...overcome some of Cas9’s shortcomings. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in...directional gene transfer, analogous to traditional restriction enzyme cloning. Sticky-end mediated gene transfer...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...editing in AT-rich genomes. Additionally, whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1...two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern and requires...rather than the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two new studies show that Cpf1...

... Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively than Cas9 complexed ...2017. Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian... epitope tagging of a large cohort of genes (e.g. tagging genes representing a protein super family). ...Synthetic cr:tracrRNA/Cas9 Ribonucleoprotein (csRNP) delivery Epitope tagging of mammalian genes using CRISPR ...knock-in for 96 different genes in a single go (Figure 3). We were able to tag >30 genes/plate in the first ...designed for ~250 genes, we have had about 30% tagging success, although many of these genes were expressed...Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease,...

...available through Addgene. Let’s take a look at lenti-Cas9-VQR-Blast, which has the Addgene ID #87155. The...format for lenti-Cas9-VQR is the URL “http://n2t.net/addgene:87155” where the Addgene registered prefix...lenti-Cas9-VQR-Blast was a gift from Daniel Bauer (Addgene plasmid # 87155 ; http://n2t.net/addgene:87155...Bauer (Addgene plasmid # 87155 http://n2t.net/addgene:87155)” Cite plasmid using RRID “lenti-Cas9-VQR-Blast...lenti-Cas9-VQR is represented as “RRID:Addgene_87155” and the following URL (https://scicrunch.org/resolver...resolver/RRID:Addgene_87155) will take you directly to the lenti-Cas9-VQR plasmid page. Despite their differences...

...Wenning Qin and Haoyi Wang. CRISPR/Cas9 is revolutionizing the mouse gene-targeting field. Mice have long... so genes. It would be great if we could just go in and quickly manipulate individual mouse genes and ...insertion of a reporter gene, or replacement of the murine sequence with a human gene. Advantages of using...conventional gene targeting. CRISPR tools were recently used to introduce a 25 kb human gene into the mouse...design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nat Biotechnol 32:1262–1267..., Nakauchi H (2018) Intra-embryo Gene Cassette Knockin by CRISPR/Cas9-Mediated Genome Editing with Adeno-Associated... Efficient Production of Gene-Modified Mice using Staphylococcus aureus Cas9. Sci Rep 6: . https://doi.org...

...protein-coding genes thanks to systems like CRISPR/Cas9 that allow them to manipulate full genes and observe...nearby genes and its role in gene expression, though this may still unexpectedly affect nearby genes (Figure...The CRISPR/Cas9 system is often used to knock-out, or permanently disable, protein-coding genes by generating...JS. Fitness effects of CRISPR/Cas9-targeting of long non-coding RNA genes. Nature biotechnology. 2020;38...CRISPR plasmids at Addgene! Knock-in: Introducing tags or stop signals Once Cas9 generates double-strand breaks...powerful tools. CRISPRi uses a nuclease-dead Cas9 protein (dCas9) and a sgRNA to temporarily block the transcription...only 2% of our genome is made of protein-coding genes, and you might be wondering what the rest of our...

...effectively serving their function of guiding Cas9 to generate a targeted break. These advances in gRNA technology...surprise then that stability of the RNA used in CRISPR-Cas9 experiments – the “gRNA” – suffers from the same...modifications The recognition sequence for CRISPR-Cas9 targeting is led by a ~20 bp gRNA which is part ...trans-activating crRNA (tracrRNA) which interacts with the Cas9 protein. These RNA molecules are vulnerable to attack...partially DNA gRNA is surprisingly well-tolerated by the Cas9 protein and can increase on-target efficiency. In...modifications listed above were developed before CRISPR-Cas9, for use in applications like siRNA and antisense...On-off switches for gRNAs A major drawback of CRISPR-Cas9 technology is that most expression systems are constantly...

...which I use to study gene regulation during developmental processes, CRISPR/Cas9 allows us to delete sequences...co-conversion markers. Expanding beyond Cas9 Since the development of CRISPR/Cas9 editing methods, other Cas proteins...mammalian and yeast cells using modified Cas9 proteins to regulate gene expression (CRISPRi/CRISPRa), visualize...Forstemann K (2014) Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination...Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics 198:837–846 ....Editing Using CRISPR-Cas9. Genetics 211:431–457 . https://doi.org/10.1534/genetics.118.301775 Ghanta KS...elegansvia a CRISPR–Cas9 System. Genetics 195:1181–1185 . https://doi.org/10.1534/genetics.113.156075 Ward...

