...loci in living human cells by an optimized CRISPR/Cas system. Chen B, Gilbert LA, Cimini BA, Schnitzbauer... 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...well-known being transcriptional activation/repression. dCas9 has also previously been adapted to fluorescently...required by each ortholog, a gRNA designed for one dCas9 should be specific to that ortholog and not cross-talk...

...puromycin resistance cassette, such as PX459). After you co-transfect the CRISPR/Cas system with your homologous...article list, the Genome Engineering Using CRISPR/Cas Systems has expanded out of control. To further consolidate...focus on genome editing using the Zhang lab's CRISPR/Cas plasmids. Designing your CRISPR genome editing experiment...that can be inserted into the genome using CRISPR/Cas forHomologous Recombination (HR)? How long should... crispr cas...the Cas9 nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does...questions and answers. Here are just 4 of the CRISPR-Cas9 FAQs. If you still have more questions after reading...

...CRISPR-Cas system for mammalian use Stanley Qi’s lab has developed a miniature Cas system (CasMINI) engineered...Miniature CRISPR-Cas systems SEND plasmids Prime editing improvements Miniature CRISPR-Cas systems By Brook... yet active Cas system starting with the same Un1Cas12f1 (Cas14). They noted that Cas12f1 had an extra-long...  They did not modify the Cas12f1 enzyme. The result is a highly efficient Cas system with very compact...miniature CRISPR-Cas12f nuclease Quanjiang Ji’s lab describes an effective miniature Cas system that utilizes...the uncultivated archaea Cas12f (Un1Cas12f1, or Cas14a1) system. The CasMINI system was developed by engineering... enzyme with a fully active Cas9 enzyme. PE-Cas9 comprises an active Cas9 fused to the reverse transcriptase...

...Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes." Mol Cell. 66(3) (2017):373-83. PubMed PMID...with the Cas13 scaffold, testing a whopping 21 Cas13a orthologs, 15 Cas13b orthologs, and 7 Cas13c orthologs...identified Cas13a (C2c2) (Abudayyeh et al., 2016) as a RNA-targeting CRISPR enzyme, they adapted Cas13b for ...target locus, making them more flexible than Cas9/Cpf1. Some Cas13 enzymes prefer targets with a given single...sequence, but orthologs like LwaCas13a do not require a specific PFS. Cas13 enzymes do not contain the ...knockdown. In initial tests, Cas13b from Prevotella sep. P5-125 (PspCas13b) yielded 62.9% average knockdown...to deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the cytosine deaminase...

...variety of related bacteria with similar Cas systems. Some of these Cas systems have enzymes small enough to...knockdown efficiencies. The ability to identify related Cas systems opens up the potential of further development...CRISPR effector protein they named Cas7-11. Testing showed that Cas7-11 cut RNA with unprecedented specificity...luciferase RNA knockdown assays using Cas7-11   Use The Cas7-11 targeting system contains two pieces...computationally described as a fusion of four Cas7 proteins and a putative Cas11-like protein, and a guide RNA. The...efficacy rates of Cas7-11 were reported as up to 85%, broadly similar to shRNA and Cas13 systems in a range... both of which we're featuring in today's blog. Cas7-11 Development and Discovery As part of their quest...

...comparison of GeoCas9 vs. SpCas9, see the table below and read on to learn more about GeoCas9.   SpCas9 GeoCas9... 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 RNP activity or longevity. GeoCas9 is active at high... Figure 1: Thermostability of SpCas9 vs GeoCas9. SpCas9 or GeoCas9 RNPs were incubated in reaction ...Figure 2: Human plasma longevity of SpCas9 vs GeoCas9. SpCas9 or GeoCas9 RNPs were incubated in human plasma...used to make eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally occurring Cas9 diversity ...at elevated temperatures. In both cases, GeoCas9 outperformed SpCas9 at high temperatures. See Figure ...et al tested GeoCas9’s editing skills by transfecting HEK293 cells with GeoCas9 or SpCas9 RNP’s and a ...

