...possibilities for Cas proteins With their enhanced specificity, eSpCas9, SpCas9-HF1, and HypaCas9 should enable...specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target...portions of Cas9 might be important for nuclease specificity. They found that eSpCas9(1.1) and SpCas9-HF1 each...sites tested when compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available from Addgene...off-target effects” have included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA sequences... target effects of CRISPR/Cas9 by altering the nuclease activity of the Cas9 nuclease itself. Design and... and testing of eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), Slaymaker...

... A multitasking Cas Most Cas effectors have one job — Cas see DNA, Cas chop DNA. Cas3, however, has two...CRISPR 101: Cas9 vs. The Other Cas(s) CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems INTEGRATE...CRISPR has brought many other Cas effectors to light. One lesser-known Cas is waiting for its chance in...and Cas11. Type I-E Cascades contains all five of these Cas proteins, while type I-C Cascades do not contain... the spotlight — Cas3. Cas3 can induce genomic alterations on a scale that other Cas effectors can’t, ...a multi-Cas complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade is brought...complex” system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas effectors to...

... for a candidate Cas for your experiment. Target requirements As we all know, a Cas guided by a gRNA ...properties for a Cas entry (SpyCas9a) from CasPEDIA.   How can I use it? CasPEDIA operates like many...Addgene and CasPEDIA have got you covered!     Fig. 1 – CasPEDIA Cas ID display information for...for a given Cas (SpyCas9a) from CasPEDIA.   Helpful resources and information For some researchers,...CRISPR 101: Which Cas9 do I choose for my CRISPR experiment? CRISPR 101: Cas9 The Other Cas(s) The PAM Requirement...Once upon a time, not so long ago, spCas9 was the only Cas enzyme widely available and applied by researchers...What are the features? CasPEDIA has collected relevant information on all the Cas enzymes you know and ...

...the nuclease Cas9, but how does it stack up to the new Cas enzymes on the block? The Cas family of proteins...targeting. RNA targeting Cas enzymes Move over Cas9, CRISPR isn’t just for DNA anymore! Cas enzymes have been... facts on Cas family proteins and genome editing abilities   Choosing a Cas While Cas9 may be the...the most adaptable Cas currently available, the other Cas proteins should not be overlooked. The unique...DNA edits.   Cas3 – The shredder What sets Cas3 apart from Cas9 and almost all other Cas enzymes is its...delivery.   Cas14 – Mini, but single strand mighty Unlike many other Cas enzymes, Cas14 targets ssDNA...lesser-known Cas enzymes while also highlighting their similarities and differences to Cas9. DNA targeting...

...Separate Cas proteins then express and process the CRISPR loci to generate RNA, which guide a Cas nuclease.... Many CRISPR/Cas moieties carry out these functions using slightly different Cas proteins and PAM ...CRISPR/Cas defense by generating a single point mutation in the PAM sequence, preventing the Cas nucleases... information on CRISPR/Cas, check out Addgene’s CRISPR Guide or find CRISPR/Cas plasmids for your research... bacterial genome. Cas genes, which code for polymerases, nucleases, and helicases, were also disrupted...clustered regularly interspaced palindromic repeats) Cas (CRISPR associated genes) system. This popular genome...look at how prokaryotes originally utilized CRISPR/Cas as a powerful and adaptive defense strategy against...

... Jennifer Tsang. CRISPR-Cas technology is constantly evolving. Variants of Cas proteins can be used for...missing: a way to shut off Cas’s activity. The concern is that the longer Cas remains active in a cell,...protein inhibitors of CRISPR-Cas systems that help phages evade the CRISPR-Cas immune system of bacteria.... II-A CRISPR-Cas systems. A full list of Acr proteins organized by the type of CRISPR-Cas system they ...that interact with CRISPR-Cas systems such as Cascade-Cas3, Cas9, Cas12, and Cas13 (Marino et al., 2020)....AcrIIA4 blocks Cas9’s interaction with the PAM site (Dong et al., 2017), and AcrIIC3 causes Cas proteins to...interactions with Cas9 interferes and prevents the correct assembly of the crRNA-Cas complex (Zhu et al...

