Cas9 RNA Research Articles

Approaches to genome editing in agricultural animals

The results of research into genetic methods of breeding agricultural animals with improved characteristics are presented. By now a significant number of animals with an edited genome have been selected. Methods of genome modification in cattle are constantly improving. The approaches of gene editing of bovine embryos, delivery of edited constructs and improvement of embryo survival after introduction of editing systems have been studied. The studies were performed on cattle embryos. BLG and SD209 gene editing systems were developed and validated. Delivery options of the editing system into cattle cells were studied: microinjection into the zygote of plasmid DNA encoded sequence with CRISPR/Cas9 c sgRNA, the method of viral vectors (adeno-associated AAV viruses, serotypes AAV1, AAV2, AAV6, AAV9, AAVDJ), co-injection of plasmid DNA and sperm into the oocyte at the MII stage, and microinjection of Cas9 and guide RNAs. Different techniques for performing microinjections have been investigated and refined, and different preparation of Cas9 RNA and guide RNA mixtures have been tested. Based on these results, the protocol for performing microinjection of the editing system was optimized and a model experiment was performed on 160 oocytes, with 80 cells per each construction. The findings have shown that the efficiency of editing has generally improved. When injected with guide RNA against BLG gene and spCas9 mRNA, 84% of the surviving cells initiated cleavage, blastulation was 20%, and BLG knockout was 69.2%. When injected against the CD209 gene and spCas9 mRNA, 44.4% of the surviving embryos started cleavage, blastulation was 16.7%, with CD209 knockout at 44.4%. The novelty of the work lies in obtaining data on the development of editing systems with specific target genes, in improving the delivery system and cultivation of bovine embryos.

Generation of Genome-Edited Mice by Cytoplasmic Injection of CRISPR-Cas9 RNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) gene editing enables rapid production of genome-edited animals. The Cas9/guide RNA (gRNA) component can be introduced into zygotes in several ways. Here, we provide an instructional guide for the generation of knockout mice using cytoplasmic injection of in vitro transcribed Cas9 RNA and gRNA.

Single Molecule FRET Analysis of CRISPR Cas9 Single Guide RNA Folding Dynamics.

CRISPR Cas9 is an RNA guided endonuclease that is part of a bacterial adaptive immune system. Single guide RNA (sgRNA) can be designed to target genomic DNA, making Cas9 a programmable DNA binding/cutting enzyme and allowing applications such as epigenome editing, controlling transcription, and targeted DNA insertion. Some of the main hurdles against an even wider adoption are off-target effects and variability in Cas9 editing outcomes. Most studies that aim to understand the mechanisms that underlie these two areas have focused on Cas9 DNA binding, DNA unwinding, and target cleavage. The assembly of Cas9 RNA ribonucleoprotein complex (RNP) precedes all these steps and includes sgRNA folding and Cas9 binding to sgRNA. We know from the crystal structure of the Cas9 RNP what the final sgRNA conformation is. However, the assembly dynamics has not been studied in detail and a better understanding of RNP assembly could lead to better-designed sgRNAs and better editing outcomes. To study this process, we developed a single molecule FRET assay to monitor the conformation of the sgRNA and the binding of Cas9 to sgRNA. We labeled the sgRNA with a donor fluorophore and an acceptor fluorophore such that when the sgRNA folds, there are changes in FRET efficiency. We measured sgRNA folding dynamics under different ion conditions, under various methods of folding (refolding vs vectorial), and with or without Cas9. sgRNA that closely mimics the sgRNA construct used for high resolution structural analysis of the Cas9-gRNA complex showed two main FRET states without Cas9, and Cas9 addition shifted the distribution toward the higher FRET state attributed to the properly assembled complex. Even in the absence of Cas9, folding the sgRNA vectorially using a superhelicase-dependent release of the sgRNA in the direction of transcription resulted in almost exclusively high FRET state. An addition of Cas9 during vectorial folding greatly reduced a slow-folding fraction. Our studies shed light on the heterogeneous folding dynamics of sgRNA and the impact of co-transcriptional folding and Cas9 binding in sgRNA folding. Further studies of sequence dependence may inform rational design of sgRNAs for optimal function.

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.

