..., Ru, G., Meng, H., Yan, Y., Hong, M., Zhang, D., Luan, C., Zhang, S., Wu, H., Gao, H., Bai, S., Li, C...Yin, S., Yang, L., Hu, H., Han, H., Li, Y., Wang, L., Chen, G., Ma, X., Geng, H., Huang, W., Pang, X.,...confirmed that the fusion protein retained the PAM properties of the individual Cas9. They also mutagenized..., a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold...other methods. In a head-to-head comparison with Cas9, Cas9 modified 9/12 known off-targets with a 14% indel...applicability of base editing through protein engineering and protein delivery. Nature Communications, 8(...Gaudelli, N. M., Komor, A. C., Rees, H. A., Packer, M. S., Badran, A. H., Bryson, D. I., & Liu, D. R. (2017...

...equally potent Cas9 protein, the Zhang lab took a different approach. They analyzed over 600 Cas9 orthologs... gene and/or protein is reconstituted. One way to reconstitute a split protein like Cas9 is to use split...one AAV, Cas9 must be made smaller. Previous attempts to “shrink” Cas9 include the use of St1Cas9 (~3.3 ...amino acids in length, Cas9 from Campylobacter jejuni (CjCas9) is the smallest Cas9 ortholog characterized...vehicle for Cas9. However, to allow for more room for regulatory sequences while still fitting Cas9 and its...polypeptides that, when at the ends of two proteins, mediates protein trans-splicing, similar to an intron ...White DE, Brown MT, Deshmukh H, Kemp ML, Bao G (2015) Trans-spliced Cas9 allows cleavage of HBB and CCR5...

... process. When using zinc finger proteins or TALE proteins, proteins had to be re-engineered for each ...through creating a dCas9/VP64 fusion protein engineered with aptamers that bind to MS2 proteins. These MS2 proteins.../10.1016/j.cell.2014.09.039 Zhou H, Liu J, Zhou C, Gao N, Rao Z, Li H, Hu X, Li C, Yao X, Shen X, Sun ...Ying W, Zhang J, Tang C, Zhang X, Xu H, Shi L, Cheng L, Huang P, Yang H (2018) In vivo simultaneous transcriptional...CRISPR activation CRISPR activation uses dCas9, a CRISPR protein variant lacking its endonuclease ability...simplicity of gene activation: rather than requiring protein engineering for each loci, CRISPR/Cas systems only...transcriptional activators: some methods rely on fusion proteins while others re-engineer components of Cas systems...

...transduced using only protein. Read on for a description of the lab’s iTOP protein-only transduction method...its potential applications to CRISPR/Cas9 genome editing.   Protein-only transduction? Niels Geijsen’s...common protein-only transduction strategy is to fuse a cell penetrating peptide (CPP) to a protein of interest...in the buffer used for protein purification must be helping the untagged protein enter cells. Subsequent...would be compatible with CRISPR/Cas9 genome editing. Although CRISPR/Cas9 is all the rage, they had another...to determine how much protein iTOP causes a cell to take up, and continued protein expression would require...in which transient protein expression has a long-lasting effect, such as Cre- or Cas9-mediated recombination...

...anti-CRISPR proteins or running into other proteins naturally bound to the DNA. Cas3 or Cas9? Large genomic...deletions. Many of these methods rely on Cas9 or a derivative of Cas9, but the ever-expanding repertoire of...achieved using Cas9, but Cas3 may be a better option. To get large deletions using Cas9, two or more gRNAs...the blog CRISPR 101: Cas9 vs. The Other Cas(s) CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas....2023.01.024 Morisaka, H., Yoshimi, K., Okuzaki, Y., Gee, P., Kunihiro, Y., Sonpho, E., Xu, H., Sasakawa, N.,...Type I-E Cascades contains all five of these Cas proteins, while type I-C Cascades do not contain Cas6. ...considered weak, especially compared to the power of Cas9. However, by continually passing the ssDNA past ...

...the basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...nuclease folds found in other proteins. The C-terminal domain (CTD) of the protein also belongs to the NUC ...   Unbound Cas9 Now that we know what we’re looking at, let’s peek at unbound—“apo”—Cas9 from Streptococcus...confirming its sequence. This region of the protein varies between Cas9 enzymes from different species, explaining...DNA-binding protein portion of the CTD, ready to check for PAM sequencines. This helps explain why Cas9 can’t...fit. Stabilization by this region of Cas9 also allows the protein to tolerate RNA:DNA mismatches between...Figure 8: Comparison of Cas9:RNA crystal structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy...

... of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein (dCas9) fused to CIB1;...active saCas9 protein. Stop light! Photoactive anti-CRISPRs halt Cas9 activity Anti-CRISPR proteins are ...transcribes N-Cas9. The N- and C-Cas9 fragments then dimerize to form a functional protein due to being...chimeric protein consisting of Cas9 and a photo-dissociable dimeric green fluorescent protein (pdDronpa...Shao J, Li H, Chen Y, Ping Y, Li D, Ye H (2020) Engineering a far-red light–activated split-Cas9 system for...to create photoswitchable spCas9, dCas9, and S. aureus Cas9 (saCas9) proteins, showing the versatility ...N. crassa Split Cas9, Split Cas9 nickase, Split dCas9 Genome editing by NHEJ and HDR, repression of gene...