... the Addgene Podcast.     Additional Resources on the Addgene Blog Listen to Our Podcast Interview with...plasmid info with a new segment on the Addgene podcast - The Hot Plasmids Segment. Click on the player...below to listen to a quick (~5 min) Hot Plasmids podcast that introduces you to 4 new plasmid technologies... the latest and greatest plasmids through this podcast segment. Below, we break down each of the Hot Plasmids...If you have any comments about the Hot Plasmids Podcast Segment, or have recommendations for new Hot Plasmids... podcast...

...single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227. ... P., et al. "Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells." Nature Biotechnology...Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674(15)01200...An updated evolutionary classification of CRISPR-Cas systems. 5. Nat Rev Microbiol. 2015 Sep 28. doi: ...than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 efficiency...rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins...overcome some of Cas9’s shortcomings. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in...

...Joung JK (2016) Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34:...Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016...them more useful than SpCas9. For example, Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase... in length, Cas9 from Campylobacter jejuni (CjCas9) is even smaller than SaCas9 and is also compatible...blunt ends created by Cas9. Cas12a  displays lower off-target editing than SpCas9 (Kim et al., 2016).  ...as a binding signal for Cas9 and this sequence is a strict requirement for Cas9-mediated DNA cleavage. ...of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from...

...the PCR products prior to cloning, which in most cases left the functional light chain intact, the authors...

...than SpCas9, in this case, NNGRRT and NNNNGATT, respectively. Researchers also engineered SpCas9 variants...enrich for Cas9 variants with activity on non-NGG PAMs, generating xCas9 3.0-3.13. To test xCas9 3.0-3.13...Characterizing xCas9 in mammalian cells Hu et al. compared xCas9 activity with wt SpCas9 in mammalian cells...actually lower for xCas9 3.6/3.7 than for SpCas9. For some non-promiscuous gRNAs, xCas9 3.7 displayed more...using the xCas9 scaffold. With an NGG PAM, xCas9 3.7-VPR constructs modestly outperformed SpCas9-VPR in ...flexibility of xCas9 3.7. Base editor variants with xCas9 are similarly flexible. Like the xCas9 3.7 nuclease...’t know about xCas9. “I’m not 100% sure xCas9 is going to be flat out better than SpCas9. I want everyone...

...applications. That said, SpCas9 is not the only game in town, and other Cas proteins like SaCas9 and Cpf1 can circumvent...respectively. Amplified nucleotides are combined with Cas 13a in complex with crRNA targeting the sequence ...Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes. " Mol Cell. 66(3) (2017):373-83. PubMed ...functional characterization of diverse class 2 CRISPR-Cas systems." Molecular cell 60.3 (2015): 385-397. PubMed...applications of Cas13a. The origins of Cas13a: An RNA cleaving CRISPR nuclease Cas13a was originally ...Perhaps the biggest difference between Cas13a and Cas9 is that Cas13a binds and cleaves RNA rather than DNA...As with Cas9, mutating key residues in the Cas13a molecule results in a “nuclease dead” Cas13a (dCas13a...

...Doudna, J. A. (2022). Structural biology of CRISPR–Cas immunity and genome editing enzymes. Nature Reviews...of CRISPR  xCas9: Engineering a CRISPR Variant with PAM Flexibility Cas9 vs the Other Cases Further reading...the basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...   Unbound Cas9 Now that we know what we’re looking at, let’s peek at unbound—“apo”—Cas9 from Streptococcus...in this case) is only bound by the phosphate backbone. These PAM bases must be present for Cas9 to unwind...Figure 8: Comparison of Cas9:RNA crystal structure (left, PDB ID 4ZT0) with Cas9:RNA:DNA electron microscopy.... And this article only covered Cas9! Other CRISPR enzymes like Cas12 have different structures and different...