Development of CRISPR-CAS9 based RNA drugs against Eimeria tenella infection

Parasitic diseases are major trouble in many parts of the world. We consider that if a chemical can break a DNA barcode sequence, it might be used to develop a species-specific anti-parasitic agent. To examine this hypothesis, we constructed sgRNAs that target both the control (5.8S rDNA) and a DNA barcode (ITS) sequence in Eimeria tenella. In vitro experiment showed that Cas9 mRNA combined with sgRNAs could reduce the sporulation percentage of oocysts and the survival rate of sporulated oocysts and sporozoites. Quantitative real-time PCR showed that the DNAs of parasites exposed to Cas9 mRNA and sgRNAs were significantly affected, regardless of whether they were exposed to a combination of two sgRNAs or just a single sgRNA. The DNA sequencing also indicated that the experimental group exposed to two sgRNAs mixed with Cas9-induced deletion of large parts and a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments. In vivo trial, the effect of sgRNA and Cas9 RNA on the pathogenicity of E. tenella in chicken showed less lesion score and oocysts score (P < 0.05) in experimental groups than control groups. The results and concepts presented in this research can lead to discovering novel nucleic acid therapeutic drugs for Eimeriasis and other parasitic infections, which provide insights into the development of species-specific anti-parasitic agents.

Rational design of minimum CRISPR guide RNA by site-specific Cas9-RNA conjugation.

The CRISPR-Cas9 system enables facile and efficient genome engineering in living cells and organisms. We report a Cas9-RNA conjugation strategy to afford minimal crRNA containing only the guide sequence for the target gene, which may simplify and reduce the cost for large-scale and high-throughput crRNA synthesis and lead to new insights into the design of CRISPR family complexes for diverse purposes.

Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy

The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9–RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9–RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9.

26 THE USE OF PAIRED CRISPR GUIDE RNAs AND THE Cas9 SYSTEM DOES NOT ALWAYS PRODUCE SITE SPECIFIC DELETIONS OF GENE SEQUENCE IN PORCINE CELL AND EMBRYO CULTURE

The use of the CRISPR/Cas9 system has become increasingly popular for creating gene edits in both cell and embryo culture. High specificity and efficiency of editing as well as low cost and ease of use has helped to promote its use. We hypothesised that by using multiple CRISPR guides at one time, we could quickly create exact deletions spanning greater areas of sequence. A total of 5 candidate genes (A, B, C, D, E) were targeted for deletions ranging in size of 74 to 551 bp. All modifications were created through the co-injection of 2 CRISPR guide RNAs with Cas9 RNA into in vitro-produced presumptive porcine zygotes. The CRISPR guides were created using gBlocks containing the T7 promoter sequence, 18–24 bp of CRISPR guide RNA, and 85 bp of tracer RNA. The RNA structure of each guide was reviewed using RNA Folding Form as well as offsite cutting using NCBI Blast. CRISPR guide RNA pairs (20 ng μL−1) and Cas9 RNA (20 ng μL−1) were co-injected (1–3 ρl) into the cytoplasm of IVF produced porcine zygotes using the FemtoJet 4i injector. Following injections, the zygotes were cultured in vitro for 5–6 days, and viable blastocyst or morula were selected for embryo transfer into recipient gilts. Resulting pigs were assayed for expected modifications using PCR. Pigs were considered modified if an insertion or deletion was measured by gel electrophoresis and DNA sequencing. Only one pair of CRISPR guides was injected per zygote, resulting in an individual PCR assay for the gene of interest. In total, 42 live piglets were born, 24 of which were edited, yielding 57% modification. When expected modifications v. observed were analysed, only 4 of 24 pigs (16%) produced the predicted modification on at least one allele. Of the remaining 20 pigs, several showed more than one form of modification. Insertions of ranging from 1 to 400 bp were detected in 10 pigs, 9 pigs formed biallelic modifications, 6 pigs produced altered sequence for greater than 2 alleles (mosaic), 6 pigs had deletions larger than the expected ranging from 11 to 1739 bp, and 14 had deletions smaller than the expected. Due to the absence of plasmid during injections, the insertions observed contained repetitive elements from the gene being modified as well as random additional bases. Additionally, CRISPR pairs were used in cell culture of porcine fibroblast modifying gene F, where they produced 6 different deletions ranging from the expected 63b to 617 bp. We recognise that the cutting efficiency of each CRISPR guide was not measured, as our goal was to create the expected deletions from pairs of CRISPR guides. We acknowledge our hypothesis was incorrect, as this data indicates that the CRISPR/Cas9 system is a very useful tool for gene editing, however it can induce unexpected modifications when used in pairs, in cell and embryo culture. Study was supported by funding from Food for the 21st Century and NIH (U42OD011140).

Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9.

