Efficient CRISPR/HDR-mediated knock-in system and method of use
Inventors
Wang, Jia-Wang • Lockey, Richard F.
Assignees
US Department of Veterans Affairs • University of South Florida St Petersburg
Publication Number
US-11530424-B1
Publication Date
2022-12-20
Expiration Date
2036-09-19
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Abstract
The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, can efficiently generate gene knockouts of variably sizes. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. HDR-mediated gene knock-in experiments are inefficient, with no reports of successful gene knock-in with DNA fragments larger than 4 kb. Targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors was performed and indicate that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected.
Core Innovation
The invention relates to an efficient CRISPR/HDR-mediated knock-in system and its method of use for genome editing. It specifically enables the targeted insertion of large DNA fragments, ranging from about 5.8 kb to 7.4 kb, into the genomes of mouse embryonic stem (ES) cells and zygotes without the use of non-homologous end joining (NHEJ) inhibitors. CRISPR-mediated homology-directed repair (HDR) is used for precise genome editing to replace or insert desired DNA fragments at target gene loci, achieving high efficiency equivalent to that of CRISPR/HDR with NHEJ inhibitors and CRISPR/NHEJ knockout efficiency in human ES cells.
The problem being addressed is the inefficiency of HDR-mediated gene knock-in, especially for large DNA fragments greater than 4 kb. Traditional homologous recombination methods have low efficiency (generally undetectable to 0.1%), and CRISPR/HDR targeting efficiencies reported are typically 0.5-20%, which is low compared to CRISPR/NHEJ knockout levels. The background highlights that while NHEJ inhibition can increase HDR efficiency, NHEJ is essential for DNA repair and embryonic viability, and inhibiting NHEJ may cause deleterious effects, including toxicity. Additionally, prior art has only tested small inserts or a few loci with NHEJ inhibitors. The invention provides a method achieving high-efficiency knock-in of large DNA fragments without relying on NHEJ inhibitors.
The method involves providing at least one CRISPR system comprising a single guide RNA (sgRNA) complementary to the target sequence and a CRISPR endonuclease (e.g., Cas9 or Cpf1). A donor DNA sequence containing the repair gene fragment flanked by two homologous arms (each between about 3.4 and 9 kb) corresponding to the genomic target site is also provided. The CRISPR system is inserted into cells such as mouse ES cells or zygotes, allowing cleavage of the target DNA and subsequent homology-directed repair to insert the large DNA fragment at the target site with high efficiency. The invention also discloses that NHEJ inhibitors and homologous arms longer than approximately 2 kb are not prerequisites for high-efficiency knock-in. The approach further demonstrates that lower concentrations of CRISPR reagents (approximately one quarter of standard concentrations) can achieve high efficiency with reduced toxicity in zygotes.
Claims Coverage
The patent contains two independent claims covering methods of genome editing to knock in large DNA fragments into a host gene without using NHEJ inhibitors. These claims collectively disclose the composition of CRISPR systems and donor DNA vectors, the method of insertion, and conditions to achieve efficient knock-in.
CRISPR system for large fragment knock-in without NHEJ inhibitors
A method using at least one CRISPR system comprising a plasmid with DNA encoding a single guide RNA (sgRNA) targeting a host gene and a DNA encoding Cas9 endonuclease, and a donor DNA plasmid having a fragment at least 4 kb in length flanked by two homologous arms between 3.4 kb and 9 kb. This is used to insert the large DNA fragment into the target gene without employing any non-homologous end joining inhibitor.
Reduced CRISPR reagent concentrations for microinjection
The CRISPR components (sgRNA, Cas9 endonuclease, donor DNA) are used at approximately one fourth of standard concentrations during cell insertion to reduce toxicity while maintaining high knock-in efficiency.
CRISPR system insertion by electroporation or microinjection
The method of introducing the CRISPR system into cells involves electroporation or microinjection, including specific protocols where the CRISPR system is mixed with a microinjection buffer to form a solution for injection into cells such as zygotes or stem cells.
CRISPR systems with different endonucleases
The method includes use of Cas9 or Cpf1 endonucleases, with the sgRNA and endonuclease encoded in one or two nucleic acid vectors without need for tracrRNA in the case of Cpf1.
The independent claims cover an efficient genome editing method to insert large DNA fragments into target genes without NHEJ inhibition, employing CRISPR systems with defined components, delivery methodologies, and reagent concentrations, thus achieving high-precision homology-directed repair knock-ins of large sequences.
Stated Advantages
High efficiency of knocking in large DNA fragments into genomes without the need for NHEJ inhibitors.
Reduced toxicity and higher survival rates in zygotes by using lower concentrations of CRISPR reagents (about one fourth of standard concentration).
Equivalence of knock-in efficiencies between CRISPR/HDR without NHEJ inhibition and CRISPR/HDR with NHEJ inhibitors, avoiding potential deleterious effects of inhibiting DNA repair pathways.
Simplification of donor construct design by demonstrating that homologous arms longer than 2 kb are not required for efficient large fragment knock-in.
High frequency of on-target homologous recombination with minimal random integration.
Documented Applications
Genomic editing of mouse embryonic stem cells and zygotes by knocking in large DNA fragments using CRISPR/HDR without NHEJ inhibitors.
Potential treatment or prevention of genetic disorders including cancer, autoimmune disorders, and Lrba-associated diseases through targeted gene correction or insertion using CRISPR/HDR knock-in system.
Genome editing targeting tumor suppressor proteins (e.g., p53, PTEN), oncogenes (e.g., Ras, myc), and cell cycle checkpoint proteins for genetic research or therapy.
Gene editing applications addressing immune diseases caused by mutations in genes encoding antibodies, MHC proteins, or related immune components.
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