CRISPR-Cas effector polypeptides and methods of use thereof
Inventors
DOUDNA, Jennifer A. • Al-Shayeb, Basem • Banfield, Jillian F. • Pausch, Patrick
Assignees
University of California San Diego UCSD
Publication Number
US-12365887-B2
Publication Date
2025-07-22
Expiration Date
2040-03-05
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Abstract
The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided CRISPR-Cas effector protein of the present disclosure and a guide RNA. The present disclosure provides methods of modulating transcription of a target nucleic acid.
Core Innovation
The present disclosure provides RNA-guided CRISPR-Cas effector proteins referred to as Cas12J or CasΦ polypeptides, nucleic acids encoding these proteins, and compositions comprising them. The disclosed Cas12J proteins form ribonucleoprotein complexes with guide RNAs that provide sequence specificity, enabling site-specific binding and cleavage of target nucleic acids, including double-stranded and single-stranded DNA, but not RNA, distinguished from other Cas proteins by their small size and divergent features.
The problem addressed is the need for compact, efficient, programmable RNA-guided nucleases with minimal component requirements for genome editing and nucleic acid modification. Current CRISPR-Cas systems typically require multiple proteins or accessory RNAs, limiting their versatility and use especially in contexts demanding smaller constructs or simplified molecular machinery.
Claims Coverage
The claims cover methods and compositions comprising Cas12J polypeptides and guide nucleic acids for precise targeting and cleavage of double-stranded DNA (dsDNA) in cells with defined PAM sequence requirements, delivery means, and molecular modifications.
RNA-guided dsDNA cleavage with sequence-specific PAM recognition
A method of cleaving dsDNA in a cell by contacting it with a nucleic acid encoding a polypeptide having at least 95% amino acid identity to any one of SEQ ID NOs: 109-126, and a guide nucleic acid including a repeat and a spacer sequence that hybridizes to a target sequence adjacent to a 5′-NTTN-3′ PAM located on the non-target strand. The polypeptide and guide RNA form a ribonucleoprotein complex that binds and cleaves dsDNA at the target site.
Use of ribonucleoprotein complexes that cleave both strands of dsDNA
The RNP complex formed by the Cas12J polypeptide and guide RNA cleaves both strands of the dsDNA target sequence resulting in a double-strand break.
High degree of sequence identity and inclusion of nuclear localization signals
The polypeptide comprises at least 97% amino acid identity to SEQ ID NOs: 109-126 and can be fused to nuclear localization signals to enable efficient nuclear targeting in eukaryotic cells.
Lipid nanoparticle mediated delivery
The nucleic acid encoding the Cas12J polypeptide and the guide RNA can be contained in a lipid nanoparticle (LNP) for delivery to cells.
Broad cell type applicability
The methods and compositions apply to human cells, including blood and liver cells, and utilize spacer sequences in the guide RNA of 14 to 24 nucleotides in length.
Inclusion of messenger RNA and donor nucleic acids
The nucleic acid encoding the Cas12J polypeptide can be messenger RNA (mRNA). Methods may include contacting the cell with a donor nucleic acid, optionally delivered via adeno-associated viral vectors.
Modification of target DNA by nucleotide deletion
Methods can remove one or more nucleotides from the target sequence via Cas12J mediated cleavage and repair mechanisms.
The claims cover versatile composition and method aspects of using Cas12J polypeptides complexed with guide nucleic acids for programmable, PAM-dependent, RNA-guided cleavage of dsDNA in cells, including delivery and modulation features applicable to human and other cells for genome editing purposes.
Stated Advantages
Cas12J proteins are small and compact, enabling simpler delivery and potential use in contexts where size constraints limit other CRISPR systems.
Cas12J complexes provide efficient, specific cleavage of double-stranded DNA without requiring accessory RNAs, simplifying system components.
Cas12J proteins have broad PAM sequence tolerance supporting versatile targeting.
Cas12J exhibits processing of its own crRNA via its RuvC domain, consolidating RNA and DNA cleavage functions in a single active site.
Cas12J has demonstrated efficient genome editing in human cells comparable to Cas9 and Cas12a, suggesting powerful biotechnological applications.
Documented Applications
Targeted genome editing in prokaryotic and eukaryotic cells, including human cells, through programmable RNA-guided DNA cleavage.
Programmable DNA modification via RNA-guided cleavage and potentially base editing when fused to editing domains.
Detection of specific DNA sequences using the trans-ssDNA cleavage activity of Cas12J activated by target DNA binding.
Phage defense applications by targeting competing mobile genetic elements through RNA-guided DNA cleavage.
Modulation of transcription and translation by Cas12J fusion proteins including transcriptional activators or repressors.
Biotechnological use of compact CRISPR systems for therapeutic gene editing and molecular diagnostics.
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