US-20260125662-A1 - METHOD FOR IMPROVING EFFICIENCY AND ACCURACY OF GENE KNOCK-IN USING NON-RESIDENCE END OF CPF1

Abstract

A method for improving the efficiency and accuracy of gene knock-in using the non-residence end of Cpf1, where the free end of a target DNA and the free end of a donor DNA are generated using single or paired Cpf1, and combined with a complementary 5′-sticky end generated by Cpf1, so as to realize a more efficient and more accurate gene knock-in based on c-NHEJ. The method can perform NHEJ repair more efficiently and more accurately, and the accuracy can be increased from 0% to 100%. The free end of Cpf1 binds to and is protected by NHEJ core factor KU70/KU80, thereby making the ligation via NHEJ more efficient and accurate, which provides a foundation for the use of the free end of Cpf1 to improve the efficiency and accuracy of gene knock-in based on NHEJ, realizes gene knock-in or gene correction, and is suitable for non-dividing cells.

Inventors

• Anyong XIE
• Ruodan CHEN
• Yi Yang

Assignees

• Yimuhe Hangzhou Biotechnology Co., Ltd.

Dates

Publication Date

20260507

Application Date

20251222

Priority Date

20230621

Claims (5)

1. 1 . A method for improving the efficiency and accuracy of gene knock-in using the non-retained end of Cpf1, comprising: using single or paired Cpf1 to generate free target DNA ends and free donor DNA ends, and combining with the complementary 5′-cohesive ends produced by Cpf1 to achieve more efficient and more accurate gene knock-in based on c-NHEJ; if it is needed to improve the efficiency and accuracy of knocking the target gene into one end of the recipient genome, the end of the recipient genome target that requires high ligation accuracy after cleavage shall be a free end, and at least ensure that the end of the donor target that needs to be correspondingly ligated after cleavage is a free end, with the free end of the donor complementarily ligated to the free end of the recipient genome requiring high accuracy; if it is necessary to improve the efficiency and accuracy of knocking the target gene into both ends of the recipient genome, both ends of the donor target after cleavage and the corresponding two ends of the recipient genome after cleavage shall be complementary free ends, and in this case, paired Cpf1 are required for both donor and recipient genome cleavage.
2. 2 . The method for improving the efficiency and accuracy of gene knock-in using the non-retained end of Cpf1 according to claim 1 , wherein the method for improving the efficiency and accuracy of N-terminal target gene knock-in is as follows: the PAM of recipient Cpf1 target gene is located on W strand; the upstream Cpf1 target PAM of the donor DNA precursor is located on either W strand or C strand, while the downstream Cpf1 target PAM of the donor DNA precursor is located on C strand, and the corresponding 5′-cohesive ends of the recipient gene and the donor upon Cpf1 cleavage are completely complementary.
3. 3 . The method for improving the efficiency and accuracy of gene knock-in using the non-retained end of Cpf1 according to claim 1 , wherein the method for improving the efficiency and accuracy of C-terminal target gene knock-in is as follows: the PAM of recipient Cpf1 target gene is located on C strand; the upstream Cpf1 target PAM of the donor DNA precursor is located on W strand, while the downstream Cpf1 target PAM of the donor DNA precursor is located on either C strand or W strand, and the corresponding 5′-cohesive ends of the recipient gene and the donor upon Cpf1 cleavage are completely complementary.
4. 4 . The method for improving the efficiency and accuracy of gene knock-in using the non-retained end of Cpf1 according to claim 1 , wherein the method for improving the efficiency and accuracy of N-terminal or C-terminal knock-in of a DNA fragment or gene tag using Cpf1 non-retained end is carried out according to the following steps: (1) based on the requirement for N-terminal or C-terminal tag knock-in, selecting a testable Cpf1 target in the targeted gene according to the strand where the Cpf1 PAM is located; since N-terminal tag knock-in requires precise ligation between the inserted tag and the junction of the downstream target gene, the downstream end of the two ends of the DSB generated by Cpf1 target cleavage should be a free PAM-distal end, i.e., the PAM of the Cpf1 target in the target gene is located on Watson strand; if it is C-terminal tag knock-in, precise ligation between the inserted tag and the junction of the upstream target gene is required, therefore the DSB generated by Cpf1 target cleavage should be a free PAM-distal end, i.e., the PAM of the Cpf1 target in the target gene is located on Crick strand; (2) after selecting the Cpf1 target in the target gene, using T7E1 assay to test the cleavage efficiency of the selected target gene Cpf1 target in the target cells, and selecting the Cpf1 target that can be cleaved with high efficiency; (3) designing the donor DNA for N-terminal knock-in: both sides of the donor DNA precursor should contain a Cpf1 target designed based on the target sequence of the target gene, the PAM of the upstream target can be located on either Watson strand or Crick strand, while the PAM of the downstream target must be located on Crick strand, so that paired Cpf1-sgRNA cleaves the donor DNA precursor to generate donor DNA with two 5′-cohesive ends, each of which is completely complementary to the corresponding end of the recipient gene, and the downstream end of the cleaved donor DNA is a free PAM-distal end; (4) designing the donor DNA for C-terminal knock-in: both sides of the donor DNA precursor should contain a Cpf1 target designed based on the target sequence of the target gene, the PAM of the upstream target must be located on Watson strand, while the PAM of the downstream target can be located on either Watson strand or Crick strand, so that paired Cpf1-sgRNA cleaves the donor DNA precursor to generate donor DNA with two 5′-cohesive ends, each of which is completely complementary to the corresponding end of the recipient gene, and the upstream end of the cleaved donor DNA is a free PAM-distal end.
5. 5 . The method for improving the efficiency and accuracy of gene knock-in using the non-retained end of Cpf1 as according to claim 1 , wherein the method for improving the efficiency and accuracy of knocking in a DNA fragment or gene using Cpf1 non-retained end is carried out according to the following steps: (1) based on the requirement for the genomic target of DNA fragment or gene knock-in, identifying two adjacent testable Cpf1 genomic targets; the PAM of the upstream target shall be located on Crick strand, and the PAM of the downstream target shall be located on Watson strand; (2) using target-specific PCR amplification to test the efficiency of Cpf1 in simultaneously cleaving the paired Cpf1 targets in the target cells, and selecting the paired targets that can be cleaved simultaneously with high efficiency; (3) designing the donor DNA precursor for DNA fragment or gene knock-in: both sides of the donor DNA precursor contain two Cpf1 targets, the PAM of the upstream Cpf1 target is located on Watson strand, while the downstream PAM is located on Crick strand; paired Cpf1-sgRNA cleaves the donor DNA precursor to generate donor DNA with two 5′-cohesive ends, both of which are free PAM-distal ends; the upstream 5′-cohesive end shall be completely complementary to the upstream free end of the genomic Cpf1 target, and the downstream 5′-cohesive end shall be completely complementary to the downstream free end of the genomic Cpf1 target.