...et al., 2016). For editing the genome, the CRISPR/Cas system makes use of 3 components, a guide RNA (gRNA...(2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:819–823 . https://doi.org/10.1126...Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell 154:1370–1379 ....Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 153:910–918 . https...alternative Cas9, from Streptococcus aureus, is as effective at editing mouse embryos as Cas9 from S. pyogenes...Nakauchi H (2018) Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-...guest bloggers, Wenning Qin and Haoyi Wang. CRISPR/Cas9 is revolutionizing the mouse gene-targeting field...

...scientific collaboration across the globe. Listen here! Podcast Breakdown (Time: Topic) 0:00-1:00 : Intro 1:01-...the audio engineer and producer for the Addgene Podcast.     Additional Resources Learn about Seeding ...the Addgene Blog Listen to Additional Addgene Podcasts Learn How You Can Contribute to the Addgene Blog...

... CRISPR/Cas systems only require changing the programmable guide RNA. Gene activation by dCas9, also referred...Prior to the discovery of CRISPR/Cas systems, gene activation across multiple loci was an arduous process...seem next to impossible. The development of CRISPR/Cas systems, however, greatly improved the simplicity...al., 2013). To target specific sequences, CRISPR/Cas systems rely on a guide RNA complementary to the ... proteins while others re-engineer components of Cas systems themselves. SunTag, SAM, and VPR have all...bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research 41:7429–7437 . https...Crawford GE, Reddy TE, Gersbach CA (2013) RNA-guided gene activation by CRISPR-Cas9–based transcription factors...

...Considerations when choosing your Cas nuclease When choosing the Cas9 nuclease for your experiments, there...PAM sequence that the Cas nuclease recognizes determines site accessibility. Cas9s with a broader PAM specificity...Since the first use of Cas9 to engineer the genome, there are more and more Cas enzymes and variants to...choosing a Cas protein for your experiment. References Hu, Johnny H., et al. "Evolved Cas9 variants with...knowledge of both the advantages and limitations of each Cas enzyme can help determine which enzyme to use. Remember...CRISPR 101 blog posts Find more information about Cas proteins Download the CRISPR 101 eBook Resources...frequently used Cas9, spCas9 from Streptococcus pyogenes, this sequence is 3’-NGG. But Cas9s found in other...

... Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes when encoding multiple guides. ... large size of CRISPR-Cas effectors and fusion proteins, such as Cas9 and Cas12a, creates challenges for...and cell engineering applications. CasMINI, a new miniature CRISPR-Cas system for editing and gene activation...State Protein Ladder Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing .../s41598-021-96051-x Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing...of either Cas9 or Cas12a. Despite its small size, CasMINI is comparable in efficacy to dCas12a systems ...expression cassette is advantageous to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally...

...There are many fundamental questions about CRISPR/Cas biology still left to answer as well, she says, and.... [6].    References 1. Zhang B. 2021. “CRISPR/Cas gene therapy.” Journal of cellular physiology.  https...0460.1000439 6. Wu et al. 2020. . “Advances in CRISPR/Cas-based Gene Therapy in Human Genetic Diseases.” Theranostics...Northeastern University.  There can be no doubt that CRISPR/Cas9 technology has been a breakthrough for the genome-editing...lab are researching a way to treat AD using CRISPR/Cas9. They are targeting the genes responsible for both... AD. In this study, the group showed that CRISPR/Cas9 could be used to edit several autosomal-dominant...that play into late onset AD (5).     CRISPR/Cas9 corrects Alzheimer’s disease mutation in basal forebrain...

...Expanding beyond Cas9 Since the development of CRISPR/Cas9 editing methods, other Cas proteins have been...experimental options. Expanding and improving the CRISPR/Cas editing toolbox for C. elegans Recent work has further...development of toolkits such as the SapTrap CRISPR/Cas Toolkit and SapTrap-SEC kit for C. elegans genome... some cases, injection of Cre is required, however the development of the self-excising cassette (Dickinson...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 ...