... 2016). The protein capsid encapsulating the genome is majorly composed of three proteins that can be ...of terminal proteins capping the ends of linear double-stranded AdV genomes. Such protein-DNA structures...DNA Adenoviral CRISPR/Cas9 genome editing tools can also be used to deliver Cas9 and DNA templates in ... CRISPR/Cas9 research! To find more information about the adenoviral delivery of CRISPRS/Cas9 using the...Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses. Human...j.jconrel.2019.02.009 Wang D, Mou H, Li S, Li Y, Hough S, Tran K, Li J, Yin H, Anderson DG, Sontheimer EJ, ... out the plasmids at Addgene: pAdSh.PGK.Cas9 (expresses S. pyogenes Cas9 from the PGK promoter) and U6...

...expression of S. pyogenes Cas9 or Cas9 variants (hereafter referred to as Cas9) and a single stranded guide... gRNA backbones also contain Cas9, which enables expression of both Cas9 and the gRNA off of the same ... CRISPR–Cas9. Nat Plants 6:773–779 . https://doi.org/10.1038/s41477-020-0704-5 Santana I, Wu H, Hu P, ...Nuclease dead Cas9 (dCas9) based activators (such as dCas9-VP64) or repressors (dCas9-KRAB or dCas9-SRDX) can...doi.org/10.1038/nature.2016.19754 Zhang H, Demirer GS, Zhang H, Ye T, Goh NS, Aditham AJ, Cunningham FJ...get more information on multiplexing gRNAs here). Cas9 is commonly tagged with a nuclear localization sequence...targeting to the nucleus, and several codon optimized Cas9 variants have been created in an effort to increase...

...for HR is to fuse an HR protein to the nuclease. Early HR factors fused to Cas9 have been shown to increase...Charpentier, M., Khedher, A. H. Y., Menerot, S., Brion, A., et al. CtIP fusion to Cas9 enhances transgene integration...2018). 1 10.1074/jbc.TM118.000372 Yang, H., Ren, S., Yu, S., Pan, H., et al. Methods favoring homology-directed...Nat Chem Biol). Mutate the PAM or sgRNA sequences Cas9 likely goes through multiple rounds of cleavage ...guarantee there won’t be re-targeting after HDR. If your Cas9 cuts in the coding region of a gene, make sure that...mutation, so you don’t accidentally change your protein of interest.   Fig. 2: Stop re-cutting ...inhibitors to your culture media prior to and after Cas9 introduction increases HDR, just make sure to remove...

...using CRISPR/Cas9 to edit a gene, the expression of Cas9 is no longer necessary. However, Cas9 can be toxic...K., Bartholomae, C., Hubank, M., Kempski, H., Brugman, M. H., Pike-Overzet, K., Chatters, S. J., de Ridder...like creating stable cell lines, delivering CRISPR/Cas9 to cells for genome editing, and introducing inducible...their life cycle to continue. They rely on a viral protein called reverse transcriptase to convert their RNA... are proteolytically processed from the larger protein: PR (protease), RT (reverse transcriptase), and...typically encodes the vesicular stomatitis virus G protein (VSV-G), which facilitates viral entry into the... integrated into the host genome via the viral proteins packaged during production (read our blog post...

...they call CRISPR/Cas9 Synergistic Activation Mediator or SAM. SAM is an engineered protein complex for the... Crystal structure of Cas9 in complex with guide RNA and target DNA. Nishimasu H, Ran FA, Hsu PD, Konermann...Silvana Konermann, the first generation of CRISPR-Cas9 plasmids were not designed with gene activation ...question for her then was this: Can you engineer CRISPR-Cas9 activators that work well enough on any gene that... three components: 1. A nucleolytically inactive Cas9-VP64 fusion; 2. A sgRNA incorporating two MS2 RNA... plasmid which expresses the activation helper protein. SAM can be combined with a human genome-wide library...system, Konermann explained, was new knowledge about Cas9’s crystal structure. “I thought maybe we could use...

...types. They have also fused LSD1 to x-Cas9(3.7). x-Cas9(3.7) is a SpCas9 mutant that exhibits increased PAM...Staphylococcus aureus Cas9 (Sa-Cas) and deposited plasmid IF311: pMAGIC (R4-R3) NLS-Sa dCas9-NLS-LSD1. LSD1 ...covalent modifications to DNA or histones, the proteins that help package DNA, were associated with gene...With the help of guide RNAs, fusions between Cas proteins and the epigenetic modifier could be targeted ...cytosine demethylation of DNA. However, several proteins in the DNA oxidation and repair pathways work ...LSD1 is a particularly large protein, and this fusion allows for packaging into viral vectors and targeted...PAM flexibility, and this fusion protein allows for LSD1 targeting to a wider set of genomic targets. ...

