Generation of knock-in lampreys by CRISPR-Cas9-mediated genome engineering

The lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience. However, no knock-in technique for this animal has been established yet, preventing application of advanced genetic techniques. Here, we report efficient generation of F0 knock-in lampreys by CRISPR-Cas9-mediated genome editing. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) for genome digestion, a sgRNA for donor plasmid digestion, and Cas9 mRNA. Targeting different genetic loci, we succeeded in generating knock-in lampreys expressing photoconvertible protein Dendra2 as well as those expressing EGFP. With its simplicity, design flexibility, and high efficiency, we propose that the present method has great versatility for various experimental uses in lamprey research and that it can also be applied to other “non-model” organisms.

Specific Correction of the Intron-22 Inverted Factor VIII Gene in Autologous Blood Outgrowth Endothelial Cells from Patients with Severe Hemophilia A

A heterogeneous collection of >1500 distinct causative factor (F) VIII gene (F8) mutations have been identified thus far in unrelated severe Hemophilia A (HA) patients, who have less than 1% of normal FVIII activity in their plasmas and experience recurrent bleeding that often results in crippling arthropathies and can be life threatening. Curative gene therapies are being pursued intensely as the current standard of care, which involves 2-3 infusions/week of therapeutic FVIII (tFVIII) proteins throughout a patient's life, is extremely expensive and very demanding. Moreover, ~25% of patients with severe HA (PSHA) develop anti-tFVIII antibodies that neutralize the efficacy of their tFVIII proteins. Despite remarkable progress in clinical trials of various adeno-associated viruses (AAVs) as vectors for in vivo delivery of therapeutic F8 genes, it is not clear how widespread viral-mediated gene replacement therapy (GRT) will become due to current limitations that include the: (1) presence of existing immunity to the AAV capsid protein (CP) in ~30-70% of PSHA for whom GRT is contraindicated; (2) immunity to AAV-CP induced in all PSHA during the initial GRT that precludes subsequent dosing; (3) use of heterologous promoters which drive F8 expression in non-physiologic cells that may increase the encoded tFVIII protein's immunogenicity; and 4) episomal location of AAV-genome replication, which, together with "(2)" and "(3)", precludes GRT in children. These important unmet needs require new gene-based therapeutic strategies for HA. Our goal was to develop a virus-free, ex vivo personalized gene repair therapy that minimally manipulates the mutant F8 in autologous patient-derived blood outgrowth endothelial cells (BOECs)-the physiologically relevant cell type for FVIII production in vivo-followed by their expansion and reinfusion into the same individual patient. We chose to focus initially on the intron (I) 22 inversion (I22I) mutation initially as it is causative in >40% of all PSHA. CRISPR/Cas9 guide RNAs were designed to target the 3' end of F8 exon (E) 22. For initial experiments in K562 cells, a donor plasmid containing a restriction enzyme site was nucleofected with the CRISPR system encoding plasmid, which triggered successful homology-directed repair (HDR) at the target site (efficiency of 18.2%, n=2). Subsequently, a cDNA-based therapeutic donor plasmid was constructed containing all F8 coding sequences in E23-E26 followed by a bGH polyA signal. After appropriate informed consent was obtained, BOECs were cultured from the blood of 3 severe pediatric HA patients with the I22I (ages 6 years, 7 years, and 13 years). After nucleofecting the BOECs with the F8-specific CRISPR and HDR constructs, site-specific knock-in of the cDNA at the 3' end of E22 was confirmed via PCR (n=3). Direct Sanger sequencing of the resultant amplicons from the repaired I22-inverted F8 locus in treated BOECs from one representative patient confirmed complete and seamless knock-in of the therapeutic cDNA at the endogenous site of the mutant F8. Current efforts are to isolate clonal populations of the repaired BOECs and characterize their ability to secrete active FVIII in vitro. Similar experiments are underway using canine BOECs in the canine model of HA. The use of clonal populations of gene corrected autologous BOECs as the infused therapy allows whole genome sequencing analysis to be performed to confirm that no off-target cutting or integration occurred in the therapeutic cell preparation prior to infusion into the recipient patient, further strengthening the safety profile of this proposed autologous cell therapy. Overall, these current results lay promising proof-of-concept data for a potential new curative therapeutic alternative approach for HA which should overcome drawbacks of the current generations of AAV-based treatments. Disclosures Dinh: Haplogenics Corporation: Current Employment. Luu:Haplogenics Corporation: Current Employment. Mead:CSL Behring: Current Employment. Escobar:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Powell:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees. Howard:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees.

Creation of Golden Gate constructs for gene doctoring

Background Gene doctoring is an efficient recombination-based genetic engineering approach to mutagenesis of the bacterial chromosome that combines the λ-Red recombination system with a suicide donor plasmid that is cleaved in vivo to generate linear DNA fragments suitable for recombination. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. However, generation of donor plasmids typically requires multiple cloning and screening steps. Results We constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. Successful constructs can easily be identified through blue-white screening. We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli. We also provided related genetic parts to assist in the construction of mutagenesis cassettes with a tetracycline-selectable marker. Conclusions Our plasmid greatly simplifies the construction of Gene Doctoring donor plasmids and allows for the assembly of complex, multi-part insertion or deletion cassettes with a free choice of target sites and selection markers. The tools we developed are applicable to gene editing for a wide variety of purposes in Enterobacteriaceae and potentially in other diverse bacterial families.

Response to Comment on “RNA-guided DNA insertion with CRISPR-associated transposases”

Rice et al. suggest that the CRISPR-associated transposase ShCAST system could lead to additional insertion products beyond simple integration of the donor. We clarify the outcomes of ShCAST-mediated insertions in Escherichia coli, which consist of both simple insertions and integration of the donor plasmid. This latter outcome can be avoided by use of a 5′ nicked DNA donor.

Comment on “RNA-guided DNA insertion with CRISPR-associated transposases”

Strecker et al. (Research Articles, 5 July 2019, p. 48) described a system for exploiting a Tn7-type transposon-encoded CRISPR-Cas system to make RNA-guided, programmable insertions. Although this system has great promise, we note that the well-established biochemistry of Tn7 suggests that the particular system used may insert not only the transposon but also the entire donor plasmid.

Genomic and phenotypic analyses of six offspring of a genome-edited hornless bull

Genome editing followed by reproductive cloning was previously used to produce two hornless dairy bulls. We crossed one genome-edited dairy bull, homozygous for the dominant PC Celtic POLLED allele, with horned cows (pp) and obtained six heterozygous (PCp) polled calves. The calves had no horns and were otherwise healthy and phenotypically unremarkable. We conducted whole-genome sequencing of all animals using an Illumina HiSeq4000 to achieve ~20× coverage. Bioinformatics analyses revealed the bull was a compound heterozygote, carrying one naturally occurring PC Celtic POLLED allele and an allele containing an additional introgression of the homology-directed repair donor plasmid along with the PC Celtic allele. These alleles segregated in the offspring of this bull, and inheritance of either allele produced polled calves. No other unintended genomic alterations were observed. These data can be used to inform conversations in the scientific community, with regulatory authorities and with the public around ‘intentional genomic alterations’ and future regulatory actions regarding genome-edited animals.

High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

ABSTRACTGenetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of theEscherichia colichromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used forcadAknockout,gdhAknock-in, seamless deletion ofpepD, site-directed mutagenesis of the essentialmetKgene, and replacement ofmetKwith theRickettsiaS-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.