... 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...joins the pantheon of useful CRISPR/Cas9 techniques, we at Addgene are excited to see what’s coming next...this publication at Addgene. A protein-tagging system for signal amplification in gene expression and fluorescence... 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... chromatin organization. This fluorescent CRISPR/Cas9-based method is a great step forward in live cell...

...inducible CRISPR/Cas9 systems for transcriptional activation and genome editing by fusing Cas9 to a mutated...identify essential genes and phage host factors, and identify sequence-specific toxicity of Cas9. Read the paper...Creating knock-ins with GeneWeld vectors GeneWeld from Jeffrey Essner’s lab uses CRISPR/Cas9 and a donor vector...research! This time: GeneWeld vectors to create knock-ins CasX Drug inducible CRISPR/Cas9 activation E. coli...transcriptional regulators. In this system, Cas9 generates a double-stranded DNA break at the genomic ...averaging 19 targets per gene. The lab has previously used the library with SpdCas9 to perform pooled CRISPRi...either side of the cargo (ex: fluorescent protein gene) on the donor vector. Therefore, targeting requires...

...combining specific sgRNAs with Cas9 or Cas9 derivatives. Unmodified Cas9 causes double stranded DNA breaks...function of lowly expressed genes or genes that are more drastically affected by gene activation rather than...investigate gene function. CRISPR libraries can be used to knock out, inhibit or activate target genes by combining...CRISPRa) to regulate gene expression. Some CRISPR libraries contain many sgRNAs per gene to increase confidence...of a gold standard set of genes (1,580 essential and 927 non-essential genes). The Root and Doench lab...viability. In addition, high copy number genes may mimic viability genes as they are subject to an increase ...overexpression library identified several overlapping genes, most genes were unique to the individual libraries. These...

...transgenic organisms with Cas9 risk creating a gene drive? The simplest way to make a gene drive is to insert...endonuclease gene. As we pointed out last year2, delivering a DNA cassette encoding the cas9 gene and single...biology and genetics because the underlying advance enabling RNA-guided gene drives is Cas9 genome editing...transgenic organisms with Cas9 should be aware of the potential hazards of creating “gene drives” capable of ... creating a gene drive is also the easiest: don't use a DNA vector that encodes both Cas9 and sgRNA. What...populations that already express Cas9 from an unlinked locus, while a Cas9+sgRNA drive targeting a synthetic...sequence (save for the residual sgRNAs and possibly cas9 gene). An immunizing reversal drive is a variant that...

...development, CRISPR/Cas9 genome editing has been used to study the effect of gene knockout in vivo and...of CRISPR/Cas9 in new avenues of research. The use of multiple gRNAs targeting a single gene can increase...increase the likelihood of gene knockout, or multiple gRNAs may be used to target genes with overlapping activity... power of polycistronic gene organization, and its translation to CRISPR/Cas9 shows great potential for...recombination. To further increase the utility of CRISPR/Cas9, it will be necessary to improve its multiplexing...observe a phenotype, the modification of multiple genes is often necessary. Guide RNAs (gRNAs) are commonly...construct size, it requires coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may...

...time-consuming, as it only changes one gene at a time. CRISPR-based systems enable gene knockout in immune cells in... CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic stem cells with a lentiviral... to create mice with gene knockouts in their immune systems. However, many genes have different functions...combination with different genes. Having CRISPR systems for assessing genes in defined contexts would ... Knocks out a pair of genes pXPR_219 Violet-excited GFP (vex) Rosa26-Cas9 I-CHIME Inducible...knockout of a single gene pXPR_068 Violet-excited GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 L-CHIME Knocks...) Lineage-Cre; Rosa26-Cas9 S-CHIME Knocks out pairs of genes sequentially pXPR_220 Violet-...