...mutations arise from base/prime editing or Cas cleavage. In the case of Cas cleavage, off-target mutations can...confer specificity of the nuclease; however, many Cas and Cas-like nucleases are promiscuous. In other words...consider your Cas enzyme. Even if there are similar sequences present in the genome, certain Cas enzymes are...approaches have been developed. Cas ChIP-Seq, where only DNA sites bound by Cas are sequenced, has been tested...appropriate method for an activator or repressor Cas. Similarly, Cas ChIP-Seq would also identify all DNA binding...CRISPR 101: Which Cas9 do I choose for my CRISPR experiment? CRISPR 101: Cas9 The Other Cas(s) CRISPR 101... of Maya Kostman. The most commonly used Cas, spCas9, has gone through multiple rounds of optimization...

...type VI, each relying on a unique Cas effector — Cas9, Cas12, or Cas13. If you’d like to explore Class ...while Class 2 use single protein Cas effectors, like Cas9. The Cascade complex doesn’t usually carry out...attributed to the multi-Cas effector, Cascade. Developing tools using a Cascade complex is tricky, as you...the blog CRISPR 101: Cas9 vs. The Other Cas(s) CRISPR 101: RNA Editing with Cas13 Build your CRISPR vocabulary...Class 1 CRISPR systems use multi-Cas effectors, referred to as Cascade (CRISPR-associated complex for antiviral... subtypes use Cas10 as the primary effector complex but are defined by the accessory Cas proteins in the...which are among the smallest Cas enzymes (400 to 700 amino acids), use a Cas14 enzyme, and uniquely target...

...gene cas14. cas14 encodes for a small Cas protein (40-70 kDa), which is half the size of other Cas proteins...characteristic of CRISPR-Cas enzymes. In contrast to other Cas enzymes, Cas14 has not been found in bacterial...integrase, cas1. They discovered a family of CRISPR-Cas systems containing cas1, cas2, cas4, and a new...CRISPR-Cas systems. There are 24 variants of the cas14 gene that cluster into 3 subgroups (cas14a-c). All.... Identifying Cas14 variants In this quest to discover new and maybe more efficient Cas systems, Jennifer...of virtually any organisms on this planet, CRISPR-Cas systems were merely adaptive immune systems that ...try to determine whether simpler and maybe smaller Cas systems could exist in nature. To do this, they ...

...Protein Cas: Different CRISPR systems involve different proteins, but many of them are called Cas, short...by the Cas9 protein from the appropriate species. Some Cas proteins, like LbCas12a or AsCas12a, do not...encodes a tracrRNA (in some systems including Cas9), Cas proteins, and a CRISPR array. The CRISPR array...spacer. In bacteria, these crRNAs are bound by Cas proteins (Cas9 shown here). The repeat sequence base pairs... famous of these proteins is Cas9 nuclease, but there are many other Cas proteins as well, both natural...organized. Target strand: The strand of foreign DNA that Cas proteins recognize using an RNA guide. Non-target... to be cut, which varies between CRISPR systems. Cas proteins won’t cut DNA that doesn’t have their specific...

...-flexible Cas enzymes   The solution to the PAM problem seems simple, in theory: find a Cas enzyme or ...placement significantly over SpCas9 and more so than Cas9s with an NG PAM.   Cas PAM sequence  Additional...Expanding Beyond Cas9 xCas9: Engineering a CRISPR Variant with PAM Flexibility History of CRISPR Cas – A Tale ...are some available.   Cas proteins that don’t utilize NGG   There are plenty of Cas9 variants which utilize...etc.). Additionally, alternative Cas enzymes exist, such as Cpf1/Cas12a, which recognizes the PAM sequences... of generating Cas enzymes with alternative PAM requirements (Zhao et. al, 2023). xCas9 has been shown...Walton et. al, 2020). These Cas proteins have everything we know and love about Cas9, but with broader PAM ...

