Drosophila models used to simulate human ATP1A1 gene mutations that cause Charcot-Marie-Tooth type 2 disease and refractory seizures

Certain amino acids changes in the human Na^(+)/K^(+)-ATPase pump,ATPase Na^(+)/K^(+)transporting subunit alpha 1(ATP1A1),cause Charcot-Marie-Tooth disease type 2(CMT2)disease and refractory seizures.To develop in vivo models to study the role of Na^(+)/K^(+)-ATPase in these diseases,we modified the Drosophila gene homolog,Atpα,to mimic the human ATP1A1 gene mutations that cause CMT2.Mutations located within the helical linker region of human ATP1A1(I592T,A597T,P600T,and D601F)were simultaneously introduced into endogenous Drosophila Atpαby CRISPR/Cas9-mediated genome editing,generating the Atpα^(TTTF)model.In addition,the same strategy was used to generate the corresponding single point mutations in flies(Atpα^(I571T),Atpα^(A576T),Atpα^(P579T),and Atpα^(D580F)).Moreover,a deletion mutation(Atpα^(mut))that causes premature termination of translation was generated as a positive control.Of these alleles,we found two that could be maintained as homozygotes(Atpα^(I571T)and Atpα^(P579T)).Three alleles(Atpα^(A576T),Atpα^(P579)and Atpα^(D580F))can form heterozygotes with the Atpαmut allele.We found that the Atpαallele carrying these CMT2-associated mutations showed differential phenotypes in Drosophila.Flies heterozygous for Atpα^(TTTF)mutations have motor performance defects,a reduced lifespan,seizures,and an abnormal neuronal morphology.These Drosophila models will provide a new platform for studying the function and regulation of the sodium-potassium pump.

Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome(PRRS)is recognized as one of the most infectious viral diseases of swine.Although Cluster of differentiation 163(CD163)is identified as an essential receptor for mediating PRRS virus(PRRSV)infection,the important residues involved in infection on CD163 are still unclear.Therefore,it is very important to identify these key residues to study the mechanism of PRRSV infection and to generate anti-PRRSV pigs.In this study,we first generated immortalized porcine alveolar macrophage(IPAM)cell lines harboring 40-residues(residues 523-562,including R561(arginine(R)at position 561))deletion of CD163.PRRSV infection experiments showed that these IPAM cell lines were completely resistant to PRRSV infection.We then generated cloned pigs carrying CD163-R561A(an arginine(R)to alanine(A)substitution at position 561 of CD163).PRRSV challenge experiments in porcine alveolar macrophages(PAMs)isolated from the CD163-R561A pigs showed significantly lower susceptibility to PRRSV than that of CD163-R561 PAMs.Through this study,we show that CD163523-562 contains essential residues for mediating PRRSV infection,and that CD163 R561 significantly contributes to PRRSV infection but is not essential for infection.These functional sites can therefore serve as new targets for understanding the mechanism of PRRSV infection.Furthermore,CD163-R561A pigs can be used as an important model for improving pig germplasm with resistance against PRRSV.

Genome engineering using the CRISPR/Cas system

Recently, an epoch-making genome engineering technology using clustered regularly at interspaced short palindromic repeats(CRISPR) and CRISPR associated(Cas) nucleases, was developed. Previous technologies for genome manipulation require the time-consuming design and construction of genome-engineered nucleases for each target and have, therefore, not been widely used in mouse research where standard techniques based on homologous recombination are commonly used. The CRISPR/Cas system only requires the design of sequences complementary to a target locus, making this technology fast and straightforward. In addition, CRISPR/Cas can be used to generate mice carrying mutations in multiple genes in a single step, an achievement not possible using other methods. Here, we review the uses of this technology in genetic analysis and manipulation, including achievements made possible to date and the prospects for future therapeutic applications.

Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

In plants and mammals,non-homologous end-joining is the dominant pathway to repair DNA doublestrand breaks,making it challenging to generate knock-in events.In this study,we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana.We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems.In Arabidopsis thaliana,fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants.In addition,we demonstrated stable and heritable knock-ins in wheat in 1%of the primary transformants.Taken together,our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

Precise gene replacement in plants through CRISPR/Cas genome editing technology:current status and future perspectives

CRISPR/Cas,as a simple,versatile,robust and cost-effective system for genome manipulation,has dominated the genome editing field over the past few years.The application of CRISPR/Cas in crop improvement is particularly important in the context of global climate change,as well as diverse agricultural,environmental and ecological challenges.Various CRISPR/Cas toolboxes have been developed and allow for targeted mutagenesis at specific genome loci,transcriptome regulation and epigenome editing,base editing,and precise targeted gene/allele replacement or tagging in plants.In particular,precise replacement of an existing allele with an elite allele in a commercial variety through homology-directed repair(HDR)is a holy grail in genome editing for crop improvement as it has been very difficult,laborious and time-consuming to introgress the elite alleles into commercial varieties without any linkage drag from parental lines within a few generations in crop breeding practice.However,it still remains very challenging in crop plants.This review intends to provide an informative summary of the latest development and breakthroughs in gene replacement using CRISPR/Cas technology,with a focus on achievements,potential mechanisms and future perspectives in plant biological science as well as crop improvement.

The Application of CRISPR-Cas9 Genome Editing in Caenorhabditis elegans

Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unparalleled efficacy and facility for modifying genomes in a wide variety of organisms. Caenorhabditis elegans is one of the most convenient multicellular organisms for genetic analysis, and the application of this novel genome editing technique to this organism promises to revolutionize analysis of gene function in the future. CRISPR-Cas9 has been successfully used to generate imprecise insertions and deletions via non-homologous end-joining mechanisms and to create precise mutations by homology-directed repair from donor templates. Key variables are the methods used to deliver the Cas9 endonuclease and the efficiency of the single guide RNAs. CRISPR-Cas9-mediated editing appears to be highly specific in C. elegans, with no reported off-target effects. In this review, 1 briefly summarize recent progress in CRISPR-Cas9-based genome editing in C. elegans, highlighting technical improvements in mutagenesis and mutation detection, and discuss potential future appli- cations of this technique.

A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system

Clustered regulatory interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 nuclease(Cas9),the third-generation genome editing tool,has been favored because of its high efficiency and clear system composition.In this technology,the introduced double-strand breaks(DSBs)are mainly repaired by non-homologous end joining(NHEJ)or homology-directed repair(HDR)pathways.The high-fidelity HDR pathway is used for genome modification,which can introduce artificially controllable insertions,deletions,or substitutions carried by the donor templates.Although high-level knock-out can be easily achieved by NHEJ,accurate HDR-mediated knock-in remains a technical challenge.In most circumstances,although both alleles are broken by endonucleases,only one can be repaired by HDR,and the other one is usually recombined by NHEJ.For gene function studies or disease model establishment,biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes.Thus,there is an urgent need for an efficient biallelic editing system.Here,we developed three pairs of integrated selection systems,where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker.Flanked by homologous arms containing the mutated sequences,the selection cassettes were integrated into the target site,mediated by CRISPR/Cas9-induced HDR.Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance.We tested this novel method on the amyloid precursor protein(APP)and presenilin 1(PSEN1)loci and demonstrated up to 82.0%biallelic editing efficiency after optimization.Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.