...) to a catalytically impaired Streptococcus pyogenes Cas9. They called this editor Target-ACE (adenine...toolkit for epitope tagging Lentiviral capsid-based Cas9/gRNA ribonucleoprotein delivery Cytosine deaminase...Read the paper in bioRxiv Find Target-ACE   CRISPR/Cas9-based toolkit for epitope tagging in neurons Harold... the paper in bioRxiv Find the ORANGE toolkit   Cas9/gRNA ribonucleoprotein delivery by lentiviral capsid-based...capsid-based bionanoparticle Transient expression of CRISPR/Cas9 in cells has advantages like reduced off-target ...off-target activity and decreased immune response to Cas9. Recently, Baisong Lu’s lab developed a lentiviral capsid-based...capsid-based bionanoparticle system for packaging Cas9/gRNA ribonucleoproteins for transient expression...

...Abudayyeh et al., 2016). In the endogenous CRISPR/Cas9 system, Cas9 uses both a tracrRNA and crRNA to facilitate... 27, 2020. CRISPR, and specifically Cas9 from S. pyogenes (SpCas9), is truly an exceptional genome engineering... (Shmakov et al., 2015). They were using Cas1, a gene commonly associated with CRISPR arrays and involved...analysis, they identified 53 potential candidate genes that fell into 3 categories based on the architecture...Perhaps the biggest difference between Cas13a and Cas9 is that Cas13a binds and cleaves RNA rather than...enzymes - Cas13a contains two HEPN domains, whereas Cas9 uses HNH and RuvC domains to cleave target DNA. ...roles for HEPN domains in other proteins. As with Cas9, mutating key residues in the Cas13a molecule results...

...would be compatible with CRISPR/Cas9 genome editing. Although CRISPR/Cas9 is all the rage, they had another... both Cas9 protein and a gRNA (yes, iTOP can also be used for RNA transduction) targeting a gene associated...instance, it’s much more difficult to introduce a gene of interest into primary cells, so most primary ... method and its potential applications to CRISPR/Cas9 genome editing.   Protein-only transduction? Niels...expression. Image courtesy of Diego D'Astolfo. CRISPR/Cas9: a potential application of iTOP Having tested iTOP...expression has a long-lasting effect, such as Cre- or Cas9-mediated recombination. Both primary hESCs and immortalized... indicating that iTOP may be valuable for CRISPR/Cas9 stem cell editing. The small molecule NSDB...

... gRNAs) that targets ~4,000 E. coli genes with ~15 gRNAs per gene and includes ~10,000 gRNAs targeting...paper in Communications Biology Find the Cas9-PCV and PCV-Cas9 plasmids Selective curbing of unwanted ...essential C. albicans gene. Read the paper in mSphere Find the dCas9 and dCas9-Mxi1 plasmids If you...Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR interference in Candida albicans... AAV delivery of Nme2Cas9 with gRNA cassette Erik Sontheimer’s lab identified a Cas9 protein from Neisseria...library Covalent tethering of DNA repair template to Cas9 Homology-directed repair (HDR) can be used to make...oligodeoxynucleotide HDR template covalently attached to the Cas9-gRNA RNP complex via the fused HUH endonuclease ...

...strands bluntly with WT Cas9 and one gRNA, you can create a staggered cut using a Cas9 nickase and two gRNAs...CRISPR nickase experiment Overview of Cas9 nickase We’ll use SpCas9 nickases as examples for this post. ...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...

...-wide CRISPR/Cas9 screens are a high-throughput systematic approach for identifying genes involved in ...effects of gRNA and Cas9 expression, they fail to account for the effects of Cas9-induced dsDNA breaks...library that has 10 gRNAs per gene and targets all ~20,500 protein-coding genes. Unique, non-overlapping sites... either stably expressed Cas9 or that had been lentiviral infected with Cas9. Validating the Library The...88% of previously identified essential genes while previous Cas9 and shRNA library screens only identified... identified ricin toxicity genes. The screen also discovered several genes previously not associated with...p-values of known ricin regulation genes compared to results generated using non-targeting guides as controls...

... and the wild-type sequence persists, Cas9 will cleave the gene of interest, inducing a lethal DSB.   ...Jiang et al., 2015). pCas contains Cas9 and the phage recombination gene lambda Red. pTargetF contains the...pCRISPomyces-1 includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette...functions more similarly to CRISPR/Cas9 systems adapted for eukaryotes in that Cas9-mediated cleavage induces ...efforts to regulate gene expression were limited to inducible promoters or direct gene knockout. In contrast.... These could be genes, specific polymorphisms, including antibiotic resistance genes. This approach successfully...The system available from Addgene consists of two plasmids: pCas9: carries Cas9 and chloramphenicol resistance...