Binding specificity of Cas9–guide RNA complexes to DNA is important for genome-engineering applications; however, how mismatches influence target recognition/rejection kinetics is not well understood. Here we used single-molecule FRET to probe real-time interactions between Cas9–RNA and DNA targets. The bimolecular association rate is only weakly dependent on sequence; however, the dissociation rate greatly increases from <0.006 s−1 to >2 s−1 upon introduction of mismatches proximal to protospacer-adjacent motif (PAM), demonstrating that mismatches encountered early during heteroduplex formation induce rapid rejection of off-target DNA. In contrast, PAM-distal mismatches up to 11 base pairs in length, which prevent DNA cleavage, still allow formation of a stable complex (dissociation rate <0.006 s−1), suggesting that extremely slow rejection could sequester Cas9–RNA, increasing the Cas9 expression level necessary for genome-editing, thereby aggravating off-target effects. We also observed at least two different bound FRET states that may represent distinct steps in target search and proofreading.

Use of CRISPR–cas9 gene targeting for genome-scale CRISPR screening in a glioma stem-cell line

BackgroundThe bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–cas9 system has recently emerged as a highly efficient gene-editing technique, in which the enzyme cas9 cleaves a genetic target under guidance from a CRISPR guide RNA. Glioma stem-cells are a useful model for the study of glioblastomas, but whether CRISPR–cas9 can be efficiently applied in this model is unknown. We aimed to derive and test a single cas9-expressing clone from a glioma stem-cell line. MethodsAn established human glioblastoma stem-cell line, NCH421K, was stably cultured in neural stem-cell conditions as neurospheres. A cas9 vector was designed with piggybac repeats for genome integration, and a neomycin-resistance cassette. The cas9 vector and a piggybac transposase vector were transfected into the glioma stem cells and selected with G418. The neurospheres were then dissociated into single cells, and flow sorting was used to transfer one cell to each well of a 96-well plate. FindingsWith this approach, expression of cas9 RNA in the transfected glioma stem-cell line was over 100 times higher than in wild-type cells, as demonstrated by real-time PCR. Repeat testing showed that the level of cas9 expression did not decrease over 2 months, indicating stable expression. Between 2 and 4 weeks after single-cell dissociation, single clonal neurospheres were isolated. Expansion of three such clones revealed cas9 expression in all of them. Functional testing with green fluorescence protein (GFP) expression (revealed by flow cytometry) demonstrated that transfection with a CRISPR guide RNA against GFP caused complete loss of GFP expression in over 90% of cloned glioma stem cells. InterpretationOur results demonstrate that isolation of a single glioma stem-cell clone engineered to express cas9 can generate highly efficient gene knock-outs. This model will allow unprecedented functional genomic screens of glioma stem cells on a genome-wide scale to identify novel cancer genes. FundingWellcome Trust Clinical Research Training Fellowship.

A Report for Applying CRISPR/Cas9 Genome Editing Technologies on Mouse Zygotes from Taiwan Transgenic Mouse Model Core Facility (TMMC)

Recent advances have provided the possibility to produce genetically modified animals by directly applying on zygotes using genome editing technologies such as Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Here, we showed the results of Taiwan Transgenic Mouse Models Core Facility (TMMC) for applying CRISPR/Cas9 for genetic engineering on mouse zygotes. In CRISPR/Cas9 system, we injected Cas9 plasmid or Cas9 RNA into either nucleus or cytoplasm of mouse zygoutes. Injection of Cas9 RNA with sgRNA into nucleus of zygotes was more efficient to induce indel than injection of Cas9 plasmid DNA (20% versus 12.5%). Moreover, injection of Cas9 RNA with sgRNA into cytoplasm increased indel rates to the average 38.1% in pups (the maximum is 89.4%), respectively. Further, the knock-in efficiency of injection RNA into cytoplasm was 15.3% in average (the maximum is 43.5%). In conclusion, injection of RNAs into mouse zygotes were more efficient to induce indel through non-homology end joining (NHEJ) or Knock-in through homology-directed recombination (HDR) on target genes, but might have cell toxicity when injected into nucleus than cytoplasm. The optimizations of procedure were needed to enhance efficiency and reduce toxicity.

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

Effective gene targeting in rabbits using RNA-guided Cas9 nucleases

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

The CRISPR-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA:DNA base-pairing to target foreign DNA in bacteria. Cas9:guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9:RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9:RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9:RNA. DNA strand separation and RNA:DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 employs PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate dsDNA scission.

A CRISPR Approach to Gene Targeting

It is getting easier and easier to determine complete genome sequences—of model organisms, animals and plants of commercial importance, and humans: Craig Venter, Jim Watson, the 1000 Genome Project, soon you and me. Now that researchers have all this information at hand, the focus has shifted in many cases to manipulating particular sequences to determine their function or to alter their impact. A new study by Jinek et al.1 proposes a new approach—based on the oldest of DNA recognition principles—to the design of reagents that can target specific genomic sequences.