Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.     

Expanding the plant genome editing toolbox with recently developed CRISPR–Cas systems

Since its first appearance, CRISPR–Cas9 has been developed extensively as a programmable genome-editing tool, opening a new era in plant genome engineering. However, CRISPR–Cas9 still has some drawbacks, such as limitations of the protospacer-adjacent motif (PAM) sequence, target specificity, and the large size of the cas9 gene. To combat invading bacterial phages and plasmid DNAs, bacteria and archaea have diverse and unexplored CRISPR–Cas systems, which have the potential to be developed as a useful genome editing tools. Recently, discovery and characterization of additional CRISPR–Cas systems have been reported. Among them, several CRISPR–Cas systems have been applied successfully to plant and human genome editing. For example, several groups have achieved genome editing using CRISPR–Cas type I-D and type I-E systems, which had never been applied for genome editing previously. In addition to higher specificity and recognition of different PAM sequences, recently developed CRISPR–Cas systems often provide unique characteristics that differ from well-known Cas proteins such as Cas9 and Cas12a. For example, type I CRISPR–Cas10 induces small indels and bi-directional long-range deletions ranging up to 7.2 kb in tomatoes (Solanum lycopersicum L.). Type IV CRISPR–Cas13 targets RNA, not double-strand DNA, enabling highly specific knockdown of target genes. In this article, we review the development of CRISPR–Cas systems, focusing especially on their application to plant genome engineering. Recent CRISPR–Cas tools are helping expand our plant genome engineering toolbox.

Recently discovered and characterized clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR–Cas) systems allow additional applications to plant genome editing.     

CRISPR/Cas9 engineering of albino cystinuria Type A mice

Cystinuria Type A is a relatively common genetic kidney disease occurring in 1 in 7,000 people worldwide that results from mutation of the cystine transporter rBAT encoded by Slc3a1. We used CRISPR/Cas9 technology to engineer cystinuria Type A mice via genome editing of the C57BL/6NHsd background. These mice are an improvement on currently available models as they are on a coisogenic genetic background and have a single defined mutation. In order to use albinism to track Cas9 activity, we co‐injected gRNAs targeting Slc3a1 and tyrosinase (Tyr) with Cas9 expressing plasmid DNA into mouse embryos. Two different Slc3a1 mutational alleles were derived, with homozygous mice of both demonstrating elevated urinary cystine levels, cystine crystals, and bladder stones. We used whole genome sequencing to evaluate for potential off‐target editing. No off‐target indels were observed for the top 10 predicted off‐targets for Slc3a1 or Tyr. Therefore, we used CRISPR/Cas9 to generate coisogenic albino cystinuria Type A mice that could be used for in vivo imaging, further study, or developing new treatments of cystinuria.     

Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish

Customized TALENs and Cas9/gRNAs have been used for targeted mutagenesis in zebrafish to induce indels into protein-coding genes. However, indels are usually not sufficient to disrupt the function of non-coding genes, gene clusters or regulatory sequences, whereas large genomic deletions or inversions are more desirable for this purpose. By injecting two pairs of TALEN mRNAs or two gRNAs together with Cas9 mRNA targeting distal DNA sites of the same chromosome, we obtained predictable genomic deletions or inversions with sizes ranging from several hundred bases to nearly 1 Mb. We have successfully achieved this type of modifications for 11 chromosomal loci by TALENs and 2 by Cas9/gRNAs with different combinations of gRNA pairs, including clusters of miRNA and protein-coding genes. Seven of eight TALEN-targeted lines transmitted the deletions and one transmitted the inversion through germ line. Our findings indicate that both TALENs and Cas9/gRNAs can be used as an efficient tool to engineer genomes to achieve large deletions or inversions, including fragments covering multiple genes and non-coding sequences. To facilitate the analyses and application of existing ZFN, TALEN and CRISPR/Cas data, we have updated our EENdb database to provide a chromosomal view of all reported engineered endonucleases targeting human and zebrafish genomes.     

Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism

Key message

A method based on DNA single-strand conformation polymorphism is demonstrated for effective genotyping of CRISPR/Cas9-induced mutants in rice.

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has been widely adopted for genome editing in many organisms. A large proportion of mutations generated by CRISPR/Cas9 are very small insertions and deletions (indels), presumably because Cas9 generates blunt-ended double-strand breaks which are subsequently repaired without extensive end-processing. CRISPR/Cas9 is highly effective for targeted mutagenesis in the important crop, rice. For example, homozygous mutant seedlings are commonly recovered from CRISPR/Cas9-treated calli. However, many current mutation detection methods are not very suitable for screening homozygous mutants that typically carry small indels. In this study, we tested a mutation detection method based on single-strand conformational polymorphism (SSCP). We found it can effectively detect small indels in pilot experiments. By applying the SSCP method for CRISRP-Cas9-mediated targeted mutagenesis in rice, we successfully identified multiple mutants of OsROC5 and OsDEP1. In conclusion, the SSCP analysis will be a useful genotyping method for rapid identification of CRISPR/Cas9-induced mutants, including the most desirable homozygous mutants. The method also has high potential for similar applications in other plant species.

     

Gene editing the phytoene desaturase alleles of Cavendish banana using CRISPR/Cas9

Bananas are a staple food source and a major export commodity worldwide. The Cavendish dessert banana is a triploid AAA genome type and accounts for around 47% of global production. Being essentially sterile, genetic modification is perhaps the only pathway available to improve this cultivar. In this study, we used the CRISPR/Cas9 gene editing system to deliver a self-cleaving polycistronic guide RNA (gRNA) designed to target exon 1 of the Phytoene desaturase (PDS) gene in the Cavendish cultivar “Williams”. Genotyping of 19 independent events showed a 100% PDS modification rate primarily in the form of insertions (1–105 nt) or deletions (1–55 nt) (indels) at the predicted cleavage site. Tri-allelic disruptive modifications were observed in 63% of plants and resulted in both albinism and dwarfing. Pale green (16%) and wildtype green (21%) phenotypes generally correlated with in-frame indels in at least one of the three PDS alleles. Editing efficiency was dependent on both target site selection and Cas9 abundance. This is the first report of a highly effective CRISPR/Cas9 modification system using a polycistronic gRNA in Cavendish banana. Such an editing platform will be of considerable utility for the development of disease resistance and novel agro-traits in this commercially important cultivar into the future.     

CRISPR/Cas9—The ultimate weapon to battle infectious diseases?

Infectious diseases are a leading cause of death worldwide. Novel therapeutics are urgently required to treat multidrug‐resistant organisms such as Mycobacterium tuberculosis and to mitigate morbidity and mortality caused by acute infections such as malaria and dengue fever virus as well as chronic infections such as human immunodeficiency virus‐1 and hepatitis B virus. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system, which has revolutionized biomedical research, holds great promise for the identification and validation of novel drug targets. Since its discovery as an adaptive immune system in prokaryotes, the CRISPR/Cas9 system has been developed into a multi‐faceted genetic modification tool, which can now be used to induce gene deletions or specific gene insertions, such as conditional alleles or endogenous reporters in virtually any organism. The generation of CRISPR/Cas9 libraries that can be used to perform phenotypic whole genome screens provides an important new tool that will aid in the identification of critical host factors involved in the pathogenesis of infectious diseases. In this review, we will discuss the development and recent applications of the CRISPR/Cas9 system used to identify novel regulators, which might become important in the fight against infectious diseases.     

Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System

Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.     