Description

CROSS-REFERENCE OF THE RELATED APPLICATIONS This application is a continuation application of International Application No. PCT/CN2024/098243, filed on Jun. 7, 2024, which is based upon and claims priority to Chinese Patent Application No. 202310739608.5, filed on Jun. 21, 2023, the entire contents of which are incorporated herein by reference. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy is named GBZD060_SequenceListing.xml, created on Dec. 22, 2025, and is 275,638 bytes in size. TECHNICAL FIELD The present invention relates to a method for improving the efficiency and accuracy of gene knock-in using the Cpf1 non-retained end in the field of biotechnology. Specifically, the content relates to the characteristic that Cpf1 asymmetrically retains at the two ends of a cleavage target after cleaving DNA at a specific site. By using single or paired Cpf1, free endogenous genomic target DNA ends and free donor DNA ends are generated. Combined with the complementary 5′-cohesive ends induced by Cpf1, more efficient and more accurate gene knock-in is achieved through the directional non-homologous end joining (NHEJ) between the free ends of the endogenous genomic target DNA and the free ends of the donor DNA. In non-dividing cells, due to the low activity of homologous recombination (HR), it is difficult to mediate gene knock-in via the HR method. Thus, the present invention provides a novel strategy for accurate and efficient gene knock-in in non-dividing cells. BACKGROUND CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing technology originates from an immune defense mechanism in bacteria and archaea. It consists of two components: Cas nuclease and single-guide RNA (sgRNA). After Cas nuclease assembles with sgRNA to form a stable complex, it begins searching for the target sequence in the genome. Once the protospacer-adjacent motif (PAM) is identified, Cas nuclease interacts with the PAM and denatures the double strand of DNA, allowing the spacer sequence on the sgRNA to complementary pair with single-stranded target DNA and form RNA-DNA hybrids. Subsequently, the activated Cas nuclease cleaves the DNA strand in the RNA-DNA hybrid and the single-stranded non-target DNA strand respectively, generating a DNA double strand break (DSB) with two DNA ends. This DSB is mainly repaired through two evolutionarily conserved endogenous cellular repair pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). HR is primarily active during the S and G2 phases of the cell cycle, and mainly uses sister chromatids as homologous templates to repair DNA replication-coupled DSB. NHEJ operates throughout the entire cell cycle and repairs DNA by ligating the two ends of the DNA break. NHEJ is mainly executed by several core factors, including KU70/KU80, DNA-PKcs, and XRCC4/DNA ligase 4, etc. The NHEJ pathway is commonly referred to as classical NHEJ (c-NHEJ). However, when certain core NHEJ factors fail to participate promptly, NHEJ efficiency decreases, the frequency and length of deletions or insertions increase, and the ligation process further requires the assistance of microhomology (MH) sequences. By manipulating these two endogenous DSB repair pathways, the DSB repair will yield a certain proportion of desired gene-editing products, thereby achieving the goal of gene editing. Targeted gene knock-in based on DSB repair is a gene-editing strategy that relies on high-accuracy repair pathways. It can be used for accurate insertion of target DNA fragments or genes to achieve: 1) correction of point mutations or other mutation types in target genes; 2) insertion of target genes or reporter systems requiring specific expression at target genomic loci, including SafeHarbor loci; 3) N-terminal/C-terminal tagging or N-terminal/C-terminal gene fusion of target genes. A major strategy for gene knock-in uses the accurate repair mechanism of HR, which requires mediation by homologous sequences or homology arms. This strategy has been widely used in correcting defective genes, accurate insertion of target genes (including fluorescent protein genes or drug selection marker genes), and has shown application potential in generating model animals, producing therapeutic cells (e.g., CAR-T cells), and other related fields of in vivo gene therapy. However, since DSB repair in cells prefers the NHEJ pathway and the HR only occurs in the S and G2 phases of the cell cycle, HR-based gene-editing strategies not only have low efficiency that is difficult to meet requirements for gene editing, but also are not suitable for non-dividing cells lacking S and G2 phases, such as neurons and muscle cells. Therefore, NHEJ has also been developed for gene knock-in, enabling accurate CRISPR/Cas9-induced site-specific integration in various dividing and non-dividing mammalia