Improving Stability and Specificity of CRISPR/Cas9 System by Selective Modification of Guide RNAs with 2′-fluoro and Locked Nucleic Acid Nucleotides

The genome editing approach using the components of the CRISPR/Cas system has found wide application in molecular biology, fundamental medicine and genetic engineering. A promising method is to increase the efficacy and specificity of CRISPR/Cas-based genome editing systems by modifying their components. Here, we designed and chemically synthesized guide RNAs (crRNA, tracrRNA and sgRNA) containing modified nucleotides (2’-O-methyl, 2’-fluoro, LNA—locked nucleic acid) or deoxyribonucleotides in certain positions. We compared their resistance to nuclease digestion and examined the DNA cleavage efficacy of the CRISPR/Cas9 system guided by these modified guide RNAs. The replacement of ribonucleotides with 2’-fluoro modified or LNA nucleotides increased the lifetime of the crRNAs, while other types of modification did not change their nuclease resistance. Modification of crRNA or tracrRNA preserved the efficacy of the CRISPR/Cas9 system. Otherwise, the CRISPR/Cas9 systems with modified sgRNA showed a remarkable loss of DNA cleavage efficacy. The kinetic constant of DNA cleavage was higher for the system with 2’-fluoro modified crRNA. The 2’-modification of crRNA also decreased the off-target effect upon in vitro dsDNA cleavage.     

Mismatch Intolerance of 5′-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

The CRISPR/Cas9 system has recently emerged as a useful gene-specific editing tool. However, this approach occasionally results in the digestion of both the DNA target and similar DNA sequences due to mismatch tolerance, which remains a significant drawback of current genome editing technologies. However, our study determined that even single-base mismatches between the target DNA and 5′-truncated sgRNAs inhibited target recognition. These results suggest that a 5′-truncated sgRNA/Cas9 complex could be used to negatively select single-base-edited targets in microbial genomes. Moreover, we demonstrated that the 5′-truncated sgRNA method can be used for simple and effective single-base editing, as it enables the modification of individual bases in the DNA target, near and far from the 5′ end of truncated sgRNAs. Further, 5′-truncated sgRNAs also allowed for efficient single-base editing when using an engineered Cas9 nuclease with an expanded protospacer adjacent motif (PAM; 5′-NG), which may enable whole-genome single-base editing.     

Points of View on the Tools for Genome/Gene Editing

Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H₈₄₀A) and RuvC nuclease activity null mutant (D₁₀A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D₁₀A), Cas9(H₈₄₀A), and Cas9(D₁₀A, H₈₄₀A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.     

Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.     

Probing the Dynamics of Streptococcus pyogenes Cas9 Endonuclease Bound to the sgRNA Complex Using Hydrogen-Deuterium Exchange Mass Spectrometry

The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing tools. The attainment of high specificity and efficiency of Cas9 during targetted DNA cleavage is the main problem that limits the clinical application of the CRISPR–Cas9 system. A deep understanding of the Cas9 mechanism and its structural-functional relationships is required to develop strategies for precise gene editing. Here, we present the first attempt to describe the solution structure of Cas9 from S. pyogenes using hydrogen-deuterium exchange mass spectrometry (HDX-MS) coupled to molecular dynamics simulations. HDX data revealed multiple protein regions with deuterium uptake levels varying from low to high. By analysing the difference in relative deuterium uptake by apoCas9 and its complex with sgRNA, we identified peptides involved in the complex formation and possible changes in the protein conformation. The REC3 domain was shown to undergo the most prominent conformational change upon enzyme-RNA interactions. Detection of the HDX in two forms of the enzyme provided detailed information about changes in the Cas9 structure induced by sgRNA binding and quantified the extent of the changes. The study demonstrates the practical utility of HDX-MS for the elucidation of mechanistic aspects of Cas9 functioning.     

Versatile 3′ Functionalization of CRISPR Single Guide RNA

Specific applications of CRISPR/Cas genome editing systems benefit from chemical modifications of the sgRNA. Herein we describe a versatile and efficient strategy for functionalization of the 3′-end of a sgRNA. An exemplary collection of six chemically modified sgRNAs was prepared containing crosslinkers, a fluorophore and biotin. Modification of the sgRNA 3′-end was broadly tolerated by Streptococcus pyogenes Cas9 in an in vitro DNA cleavage assay. The 3′-biotinylated sgRNA was used as an affinity reagent to identify IGF2BP1, YB1 and hnRNP K as sgRNA-binding proteins present in HEK293T cells. Overall, the modification strategy presented here has the potential to expand on current applications of CRISPR/Cas systems.     

Thermodynamic Swings: How Ideal Complex of Cas9–RNA/DNA Forms

Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR–Cas9 tool. The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing systems. The attainment of high-specificity and -efficiency Cas9 during targeted DNA cleavage is the main problem that limits the practical application of the CRISPR–Cas9 system. In this study, we analyzed the thermodynamics of interaction of a complex’s components of Cas9–RNA/DNA through experimental and computer simulation methods. We found that there is a small energetic preference during Cas9–RNA/DNA formation from the Cas9–RNA and DNA/DNA duplex. The small difference in binding energy is relevant for biological interactions and could be part of the sequence-specific recognition of double-stranded DNA by the CRISPR–Cas9 system.     

Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems

Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.     

CRISPR/Cas9 Technology and Its Utility for Crop Improvement

The rapid growth of the global population has resulted in a considerable increase in the demand for food crops. However, traditional crop breeding methods will not be able to satisfy the worldwide demand for food in the future. New gene-editing technologies, the most widely used of which is CRISPR/Cas9, may enable the rapid improvement of crop traits. Specifically, CRISPR/Cas9 genome-editing technology involves the use of a guide RNA and a Cas9 protein that can cleave the genome at specific loci. Due to its simplicity and efficiency, the CRISPR/Cas9 system has rapidly become the most widely used tool for editing animal and plant genomes. It is ideal for modifying the traits of many plants, including food crops, and for creating new germplasm materials. In this review, the development of the CRISPR/Cas9 system, the underlying mechanism, and examples of its use for editing genes in important crops are discussed. Furthermore, certain limitations of the CRISPR/Cas9 system and potential solutions are described. This article will provide researchers with important information regarding the use of CRISPR/Cas9 gene-editing technology for crop improvement, plant breeding, and gene functional analyses.     

Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.     

Enhancing gene editing efficiency for cells by CRISPR/Cas9 system-loaded multilayered nanoparticles assembled via microfluidics

Most non-viral carriers for in vitro delivery of nucleic acids suffer from low efficiency of introducing mRNA and other nucleic acids,especially large mRNA.Cas9 protein is the nuclease part of the powerful gene-editing tool,CRISPR/Cas9 system,Cas9 mRNA is particularly large,thus presents a big challenge for delivery.We assembled a multilayered biodegradable nanocarrier to load Cas9 mRNA inside to protect Cas9 mRNA from degradation.We used a microfluidic chip to synthesize a small,positively charged,and degradable core to attract negatively charged Cas9 mRNA.The microfluidic assembly allows the core to be small enough to incorporate into a cationic liposome.The multilayered nanocarriers elevated the delivery efficiency of Cas9 mRNA by over 2 folds and increased the expression by over 5 folds compared to commercially used non-viral carriers.In addition,the multilayered nanocarriers do not require reduced serum medium for transfection.When using the standard complete medium for transfection,the multilayered nanocarriers could increase the expression of Cas9 mRNA by over 15 folds compared to commercially used non-viral carriers.The co-delivery of Cas9 mRNA and sgRNA via LRC elevated the gene-editing efficiency by 3 folds compared to that via commercially used non-viral carriers.Based on the higher transfection efficiency of Cas9 mRNA/sgRNA than commercially used non-viral carriers,these multilayered nanocarriers may have a good prospect as efficient commercial delivery carriers for Cas9 mRNA/sgRNA and other nucleic acids.     

Single-Step Genome Editing of Small Ruminant Embryos by Electroporation

We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos.     

Application of CRISPR/CasΦ2 System for Genome Editing in Plants

CRISPR/Cas system has developed a new technology to modify target genes. In this study, CasΦ2 is a newly Cas protein that we used for genome modification in Arabidopsis and tobacco. PDS and BRI1 of marker genes were chosen for targeting. CasΦ2 has the function to cleave pre-crRNA. In the presence of 10 mM Mg²⁺ irons concentration, sgRNA3 type guided CasΦ2 to edit target gene and generate mutation, and a mutant seedling of AtBRI1 gene with an expected male sterile phenotype was obtained. In the process of tobacco transformation, the gene editing activity of CasΦ2 can be activated by 100 nM Mg²⁺ irons concentration, and sgRNA1 type guided CasΦ2 to edit target gene. Mutant seedlings of NtPDS gene with an expected albino were obtained. The results indicate that CasΦ2 can effectively edit target genes under the guidance of different sgRNA type in the presence of Mg²⁺ ions. Together, our results verify that the CRISPR/CasΦ2 system is an effective and precise tool for genome editing in plants.     

Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.     

Knockout of Tobacco Homologs of Arabidopsis Multi-Antibiotic Resistance 1 Gene Confers a Limited Resistance to Aminoglycoside Antibiotics

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the Arabidopsis Multi-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacum MAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs–knockout seedlings of the T₁ generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants’ sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.     

Comparative Analysis of Genome Editors Efficiency on a Model of Mice Zygotes Microinjection

Genome editing is an indispensable tool for functional genomics. The caveat of the genome-editing pipeline is a prevalence of error-prone non-homologous end joining over homologous recombination, while only the latter is suitable to introduce particularly desired genetic variants. To overcome this problem, a toolbox of genome engineering was appended by a variety of improved instruments. In this work, we compared the efficiency of a number of recently suggested improved systems for genome editing applied to the same genome regions on a murine zygote model via microinjection. As a result, we observed that homologous recombination utilizing an ssDNA template following sgRNA directed Cas9 cleavage is still the method of choice for the creation of animals with precise genome alterations.