P3a site-specific and cassette mutagenesis for seamless protein, RNA and plasmid engineering

ZFN, TALENs and CRISPR/Cas9 system have been used to generate point mutations and large fragment deletions and insertions in genomic modifications. CRISPR/Cas9 system is the most flexible and fast developing technology that has been extensively used to make mutations in all kinds of organisms. However, the most mutations reported up to date are small insertions and deletions. In this report, CRISPR/Cas9 system was used to make large DNA fragment deletions and insertions, including entire Dip2a gene deletion, about 65kb in size, and β-galactosidase (lacZ) reporter gene insertion of larger than 5kb in mouse. About 11.8% (11/93) are positive for 65kb deletion from transfected and diluted ES clones. High targeting efficiencies in ES cells were also achieved with G418 selection, 46.2% (12/26) and 73.1% (19/26) for left and right arms respectively. Targeted large fragment deletion efficiency is about 21.4% of live pups or 6.0% of injected embryos. Targeted insertion of lacZ reporter with NEO cassette showed 27.1% (13/48) of targeting rate by ES cell transfection and 11.1% (2/18) by direct zygote injection. The procedures have bypassed in vitro transcription by directly co-injection of zygotes or co-transfection of embryonic stem cells with circular plasmid DNA. The methods are technically easy, time saving, and cost effective in generating mouse models and will certainly facilitate gene function studies.

Targeted insertion of large DNA fragments holds great potential for treating genetic diseases. Prime editors can effectively insert short fragments (~44 bp) but not large ones. Here we developed GRAND editing to precisely insert large DNA fragments without DNA donors. In contrast to prime editors, which require reverse transcription templates hybridizing with the target sequence, GRAND editing employs a pair of prime editing guide RNAs, with reverse transcription templates nonhomologous to the target site but complementary to each other. This strategy exhibited an efficiency of up to 63.0% of a 150-bp insertion with minor by-products and 28.4% of a 250-bp insertion. It allowed insertions up to ~1 kb, although the efficiency remains low for fragments larger than 400 bp. We confirmed efficient insertion in multiple genomic loci of several cell lines and non-dividing cells, which expands the scope of genome editing to enable donor-free insertion of large DNA sequences.

Targeted insertion of large DNA fragments holds promise for genome engineering and gene therapy. Prime editing (PE) effectively inserts short (<50 bp) sequences. Employing paired prime editing guide RNAs (pegRNAs) has enabled PE to better mediate relatively large insertions in vitro, but the efficiency of larger insertions (>400 bp) remains low and in vivo application has not been demonstrated. Inspired by the efficient genomic insertion mechanism of retrotransposons, we develop a template-jumping (TJ) PE approach for the insertion of large DNA fragments using a single pegRNA. TJ-pegRNA harbors the insertion sequence as well as two primer binding sites (PBSs), with one PBS matching a nicking sgRNA site. TJ-PE precisely inserts 200 bp and 500 bp fragments with up to 50.5 and 11.4% efficiency, respectively, and enables GFP (~800 bp) insertion and expression in cells. We transcribe split circular TJ-petRNA in vitro via a permuted group I catalytic intron for non-viral delivery in cells. Finally, we demonstrate that TJ-PE can rewrite an exon in the liver of tyrosinemia I mice to reverse the disease phenotype. TJ-PE has the potential to insert large DNA fragments without double-stranded DNA breaks and facilitate mutation hotspot exon rewriting in vivo.

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. 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. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show 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. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

Application of the CRISPR–Cas System for Efficient Genome Engineering in Plants

At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 ‘transgene-free’ homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean.

BackgroundThe precise insertion of large DNA fragments (>3–5 kb) remains one of the key obstacles in establishment of genetically modified murine models.MethodsA 21 kb large DNA fragment containing three tandemly linked copies of the human HRAS gene was inserted into the genome of C57BL/6J mouse, generating a mouse model designated as KI.C57‐ras (or named NF‐hHRAS). Whole‐genome sequencing and Sanger sequencing were utilized to it confirm precise insertion and copy number. The stability of transgene expression among different generations was verified from multiple aspects using by digital PCR, western blot and DNA sequencing. To assess tumor susceptibility in the mouse model, N‐Nitroso‐N‐methylurea (MNU) was administered at a dosage of 75 mg/kg. Histopathological examinations were conducted using hematoxylin and eosin (H&E) staining.ResultsThe HRAS DNA fragment was inserted into mouse chromosome 15E1 site, locating between 80 623 202 bp and 80 625 020 bp. NF‐hHRAS mice exhibited stable inheritance and displayed consistent phenotypes across individuals. Moreover, this mouse model exhibited a high susceptibility to carcinogens. Upon administration of MNU the earliest mortality onset was earlier than that of wild‐type littermates (day 65 vs. day 78 for male and day 56 vs. day 84 for female). Notably, 100% of the NF‐hHRAS transgenic mice developed tumors, with approximately 84% of male NF‐hHRAS mice exhibiting specific tumor types, such as squamous cell carcinoma or squamous cell papilloma, which was consistent with the previously reported carcinogenic rasH2 mouse model. The types of tumors and the target organs exhibited diversity in NF‐hHRAS mice, while the spontaneous tumor incidence remained low (1/50).ConclusionsThe NF‐hHRAS mice demonstrated excellent genetic stability, a reproducible phenotype, and high susceptibility to carcinogens, indicating their potential utility in non‐clinical safety evaluations of drugs as per the S1B guidelines issued by the ICH (The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use).

