... 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...

...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...

...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...

...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...

...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...

...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...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...background. Their new method uses a selection cassette containing genes for hygromycin resistance and the...heat-shock inducible Cre to generate a self-excising cassette (SEC). To make it possible fluorescently label...into the genome. Figure 1: Self-excising cassette schematic from Dickinson et al. (2015). Used with...

...PubMed Central PMCID: PMC5947990. 3. Jarvik JW, Telmer CA. 1998. Epitope tagging. Annu Rev Genet 32: 601-618... Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively than Cas9 complexed ...Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease,...target site. With the widespread adoption of CRISPR/Cas9 technology and rapid improvements in the field, ...set out to develop a hassle-free, highly efficient Cas9 protein-based method for epitope tagging in mammalian...genes at a time using Custom Synthetic cr:tracrRNA/Cas9 Ribonucleoprotein (csRNP) delivery Epitope tagging...delivery of three reagents to cells: a guide RNA, Cas9 protein, and a donor DNA that contains the tag sequence...

...other Cas proteins Read all of our CRISPR blog posts Read about GeoCas9, another thermostable Cas9 Resources...indicates that like GeoCas9 and ThermoCas9, IgnaviCas9 is a type II-C Cas9. IgnaviCas9 is most similar to...thermostable Cas9 enzymes have previously been characterized: GeoCas9 from the Doudna lab and ThermoCas9 from ...Find IgnaviCas9 here! What can you do with IgnaviCas9? Why would a thermostable Cas9 be useful? It can enable...launched in the Quake Lab. Identifying IgnaviCas9, a hyperthermostable Cas9 This year it was wonderful to learn... Ignavibacteriae phylum genome. IgnaviCas9 is a hyperthermostable Cas9 CRISPR nuclease that is active ...assembled genomes, but also led to the discovery of IgnaviCas9, a new CRISPR enzyme from a sample from Mound...

...Enzymatic domains tethered to dCas9 often interact with DNA independently of dCas9 targeting (Galonska 2018...binding is crucial for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable...has several great options: pLV hUbC-dCas9-T2A-GFP for removable dCas9 expression and pLM-CMV-R-Cre. Selection... Some enzymatic fusions are godsends. Tethering dCas9 to the histone acetyltransferase p300 or the DNA...absent before. But other enzymatic fusions, such as dCas9-TET, can, like the iron ball-and-chain that they...-based epigenetic approach (spoiler alert: it’s dCas9… with no modifications). Ready to get started?   ...dioxygenase  When I started to work on building a CRISPR/dCas9-based tool for DNA demethylation in 2015, none existed...

... to CRISPR/Cas9 Technology? Learn More: Addgene's CRISPR/Cas9 Guide History of CRISPR Cas - A tale of... use CRISPR/Cas9 to generate mice with genes knocked out in the insulin signaling cascades in only 4 months...awards that will be bestowed upon discovers of CRISPR/Cas9 genome engineering, Dr. Jennifer Doudna and Dr. ...offered the great analogy of comparing the CRISPR/Cas9 system to Norton Antivirus. Antivirus programs work... crispr/cas9...

...advanced genome editing machinery such as CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out...explorations of the use of AdV for in vivo CRISPR-Cas delivery utilized the Non Homologous End Joining ..., Cowan CA, Rader DJ, Musunuru K (2014) Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome ...capacity is large enough to carry the Cas9 gene and a gRNA expression cassette in one single viral particle. ...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... out the plasmids at Addgene: pAdSh.PGK.Cas9 (expresses S. pyogenes Cas9 from the PGK promoter) and U6...

...versions of Cas9, nuclease and nickase, but we are now adopting dCas9, dCas9-VP64, and dCas9-KRAB in our...They went on to show that their CRISPR/Cas9-nuclease and CRISPR/Cas9-nickase vectors work successfully to...all-in-one vector construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available...one of the most advantageous properties of CRISPR/Cas9 compared to ZFNs and TALENs,” said Tetsushi Sakuma...offers ease and efficiency for all-in-one CRISPR/Cas9 vectors expressing up to seven guide RNAs (gRNAs...Figure 1: Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome ...Golden Gate cloning method to ligate expression cassettes of the gRNAs into a single vector in tandem. They...

... screened 11 other Cas9 orthologs to identify a more efficient, well tolerated, Cas enzyme. They identified...binding the DNA or extending the transcript. Different Cas orthologues and subtypes function somewhat differently...from the Lu lab found early success using CRISPR-Cas to target subsets of bacteria in population based...that leverage the remarkable properties of CRISPR-Cas systems is base editing. This process is generally...Fischetti VA, Marraffini LA (2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials...bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research 41:7429–7437 . https... Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synth Biol 4:723–728 . https://doi.org...

... the Addgene Podcast.     Additional Resources on the Addgene Blog Listen to Our Podcast Interview with...In the third episode of our Hot Plasmids podcast series, you'll learn about optogenetics tools for controlling...

...Listen to the Fire-Cas9 Podcast Segment Jerry Crabtree’s lab has developed a new CRISPR/Cas9-based system to...epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to a variety of protein modules...Inducible Recruitment for Epigenome editing by Cas9 (FIRE–Cas9), combines these approaches to enable rapid...here. No time to read? Listen to our Hot Plasmids Podcast! TRIM-Away: Targeted endogenous protein degradation...contributed by Mary Gearing Listen to the TRIM-Away podcast segment CRISPR and RNAi have helped researchers...Eric J. Perkins Listen to Endosomal pH Sensors podcast segment The first Addgene plasmid deposit from ...Communications. 2017. PubMed PMID: 28924139. FIRE-Cas9 for inducible interrogation of the epigenome Article...