Efficient multinucleotide deletions using deaminase-Cas9 fusions in human cells

CRISPR/Cas9 system is a robust genome editing platform in biotechnology and medicine. However, it generally produces small insertions/deletions (indels, typically 1–3 bp) but rarely induces larger deletions in specific target sites. Here, we report a cytidine deaminase-Cas9 fusion-induced deletion system (C-DEL) and an adenine deaminase-Cas9 fusion-induced deletion system (A-DEL) by combining Cas9 with rat APOBEC1 (rA1) and TadA 8e, respectively. Both C-DEL and A-DEL improve the efficiency of deletions compared with the conventional Cas9 system in human cells. In addition, the C-DEL system generates a considerable fraction of predictable multinucleotide deletions from 5′-deaminated C bases to the Cas9-cleavage site and increases the proportion of larger deletions at the target loci. Taken together, the C-DEL and A-DEL systems provide a practical strategy for producing efficient multinucleotide deletions, expanding the CRISPR/Cas9 toolsets for gene modifications in human cells.     

In rice splice variants that restore the reading frame after frameshifting indel introduction are common,often induced by the indels and sometimes lead to organism-level rescue

The introduction of frameshifting non-3n indels enables the identification of gene-trait associations. However, it has been hypothesised that recovery of the original reading frame owing to usage of non-canonical splice forms could cause rescue. To date there is very little evidence for organism-level rescue by such a mechanism and it is unknown how commonly indels induce, or are otherwise associated with, frame-restoring splice forms. We perform CRISPR/Cas9 editing of randomly selected loci in rice to investigate these issues. We find that the majority of loci have a frame-restoring isoform. Importantly, three quarters of these isoforms are not seen in the absence of the indels, consistent with indels commonly inducing novel isoforms. This is supported by analysis in the context of NMD knockdowns. We consider in detail the two top rescue candidates, in wax deficient anther 1 (wda1) and brittle culm (bc10), finding that organismal-level rescue in both cases is strong but owing to different splice modification routes. More generally, however, as frame-restoring isoforms are low abundance and possibly too disruptive, such rescue we suggest to be the rare exception, not the rule. Nonetheless, assuming that indels commonly induce frame-restoring isoforms, these results emphasize the need to examine RNA level effects of non-3n indels and suggest that multiple non-3n indels in any given gene are advisable to probe a gene’s trait associations.     

Investigation of CRISPR/Cas9-induced SD1 rice mutants highlights the importance of molecular characterization in plant molecular breeding

Although Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/CRISPR-associated 9(Cas9) system has been widely used for basic research in model plants,its application for applied breeding in crops has faced strong regulatory obstacles,due mainly to a poor understanding of the authentic output of this system,particularly in higher generations.In this study,different from any previous studies,we investigated in detail the molecular characteristics and production performance of CRISPR/Cas9-generated SD1(semi-dwarf 1) mutants from T_2 to T_4 generations,of which the selection of T_1 and T_2 was done only by visual phenotyping for semidwarf plants.Our data revealed not only on-and off-target mutations with small or lager indels but also exogenous elements in T_2 plants.All indel mutants passed stably to T_3 or T_4 without additional modifications independent on the presence of Cas9,while some lines displayed unexpected hereditary patterns of Cas9 or some exogenous elements.In addition,effects of various SD1 alleles on rice height and yield differed depending on genetic backgrounds.Taken together,our data showed that the CRISPR/Cas9 system is effective in producing homozygous mutants for functional analysis,but it may be not as precise as expected in rice,and that early and accurate molecular characterization and screening must be carried out for generations before transitioning of the CRISPR/Cas9 system from laboratory to field.     

Designing gene drives to limit spillover to non-target populations

The prospect of utilizing CRISPR-based gene-drive technology for controlling populations has generated much excitement. However, the potential for spillovers of gene-drive alleles from the target population to non-target populations has raised concerns. Here, using mathematical models, we investigate the possibility of limiting spillovers to non-target populations by designing differential-targeting gene drives, in which the expected equilibrium gene-drive allele frequencies are high in the target population but low in the non-target population. We find that achieving differential targeting is possible with certain configurations of gene-drive parameters, but, in most cases, only under relatively low migration rates between populations. Under high migration, differential targeting is possible only in a narrow region of the parameter space. Because fixation of the gene drive in the non-target population could severely disrupt ecosystems, we outline possible ways to avoid this outcome. We apply our model to two potential applications of gene drives—field trials for malaria-vector gene drives and control of invasive species on islands. We discuss theoretical predictions of key requirements for differential targeting and their practical implications.     