.../nwad143 Tong, H., Wang, H., Wang, X., Liu, N., Li, G., Wu, D., Li, Y., Jin, M., Li, H., Wei, Y., Li, ....2024.01.021 Tong, H., Liu, N., Wei, Y., Zhou, Y., Li, Y., Wu, D., Jin, M., Cui, S., Li, H., Li, G., Zhou,...Zhou, J., Yuan, Y., Zhang, H., Shi, L., Yao, X., & Yang, H. (2023). Programmable deaminase-free base editors...Tong, H., Wang, X., Liu, Y., Liu, N., Li, Y., Luo, J., Ma, Q., Wu, D., Li, J., Xu, C., & Yang, H. (2023...DNA N-glycosylase (UNG) enzymes. Various other proteins included in different CGBE tools allow the researcher... human AAG (remember, MPG and AAG are the same protein!) (Chen et al., 2024). They called their initial...inserted the deaminase into the C-terminal domain of Cas9, which narrowed the editing window and reduced A...

...complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade complex doesn’t usually...–C, with the beloved SpCas9 (from Streptococcus pyogenes) belonging in II-A. Cas9 was the first to be ...first time. The discovery and application of CRISPR-Cas9 kicked off over a decade of frenzied interest in...effector complex but are defined by the accessory Cas proteins in the Cascade complex. Despite a tenuous grasp... Despite being in Class 1, Cas7-11 is a single protein effector and is able to target and edit RNA in ...Class 2 Class 2 CRISPR systems rely on a single protein Cas effector and are likely the class most people...type VI, each relying on a unique Cas effector — Cas9, Cas12, or Cas13. If you’d like to explore Class...

...tagging proteins, as it can be fused to the N- or C-terminus (or even in the middle of proteins) and performs...Overview of the dual-plasmid CRISPR/Cas9 workflow in M. abscessus. A) Sth1Cas9 mediates double strand break ...mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 for gene disruption in M. abscessus  Bioluminescent... produced by our partners at the Institute for Protein Innovation (IPI), part of a new collection of epitope...short, linear epitope derived from the P and V proteins of Simian virus 5 (SV5) (Randall et al., 1987)...including popular new approaches like PROTAC targeted protein degraders. But CRBN has been difficult to study...drug ligands, and in ternary complexes with other proteins, establishing CRBNmidi as a promising tool for...

...Expanding beyond Cas9 Since the development of CRISPR/Cas9 editing methods, other Cas proteins have been adapted...in mammalian and yeast cells using modified Cas9 proteins to regulate gene expression (CRISPRi/CRISPRa...2017 uses the direct injection of a CRISPR/Cas9 ribonucleoprotein complex with linear DNA as repair templates...University of California, Santa Cruz. Emerging CRISPR/Cas9 editing technologies have transformed the palette...regulation during developmental processes, CRISPR/Cas9 allows us to delete sequences and introduce mutations...remain in the edited animal. Development of CRISPR/Cas9 editing strategies in C. elegans As brilliantly ...determined optimal PAM orientation for editing using two Cas9 targets. An orientation where the 5’ ends of the...

...are a number of drawbacks to this approach: TET proteins are not demethylases; they are dioxygenases that...rare DNA bases that are recognized by distinct proteins and result in unclear transcriptional consequences..., Charlton et al. 2020). Most importantly, TET proteins affect transcription independent of their enzymatic...may seem counterintuitive: how can a DNA-binding protein with no enzymatic activity cause demethylation?...the Epigenome  Mapping the 4D Nucleome with CRISPR/Cas9 Resources at Addgene.org Read our CRISPR Guide...Morita S, Noguchi H, Horii T et al. (2016) Targeted DNA demethylation in vivo using dCas9–peptide repeat ...DE, Ross DL, Vachhani RV, Ming G-L, King JR, Song H, Sweatt JD (2013) TET1 Controls CNS 5-Methylcytosine...

... a colorful Cas9 toolbox, Ma et al. turned to SpCas9 and its orthologs NmCas9 and St1Cas9. Each ortholog...states. dCas9 gets a colorful makeover Even without its nuclease activity, catalytically dead Cas9 (dCas9... colorful system that uses fluorescently labeled Cas9 to label multiple genomic loci in live cells. While...hybridization (FISH) or fluorescently labeled TALEs, CRISPR/Cas9’s ease of use and ability to label live cells make...ortholog was fused to a different fluorescent protein to create three colors. The specificity of these orthologs...estimate that a minimum of 150-200 fluorescent protein molecules is necessary for a detectable signal,... scaffold that can be used to recruit multiple protein molecules (and also available from Addgene!) Since...

...fluorescent and 3xFLAG-tag fusion proteins. The repair template and Cas9-sgRNA plasmid is then injected ...tagging of every protein in the C. elegans genome. Although they tested fluorescent protein fusions, this...for CRISPR/Cas9 editing in C. elegans: Engineering the Caenorhabditis elegans genome using Cas9-triggered...2013, a remarkable nine papers describing CRISPR/Cas9 genome engineering methods for C. elegans were released...even easier - now, one can generate a fluorescent protein fusion, transcriptional reporter, and loss-of-function...Multiple features of C. elegans have made CRISPR/Cas9 so successful in this species, namely, the robust...create a new and efficient selection-based CRISPR/Cas9 method to generate knock-ins on any genetic background...