...catalytically impaired Cas protein (dCas or Cas nickase) fused to a DNA-modifying enzyme, in this case a deaminase...change before the Cas domain lets go of the DNA. ABEs are somewhat slow and need a strong Cas9 domain to “hold... are only weakly compatible with many Cas domains beyond SpCas9. In order to expand the gene-editing applications...specifies the editing target site on the DNA, the Cas domain directs the modifying enzyme to the target...substitutions.  The need for speed: base editing before Cas lets go Figure 1: David Liu gives a talk at...to evolve ABEs with an increased compatibility to Cas homologs. Evolving better base editors with the help...deamination rate so that deamination can happen before Cas homologs let go. The Liu lab developed the Phage-...

...engineered variants of Cas nucleases. In doing so, they also created a modular toolkit for Cas-mediated genome...editing toolkit New Cas protein identified Jennifer Doudna’s lab recently identified Cas14, a family of small...small DNA-targeting Cas proteins. Cas14 targets and cleaves ssDNA which they have adapted for detecting...genome editing in plants While there have been many Cas nucleases developed for plants, it has been difficult...dual-nuclease Cas9-Cas9 chimeras Scot Wolfe’s lab has developed dual-nuclease Cas9-Cas9 chimeras with ...compared to monomeric Cas9’s. Read the paper in Nature Communications Find the Cas9-Cas9 chimera plasmids...Science Find the Cas14 plasmids Anti-CRISPR proteins for optogenetic control of CRISPR-Cas9 Anti-CRISPR ...

... targeting specificity). Cas9 endonuclease functions While native CRISPR/Cas systems have a variety of...sometimes sgRNA) In the native Type II CRISPR/Cas system, Cas9 is guided to its target sites with the aid...vary (for example, xCas9 is a variant with increased PAM flexibiliy and eSpCas9/SpCas9-HF1 have increased...sequence, Cas9 will neither bind nor cut the target. Cas9 homologs from different organisms or Cas9 mutants...pyogenes (SP); SpCas9 3' NGG SpCas9 D1135E variant 3' NGG (reduced NAG binding) SpCas9 VRER variant...3' NGCG SpCas9 EQR variant 3' NGAG SpCas9 VQR variant  3' NGAN or NGNG  xCas9 3' NG, GAA,...basic level, CRISPR/Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related...

...Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems. Ahmed A. Gomaa, et al. mBio. 2014 January...CRISPR/Cas9 system to precisely edit specific genes in monkeys. "Our study shows that the CRISPR/Cas9 system...for CRISPR Plasmids? CRISPR-Cas9 Plasmids available at Addgene CRISPR-Cas9 References, Protocols, and ...advances in the understanding and application of CRISPR/Cas9 technology are coming along at a fast and furious...Beisel and Rodolphe Barrangou, had applied the CRISPR/Cas9 system to developing an antibiotic that selectively...important details in their understanding of how CRISPR/Cas9s actually work. Last month, the Lawrence Berkeley... Nogales presented the first detailed picture of Cas9's three-dimensional shape via x-ray crystallography...

...multiple Cas proteins to form the Cascade targeting complex is a hallmark of Type I CRISPR-Cas systems,... (such as CRISPR-Cas9). Unlike in regular Type I CRISPR-Cas systems, however, Cascade from VchINT does...to Type I-F, but instead employ a Cas12k protein instead of three Cas proteins (Fig. 1A, bottom), and require...transposases and the target programmability of CRISPR-Cas, these systems represent the next generation of tools... hereafter VchINT) and encodes a Type I-F CRISPR-Cas system. The four major components required for RNA-guided... of DNA targeting by a transposon-encoded CRISPR–Cas system. Nature 577:271–274 . https://doi.org/10.1038...TS, Sternberg SH (2019) Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature...

... sequencing data.”  Recently, Seqera published a case study on how Addgene has used Nextflow to improve...our existing resources. You can read it, and other case studies, on the Seqera website. ...

