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US-12618054-B2 - CRISPR/Cpf1 systems and methods

US12618054B2US 12618054 B2US12618054 B2US 12618054B2US-12618054-B2

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.

Inventors

  • Mark Aaron Behlke
  • Michael Allen Collingwood
  • Rolf Turk
  • Christopher Anthony Vakulskas

Assignees

  • INTEGRATED DNA TECHNOLOGIES, INC.

Dates

Publication Date
20260505
Application Date
20210908

Claims (8)

  1. 1 . An isolated AsCpf1 crRNA, wherein the isolated AsCpf1 crRNA is active in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein endonuclease system, wherein the isolated AsCpf1 crRNA is a length-truncated AsCpf1 crRNA comprising a 5′-universal loop domain of 19 to 20 nucleotides in length and a 3′-target specific protospacer domain of 19 to 21 nucleotides in length and having at least one chemical modification at a position, counting from the 5′-end, selected from the group consisting of: RNA residues at position 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, 19, 21, 22, 23, 28, 29, 30, 32, 34, 35, 39, 40, 41, and combinations thereof.
  2. 2 . The isolated AsCpf1 crRNA of claim 1 , wherein the at least one chemical modification is selected from the group consisting of an end-group modification, 2′OMe modification, a 2′-fluoro modification and an LNA modification.
  3. 3 . A method of performing gene editing, comprising: contacting a candidate editing target site locus with an active CRISPR/Cpf1 endonuclease system having a wild-type AsCpf1 polypeptide and the isolated AsCpf1 crRNA of claim 1 .
  4. 4 . The isolated AsCpf1 crRNA of claim 1 , wherein the at least one chemical modification is 2′OMe modification.
  5. 5 . The isolated AsCpf1 crRNA of claim 1 , wherein the crRNA comprises 2′OMe modifications at position 1, 5, 6, 7, 8, 9, 10, 12, 13, 14, 16, 17, 18, and 19.
  6. 6 . The isolated AsCpf1 crRNA of claim 1 , wherein the crRNA comprises 2′OMe modifications at positions 21, 22, 23, 28, 29, 30, 32, 34, 35, 39, 40, and 41.
  7. 7 . The isolated AsCpf1 crRNA of claim 1 , wherein the 5′-universal loop domain is 20 nucleotides in length.
  8. 8 . The isolated AsCpf1 crRNA of claim 1 , wherein the 3′-target specific protospacer domain is 21 nucleotides in length.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation application of U.S. patent application Ser. No. 15/821,736, filed Nov. 22, 2017 and entitled “CRISPR/CPF1 SYSTEMS AND METHODS,” which claims benefit of priority under 35 U.S.C. 119 to U.S. Provisional Patent Application Ser. No. 62/425,307, filed Nov. 22, 2016 and entitled “CPF1 CRISPR SYSTEMS AND METHODS,” and U.S. Provisional Patent Application Ser. No. 62/482,896, filed Apr. 7, 2017 and entitled “HEK293 CELL LINE WITH STABLE EXPRESSION OF Acidaminococcus SP. BV3L6 CPF1,” the contents of which are herein incorporated by reference in their entirety. SEQUENCE LISTING The instant application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Sep. 8, 2021, is named IDT01-010-US-CON ST25.txt, and is 263,473 bytes in size. FIELD OF THE INVENTION This invention pertains to Cpf1-based CRISPR genes, polypeptides encoded by the same, mammalian cell lines that stably express Cpf1, crRNAs and the use of these materials in compositions of CRISPR-Cpf1 systems and methods. BACKGROUND OF THE INVENTION The use of clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas proteins (CRISPR-Cas system) for site-specific DNA cleavage has shown great potential for a number of biological applications. CRISPR is used for genome editing; the genome-scale-specific targeting of transcriptional repressors (CRISPRi) and activators (CRISPRa) to endogenous genes; and other applications of RNA-directed DNA targeting with Cas enzymes. CRISPR-Cas systems are native to bacteria and Archaea and provide adaptive immunity against viruses and plasmids. Three classes of CRISPR-Cas systems could potentially be adapted for research and therapeutic reagents. Type-II CRISPR systems have a desirable characteristic in utilizing a single CRISPR associated (Cas) nuclease (specifically Cas9) in a complex with the appropriate guide RNAs (gRNAs). In bacteria or Archaea, Cas9 guide RNAs comprise 2 separate RNA species. A target-specific CRISPR-activating RNA (crRNA) directs the Cas9/gRNA complex to bind and target a specific DNA sequence. The crRNA has 2 functional domains, a 5′-domain that is target specific and a 3′-domain that directs binding of the crRNA to the transactivating crRNA (tracrRNA). The tracrRNA is a longer, universal RNA that binds the crRNA and mediates binding of the gRNA complex to Cas9. Binding of the tracrRNA induces an alteration of Cas9 structure, shifting from an inactive to an active conformation. The gRNA function can also be provided as an artificial single guide RNA (sgRNA), where the crRNA and tracrRNA are fused into a single species (see Jinek, M., et al., Science 337 p 816-21, 2012). The sgRNA format permits transcription of a functional gRNA from a single transcription unit that can be provided by a double-stranded DNA (dsDNA) cassette containing a transcription promoter and the sgRNA sequence. In mammalian systems, these RNAs have been introduced by transfection of DNA cassettes containing RNA Pol III promoters (such as U6 or H1) driving RNA transcription, viral vectors, and single-stranded RNA following in vitro transcription (see Xu, T., et al., Appl Environ Microbiol, 2014. 80(5): p. 1544-52). In bacterial systems, these RNAs are expressed as part of a primitive immune system, or can be artificially expressed from a plasmid that is introduced by transformation (see Fonfara, I., et al., Nature, 2016. 532(7600): p. 517-21). In the CRISPR-Cas system, using the system present in Streptococcus pyogenes as an example (S.py. or Spy), native crRNAs are about 42 bases long and contain a 5′-region of about 20 bases in length that is complementary to a target sequence (also referred to as a protospacer sequence or protospacer domain of the crRNA) and a 3′ region typically of about 22 bases in length that is complementary to a region of the tracrRNA sequence and mediates binding of the crRNA to the tracrRNA. A crRNA:tracrRNA complex comprises a functional gRNA capable of directing Cas9 cleavage of a complementary target DNA. The native tracrRNAs are about 85-90 bases long and have a 5′-region containing the region complementary to the crRNA. The remaining 3′ region of the tracrRNA includes secondary structure motifs (herein referred to as the “tracrRNA 3′-tail”) that mediate binding of the crRNA:tracrRNA complex to Cas9. Jinek et al. extensively investigated the physical domains of the crRNA and tracrRNA that are required for proper functioning of the CRISPR-Cas system (Science, 2012. 337(6096): p. 816-21). They devised a truncated crRNA:tracrRNA fragment that could still function in CRISPR-Cas wherein the crRNA was the wild type 42 nucleotides and the tracrRNA was truncated to 75 nucleotides. They also developed an embodiment wherein the crRNA and tracrRNA are attached with a linker loop, forming a single guide RNA (sgR