CRISPR/Cas9‐mediated MSTN disruption and heritable mutagenesis in goats causes increased body mass

Genetic engineering in livestock has been greatly enhanced through the use of artificial programmed nucleases such as the recently emerged clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9) system. We recently reported our successful application of the CRISPR/Cas9 system to engineer the goat genome through micro‐injection of Cas9 mRNA and sgRNAs targeting MSTN and FGF5 in goat embryos. The phenotypes induced by edited loss‐of‐function mutations of MSTN remain to be evaluated extensively. We demonstrate the utility of this approach by disrupting MSTN, resulting in enhanced body weight and larger muscle fiber size in Cas9‐mediated gene‐modified goats. The effects of genome modifications were further characterized by H&E staining, quantitative PCR, Western blotting and immunofluorescence staining. Morphological and genetic analyses indicated the occurrence of phenotypic and genotypic modifications. We further provide sufficient evidence, including breeding data, to demonstrate the transmission of the knockout alleles through the germline. By phenotypic and genotypic characterization, we demonstrated the merit of using the CRISPR/Cas9 approach for establishing genetically modified livestock with an enhanced production trait.     

CRISPR_Cas systems for fungal research

Genome-editing CRISPR-Cas systems, using Cas9 and Cas12a endonucleases, have improved our ability to precisely edit genomes and control gene expression. We summarize here the knowledge gained from using CRISPR-Cas9 and CRISPR-Cas12a in fungal research. Also discussed are strategies developed for limiting the occurrences of off-target mutations caused by CRISPR-Cas genome editing.     

Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers

Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non‐homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ～20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25‐fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR‐Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.     

Targeted Mutagenesis in Atlantic Salmon (Salmo salar L.) Using the CRISPR/Cas9 System Induces Complete Knockout Individuals in the F0 Generation

Understanding the biological function behind key proteins is of great concern in Atlantic salmon, both due to a high commercial importance and an interesting life history. Until recently, functional studies in salmonids appeared to be difficult. However, the recent discovery of targeted mutagenesis using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system enables performing functional studies in Atlantic salmon to a great extent. We used the CRISPR/Cas9 system to target two genes involved in pigmentation, tyrosinase (tyr) and solute carrier family 45, member 2 (slc45a2). Embryos were assayed for mutation rates at the 17 somite stage, where 40 and 22% of all injected embryos showed a high degree of mutation induction for slc45a2 and tyr, respectively. At hatching this mutation frequency was also visible for both targeted genes, displaying a graded phenotype ranging from complete lack of pigmentation to partial loss and normal pigmentation. CRISPRslc45a2/Cas9 injected embryos showing a complete lack of pigmentation or just a few spots of pigments also lacked wild type sequences when assaying more than 80 (slc45a2) sequence clones from whole embryos. This indicates that CRISPR/Cas9 can induce double-allelic knockout in the F0 generation. However, types and frequency of indels might affect the phenotype. Therefore, the variation of indels was assayed in the graded pigmentation phenotypes produced by CRISPR/Cas9-slc45a2. The results show a tendency for fewer types of indels formed in juveniles completely lacking pigmentation compared to juveniles displaying partial pigmentation. Another interesting observation was a high degree of the same indel type in different juveniles. This study shows for the first time successful use of the CRISPR/Cas9 technology in a marine cold water species. Targeted double-allelic mutations were obtained and, though the level of mosaicism has to be considered, we demonstrate that F0 fish can be used for functional studies in Atlantic salmon.     

Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.     

Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein

The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9–FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.