...Cambridge, MA and Sequana Therapeutics in San Diego, CA. She earned a B.S in Biology from MIT.   Kenneth...at Credit Suisse’s Technology Group in Palo Alto, CA.  Benjie Chen is a co-founder of Addgene and received... In this episode of the Addgene podcast, we sit down with Addgene’s Founders - Melina Fan, Ken Fan, and...Listen to the interview with Addgene's founders Podcast breakdown Intro Chapter 1 - Who are Addgene's Founders...the audio engineer and producer for the Addgene Podcast.      Additional Resources from the Addgene Blog... podcast...

...shifts. This backbone model of Cas9 is based on Fuguo Jiang’s structure of Cas9 – primed for cleavage. This...researchers for the past fifteen years. On those occasions when we are fortunate to hand-deliver the model...contacted us in 2014 to ask if we could make a model of Cas9 based on the structure published by Martin Jinek...other aspects of the structure. While this first Cas9 model is certainly useful, it is also wrong in at... at least one serious way. We represented the Cas9 protein in a surface format made of rock-hard, inflexible... the amazing conformational flexibility that the Cas9 protein must have in order to scan along the double-stranded...model of a protein. We now have another model of Cas9 – a flexible alpha-carbon backbone model made of...

...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...

... 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...

...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...

...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 ...

...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...

...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...

...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...

...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...

...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...

...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...

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

...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...

... Is Cas9 not your Cas of choice?  There are plenty of other enzymes to choose from within the Cas family...Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors...using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing a Cas9 for site-specific...your experimental target, SpCas9 and its enhanced variants, eSpCas9 and HypaCas9, are great choices. Check...target two Cas9-nickases (Cas9n) to the target locus, enhancing specificity over a single SpCas9. The drawback... expanding number of Cas9s with looser PAM requirements compared to SpCas9. xCas9 has a PAM requirement... making a break.  Base editors are dead Cas9 (dCas9) or nCas9 enzymes fused to either cytosine or adenosine...

...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 ...

...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...

...lines, with an efficiency similar to that of Cas9. Like Cas9, NgAgo is also able to mediate homology-directed...at Addgene Cas9 and NgAgo head to head NgAgo has a number of unique features compared to Cas9. These include...acids in length, NgAgo is shorter than SpCas9 (1368 aa), SaCas9 (1053 aa), or Cpf1 (1228 or 1307 aa depending...the Addgene Blog Read About Cas9 Homolog Cpf1 Check Out High Fidelty Cas9 Mutants Browse All Genome Engineering... why you might choose NgAgo over CRISPR proteins Cas9 or Cpf1 - keep reading to learn more! Background...include: More targeting flexibility Unlike Cas9 and the other CRISPR enzymes, NgAgo does not require a...genomic target. NgAgo also appears to outperform Cas9 in GC-rich regions. DNA guides NgAgo uses 5’ phosphorylated...

...efficiency and throughput. In adapting the CRISPR/Cas system, what we are doing is adding in a second plasmid... slow-going process and it doesn’t work in many cases. In an effort to track down every last one of those...efficiently, they’ve recently adapted the CRISPR/Cas9 system for protein tagging. We spoke with Mendenhall...context? Mendenhall: The benefits are two-fold: CRISPR/Cas9 offers high efficiency of inducing double strand... vectors for use with their favorite selection cassette. They could also switch from FLAG to GFP or a ...technologies. Generally speaking, anyone using CRISPR/Cas9 in the lab already should be able to run this protocol... crispr/cas9...

...PubMed. More CRISPR-Cas9 Resources: CRISPR 101: a blog series on the basics of CRISPR-Cas technology CRISPR...pointed out last year2, delivering a DNA cassette encoding the cas9 gene and single guide RNAs (sgRNAs) with...populations that already express Cas9 from an unlinked locus, while a Cas9+sgRNA drive targeting a synthetic...School. Scientists making transgenic organisms with Cas9 should be aware of the potential hazards of creating...caution. Why does making transgenic organisms with Cas9 risk creating a gene drive? The simplest way to ...easiest: don't use a DNA vector that encodes both Cas9 and sgRNA. What if I want to work with gene drives...sequence (save for the residual sgRNAs and possibly cas9 gene). An immunizing reversal drive is a variant...

