombinant AsCpf1 and LbCpf1 nucleic acids and polypeptides for use in CRISPR / Cpf1 endonuclease systems and mammalian cell lines encoding recombinant AsCpf1 or LbCpf1 polypeptides. The invention includes recombinant ribonucleoprotein complexes and CRSPR / Cpf1 endonuclease systems having a suitable AsCpf1 crRNA is selected from a length-truncated AsCpf1 crRNA, a chemically-modified AsCpf1 crRNA, or an AsCpf1 crRNA comprising both length truncations and chemical modifications. Methods of performing gene editing using these systems and reagents are also provided.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Patent Application Serial No. 62 / 425,307, filed November 22, 2016, entitled "CPF1 CRISPRSYSTEMS AND METHODS" under 35 U.S.C. 119 and entitled "HEK293 CELLLINE WITH STABLE EXPRESSION OF ACIDAMINOCOCCUS SP.BV3L6CPF1", the priority of U.S. Patent Provisional Application Serial No. 62 / 482,896, the entire contents of which are incorporated herein by reference. [0003] sequence listing [0004] This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format, the entire contents of which are hereby incorporated by reference. Said ASCII copy was created in _____, is named IDT01-010-US_ST25.txt, and is _____ bytes in size. technical field [0005] The present invention relates to Cpf1-based CRISPR genes, polypeptides encoded thereby, mammalian cell lines stably expressing Cpf1, crRNA and the use of these materials in combinations of CRISPR-Cpf1 system...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/10C12N15/113C12N15/90C12Y301/00
CPCC12N15/102C12N15/113C12N15/63C12N15/90C12N15/111C12N9/22C12N2310/20C12Y301/30C12N15/8509
Inventor M·A·贝尔克M·A·科林伍德R·图克C·A·瓦库斯卡斯
Owner INTEGRATED DNA TECHNOLOGIES
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