Precise manipulation of genome structural variations holds great potential for plant trait improvement and biological research. Here we present a genome-editing approach, dual prime editing (DualPE), that efficiently facilitates precise deletion, replacement and inversion of large DNA fragments in plants. In our experiments, DualPE enabled the production of specific genomic deletions ranging from ~500 bp to 2 Mb in wheat protoplasts and plants. DualPE was effective in directly replacing wheat genomic fragments of up to 258 kb with desired sequences in the absence of donor DNA. Additionally, DualPE allowed precise DNA inversions of up to 205.4 kb in wheat plants with efficiencies of up to 51.5%. DualPE also successfully edited large DNA fragments in the dicots Nicotiana benthamiana and tomato, with editing efficiencies of up to 72.7%. DualPE thus provides a precise and efficient approach for large DNA sequence and chromosomal engineering, expanding the availability of precision genome-editing tools for crop improvement.

We describe a transgenesis platform for Drosophila melanogaster that integrates three recently developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and bacteriophage PhiC31-mediated transgenesis. The BAC is maintained at low copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. PhiC31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure/function analyses of most Drosophila genes.

The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep

A rapid and versatile site-directed method of mutagenesis for double-stranded plasmid DNA

Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the -6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1kb was 79.6%, and the editing efficiencies for 5- and 20kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.

Pichia pastoris has been widely exploited for the heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value chemicals and pharmaceuticals. Effective synthetic biology tools for genetic engineering are essential for industrial and biotechnological research in this yeast. Here, we describe a novel and efficient genome editing method mediated by the CRISPR-Cpf1 system, which could facilitate the deletion of large DNA fragments and integration of multiplexed gene fragments. The CRISPR-Cpf1 system exhibited a precise and high editing efficiency for single-gene disruption (99 ± 0.8%), duplex genome editing (65 ± 2.5% to 80 ± 3%), and triplex genome editing (30 ± 2.5%). In addition, the deletion of large DNA fragments of 20kb and one-step integration of multiple genes were first achieved using the developed CRISPR-Cpf1 system. Taken together, this study provides an efficient and simple gene editing tool for P.pastoris. The novel multiloci gene integration method mediated by CRISPR-Cpf1 may accelerate the ability to engineer this methylotrophic yeast for metabolic engineering and genome evolution in both biotechnological and biomedical applications.

We investigated the relationship between the outer membrane protein OprD2 and carbapenem-resistance in 141 clinical isolates of Pseudomonas aeruginosa collected between January and December 2013 from the First Affiliated Hospital of Anhui Medical University in China. Agar dilution methods were employed to determine the minimum inhibitory concentration of meropenem (MEM) and imipenem (IMP) for P. aeruginosa. The gene encoding OprD2 was amplified from141 P. aeruginosa isolates and analyzed by PCR and DNA sequencing. Differences between the effects of IMPR and IMPS groups on the resistance of the P. aeruginosa were observed by SDS-poly acrylamide gel electrophoresis (SDS-PAGE). Three resistance types were classified in the 141 carbapenem-resistant P. aeruginosa (CRPA) isolates tested, namely IMPRMEMR (66.7%), IMPRMEMS (32.6%), and IMPRMEMS (0.7%). DNA sequencing revealed significant diverse gene mutations in the OprD2-encoding gene in these strains. Thirty-four strains had large fragment deletions in the OprD2gene, in 6 strains the gene contained fragment inserts, and in 96 resistant strains, the gene featured small fragment deletions or multi-site mutations. Only 4 metallo-β-lactamase strains and 1 imipenem-sensitive (meropenem-resistant) strain showed a normal OprD2 gene. Using SDS-PAGE to detect the outer membrane protein in 16 CRPA isolates, it was found that 10 IMPRMEMR strains and 5 IMPRMEMS strains had lost the OprD2 protein, while the IMPSMEMR strain contained a normal 46-kDa protein. In conclusion, mutation or loss of the OprD2-encoding gene caused the loss of OprD2, which further led to carbapenem-resistance of P. aeruginosa. Our findings provide insights into the mechanism of carbapenem resistance in P. aeruginosa.

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy1,2. Twin prime editing (PE) guide RNAs (pegRNAs) have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs3–6. Here we describe a Prime Assembly (PA) approach for the insertion of large DNA donor fragments, whose ends are designed to overlap with the flaps generated by twinPE. We used PA to insert one, two, or three overlapping DNA fragments, with total insertion sizes ranging from 0.1 to 11 kilobase pairs. An inhibitor of non-homologous end joining (NHEJ) enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced and does not require co-delivery of exogenous DNA-dependent DNA polymerases. Our study demonstrates that PA can initiate “Gibson-like” assembly in cells to generate gene insertions without double-stranded DNA breaks or recombinases.