...Hazan and Bester 2021). Other Cas variants Another option is CRISPR/Cas13, which targets RNA instead of.... Newer Cas protein variants may also be useful for targeting ncRNAs. For example, the Cas12a variant ...Lal A. Interrogating lncRNA functions via CRISPR/Cas systems. RNA Biology. 2021;18(12):2097. doi:10.1080...Targeting Non-Coding RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting. Front...Addgene blog RNA Editing with Cas13 Targeting RNA with Cas13a (C2c2) Cas13d: Small RNA-targeting CRISPR ...powerful tools. CRISPRi uses a nuclease-dead Cas9 protein (dCas9) and a sgRNA to temporarily block the transcription...protein-coding genes thanks to systems like CRISPR/Cas9 that allow them to manipulate full genes and observe...

... the Addgene Podcast.     Additional Resources on the Addgene Blog Listen to Our Podcast Interview with...In the second episode of our Hot Plasmids podcast series, you'll learn about new red fluorescent proteins...plasmids webpage. Listen to the entire Hot Plasmids podcast! Hot plasmids covered in this segment Hot Plasmid...

...proteins that negatively modulate the activity of Cas enzymes, some of which are also functional in multiple...here How do Cas13d enzymes compare to other Cas13s? Like other Cas13 enzymes, the Cas13d orthologs described... for other Cas13a and Cas13b effectors. Like previously characterized Cas13 enzymes, CasRx also displays...metagenome” (AdmCas13d) and Ruminococcus flavefaciens (nicknamed CasRx), as well as EsCas13d. Find the ...transcriptional inhibition (CRISPRi), and Cas13a/Cas13b RNA knockdown. CasRx was the clear winner with median ...identified Cas13a-c orthologs, but they do include HEPN nuclease domains characteristic of the Cas13 superfamily...orthologs from Eubacterium siraeum (EsCas13d) and Ruminococcus sp. (RspCas13d), while Konermann et al. characterized...

...CRISPR nickase experiment Overview of Cas9 nickase We’ll use SpCas9 nickases as examples for this post. ...strands bluntly with WT Cas9 and one gRNA, you can create a staggered cut using a Cas9 nickase and two gRNAs...By mutating one of two Cas9 nuclease domains, researchers created the CRISPR nickase. Nickases create...targeting range: using an individual gRNA with WT Cas9, repair levels decrease rapidly 10 bp from the cut...The HDR efficiency compared favorably to using WT Cas9 with the left gRNA across the window. In a subsequent... this problem. D10A nickase outperformed both WT Cas9 sites tested by a wide margin, exhibiting >20% repair...adapted from an IDT webinar: Optimized methods to use Cas9 nickases in genome editing. For more information...

...: An overview of the general schematic of CRISPR/Cas based COVID-19 testing methods.   Rapid detection...this test varies in the sequence of steps and the Cas protein used. Here, all the components needed for...candidate who investigates cellular aspects of CRISPR/Cas immune systems in the Sashital Lab, Iowa State University...Unlike Cas12a, Cas13a does not require strict sequence preferences at the target site, while Cas12a requires...of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences, Cas12a activity gets activated...transcription and Cas13 is then used to detect the amplified region of interest. Cas13 uses a complementary...then transcribed to provide RNA substrates for Cas13. Cas13, upon finding complementarity between the gRNA...

...mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9:39 . https://doi.org/10.1186...Passricha N, Khurana SMP, Tuteja N (2016) The CRISPR/Cas Genome-Editing Tool: Application in Improvement of...Zhu H, Li C, Gao C (2020) Applications of CRISPR–Cas in agriculture and plant biotechnology. Nat Rev Mol...Nuclease dead Cas9 (dCas9) based activators (such as dCas9-VP64) or repressors (dCas9-KRAB or dCas9-SRDX) can...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 ...get more information on multiplexing gRNAs here). Cas9 is commonly tagged with a nuclear localization sequence...