Composition for genome editing using CRISPR/CPF1 system and use thereof
Inventors
Kim, Yong Sam • Ko, Jeong Heon • LEE, Jeong Mi • MOON, Su Bin
Assignees
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Publication Number
US-11667917-B2
Publication Date
2023-06-06
Expiration Date
Abstract
The present invention relates to a composition for genome editing using a CRISPR/Cpf1 system and a use thereof and, more particularly, to a composition for genome editing comprising: a CRISPR RNA (crRNA) including a guide sequence capable of hybridizing with a target nucleotide sequence, and a uridine repeat sequence connected to the 3′-end of the guide sequence, or a DNA encoding the same; and a Cpf1 protein or a DNA encoding the same, a method for genome editing using the same, a method for construction of a genetically modified organism, and a genetically modified organism. The present invention can increase an indel efficiency and decrease off-target activity in genome editing of eukaryotic cells using the CRIPSPR/Cpf1 system and thus can easily construct a genetically modified cell or genetically modified animal or plant having a desired gene inserted thereinto (knock-in) or deleted therefrom (knock-out).
Core Innovation
The invention provides a CRISPR/Cpf1 genome-editing composition comprising a CRISPR RNA (crRNA) and a Cpf1 protein, or DNA encoding the same. The crRNA includes a guide sequence complementary to a target nucleotide sequence, and the guide sequence is linked to a 3′ uridine repeat sequence. The disclosed composition is framed around eukaryotic genome editing with Cpf1 and a crRNA having a 3′ uridine repeat sequence linked to the guide.
The problem addressed is the need to increase indel (dsDNA cleavage) efficiency while reducing off-target activity in eukaryotic genome editing using Cpf1. The uridine-repeat modification of the crRNA is reported to improve editing outcomes, including knock-in and knock-out, while preserving Cpf1 specificity. The disclosure further describes that the approach enables construction of genetically modified cells, animals, and plants.
The document further includes comparison against SpCas9 and describes that the uridine-repeat design supports multi-target applicability and PAM-variant compatibility. Mechanistic and stability analyses are described as showing improved crRNA–AsCpf1 complex binding affinity, without increasing crRNA levels via transcript stability.
Claims Coverage
The independent claim covers a genome-editing composition with two principal inventive elements: a crRNA, or DNA encoding the crRNA, where a target-complementary guide sequence is linked to a 3′ uridine repeat sequence, and a Cpf1 protein, or DNA encoding the Cpf1 protein, with the guide sequence length constrained to 18 to 24 nt. Dependent claims further refine the uridine repeat structure, narrow guide length, specify Cpf1 source microorganisms by named genera, and constrain implementation details such as introduction/delivery and whether crRNA and Cpf1 are encoded on one or separate recombinant vectors.
3′ Uridine-repeat-linked crRNA guide for Cpf1 editing
A CRISPR RNA (crRNA) comprising a guide sequence complementary to a target nucleotide sequence and a uridine repeat sequence linked to a 3′ end of the guide sequence, or DNA encoding the crRNA.
Cpf1 protein in a genome-editing composition
A Cpf1 protein or DNA encoding the Cpf1 protein, combined with the uridine-repeat-linked crRNA, or DNA encoding the crRNA, for genome editing.
Guide sequence length between 18 and 24 nt
The length of the guide sequence is 18 to 24 nt.
Structured uridine repeat defined by (UaV)nUb
The uridine repeat sequence is defined as a nucleotide sequence represented by (UaV)nUb, where a and b are integers from 2 to 20, n is an integer from 1 to 5, and V is adenine, cytosine, or guanine.
Guide sequence length narrowed to 18 to 23 nt
The guide sequence length is 18 to 23 nt.
Cpf1 protein derived from specified microorganism genera
The Cpf1 protein is derived from one or more microorganisms selected from the group consisting of Candidatus genus, Lachnospira genus, Butyrivibrio genus, Peregrinibacteria, Acidominococcus genus, Porphyromonas genus, Prevotella genus, Francisella genus, Candidatus methanoplasma, and Eubacterium genus microorganisms.
Specified introduction methods for the composition
Introduction of the composition is achieved by local injection, microinjection, electroporation, or a lipofection method.
One recombinant vector or separate vectors for crRNA and Cpf1 DNA
The DNA encoding the crRNA and the DNA encoding the Cpf1 protein are inserted either in one recombinant vector or separate vectors.
Overall claim coverage focuses on a Cpf1 genome-editing composition using a crRNA whose target-complementary guide is linked to a 3′ uridine repeat sequence, paired with a Cpf1 protein or corresponding DNA, and constrained by guide length. Dependent claims further specify the uridine-repeat structure, narrow guide length, limit the Cpf1 source microorganisms by named genera, and constrain delivery and vector arrangement.
Stated Advantages
Increase indel (dsDNA cleavage) efficiency.
Reduce off-target activity in eukaryotic genome editing.
Enable knock-in and knock-out.
Preserve Cpf1 specificity while improving editing performance.
Support multi-target applicability.
Enable construction of genetically modified cells, animals, and plants.
Improve crRNA–AsCpf1 complex binding affinity without increasing crRNA levels via transcript stability.
Documented Applications
Eukaryotic genome editing enabling knock-in and knock-out.
Construction of genetically modified cells.
Construction of genetically modified animals.
Construction of genetically modified plants.
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