... PAM sequence. Cas9 Species/Variants and PAM Sequences Species/Variant of Cas9 PAM Sequence ...complex as the “gRNA”. The gRNA contains both the 20 nucleotide target sequence to direct Cas9 to a specific...express a gRNA designed to direct Cas9 to their target sequence of choice (see tips for designing a gRNA) and...the guide RNA or a PAM sequence, Cas9 will neither bind nor cut the target. Cas9 homologs from different... and the scaffolding sequence necessary for Cas9 binding. When using CRISPR/Cas9 for genome editing, researchers...synthetic guide RNA or gRNA (sometimes sgRNA) In the native Type II CRISPR/Cas system, Cas9 is guided to its...condensed into one RNA sequence known as the guide RNA (gRNA) or single guide RNA (sgRNA). Throughout the remainder...

...repression. dCas9 has also previously been adapted to fluorescently label a sequence matching its gRNA, facilitating...differences in the PAM sequences required by each ortholog, a gRNA designed for one dCas9 should be specific... et al. tested their dCas9 variants using gRNAs specific for telomeric sequences and showed that different...different pairs of chromosomes using gRNAs specific to sequences on chromosomes 9 and 13. They next turned... 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...fluorescently labeled dCas9 isoforms are efficiently directed to the proper target sequence. They were also ...

... nucleotide PAM sequence serves as a binding signal for Cas9 and this sequence is a strict requirement...requirement for Cas9-mediated DNA cleavage. The need for more PAM sequences While PAM sequences for the commonly...of novel S. pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from... ~20 additional Cas9 species PAM sequence may not be characterized Non-SpCas9's bind a variety ...variety of PAM sequences, which makes them useful when no suitable SpCas9 PAM sequence is present. Furthermore...20, 2020. Cas9 can be used to modify any desired genomic target provided that (1) the sequence is unique...and (2) the sequence is located just upstream of a Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide...

...hIRF-1 gRNA #409 (Sa) Expresses a guide RNA (gRNA) to target human IRF-1 promoter for the Sa-Cas9 system...hIRF-1 gRNA #351 (Sa) Expresses a guide RNA (gRNA) to target human IRF-1 promoter for the Sa-Cas9 system...interest 61079 gRNA-hIRF-1 #12 Expresses a guide RNA (gRNA) to target human IRF-1 promoter 61080 gRNA-hIRF-1 ...expressing dCas9-2xAM and gRNA for human IRF-1 promoter. 98041 Sa-dCas9-NLS-3xFLAG/pcDNA3.1 Expresses Sa-dCas9...interest 85586 gRNA Cloning Vector Bbs I ver. 2 An empty sgRNA expression vector. sgRNA sequence can be cloned... a catalytically inactive form of Cas9 (dCas9) plus guide RNA (gRNA) for biochemical purification (Fig...CRISPR Cas9 gRNA plasmids from Dr. Hodaka Fujii's lab. Genome...

...mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA target sequence does not...overcome some of Cas9’s shortcomings. Cpf1’s preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in...especially given that Cpf1 gRNAs are also shorter than those needed for Cas9 (42 nt vs 100 nt.) However...efficiency varies widely with gRNA sequence. Both groups used multiple methods to examine Cpf1 off-target editing... they designed gRNAs with single and double mismatches throughout the 23-base sequence. Double mismatches...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...

.... pyogenes Cas9 in the ABE complex with PAM-variant Cas9 proteins: the S. pyogenes NG-Cas9 (PAM: NG) to...create NG-ABE8.xm/d and the S. aureus Cas9 (PAM: NNGRRT) to create Sa-ABE8.x-m/d. With these variants, ABE8... combination with a synthetic library of TadA sequences to create the eighth generation of adenine base...constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The dC9-ABE8.x-m/d constructs ...frequency more than 90% compared to ABE7.10. Choosing Cas9 proteins to increase the targeting scope of adenine... base editors The most commonly used S. pyogenes Cas9 requires the target site in the genomic DNA to contain...frequency over ABE7.10. The ability to use non-standard Cas9 in the ABE8 complexes enables a broader targeting...

...insertable sequences or specific mutations, along with homologous DNA to the target sequence to be modified...continue to cleave DNA if the gRNA/PAM site remains intact. In other words, Cas9 will keep cutting and repairing...and deletions along the way. HDR uses homologous sequence as a template for repair and is thus the more ...used for repair is identical to the original DNA sequence at the DSB, or it can introduce specific mutations...non-crossover events. This means all newly synthesized sequences are retained by the original damaged DNA molecule...Homology directed repair and genome engineering CRISPR-Cas9 has revolutionized the genome engineering world ...is introduced disrupting one or both of these sequences. To prevent re-cutting and the resulting mutations...

...includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and a gRNA cassette....tuberculosis. dCas9 and a gRNA are expressed on pRH2502 and pRH2521, respectively. Both dCas9 and the gRNA are ...recombineering is unsuccessful and the wild-type sequence persists, Cas9 will cleave the gene of interest, inducing...survive have inactivating mutations in the Cas9 or spacer sequence, and these rare events are easily detectable...pMAZ-SK contains an aTet-inducible gRNA and a backbone-targeting gRNA cassette for plasmid curing through...contains Cas9 and the phage recombination gene lambda Red. pTargetF contains the specific gRNA(s), and ...functions more similarly to CRISPR/Cas9 systems adapted for eukaryotes in that Cas9-mediated cleavage induces ...

...Staphylococcus aureus Cas9 (Sa-Cas) and deposited plasmid IF311: pMAGIC (R4-R3) NLS-Sa dCas9-NLS-LSD1. LSD1 ...types. They have also fused LSD1 to x-Cas9(3.7). x-Cas9(3.7) is a SpCas9 mutant that exhibits increased PAM... specific DNA sequences. Non-editing CRISPR applications direct catalytically dead dCas9 fused to a variety...control over gene expression that go beyond genomic sequence. Dysregulation of the epigenome (the sum of epigenetic...larger pMVP/pMAGIC cloning system, which contains Cas9 fusions for several epigenetic modifiers, and is...Gersbach CA (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters...Crawford GE, Reddy TE, Gersbach CA (2017) CRISPR–Cas9 epigenome editing enables high-throughput screening...

...with paired gRNAs to lower off-target cleavage frequency. Truncated gRNAs: Truncated gRNAs display less...High fidelity Cas9s: Three high fidelity Cas9 variants , SpCas9-HF, eSpCas9, and HypaCas9 display very ...III and Cas9 enzymes, which together separate the individual crRNAs. (3) The tracrRNA and Cas9 nuclease...Figure 2: An overview of CRISPR and NHEJ/HDR. The Cas9/gRNA complex produces a DSB at the target genomic locus...Staphylococcus aureus (SaCas9) is smaller than SpCas9 and more easily packaged in AAV. Cas9 orthologs also have... unique crRNA. (4) Each crRNA:tracrRNA:Cas9 complex seeks out the DNA sequence complementary to the crRNA...beyond SpCas9, moving us closer to being able to target every locus in every genome. Multiple Cas9 orthologs...

...oligonucleotides Cas9, photocaged-gRNA Gene editing Manna et al., 2019   NPPOC-caged trimethoprim (TMP) Cas9 fused...cell, the dCas9-CIB1 fusion binds to the target DNA sequence as directed by the guide RNA (gRNA), while ...proteins from N. crassa Split Cas9, Split Cas9 nickase, Split dCas9 Genome editing by NHEJ and HDR, repression...Photoswitchable Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing ...heterodimerizing domain to each of the Cas9 fragments, the authors created a photoactive Cas9 tool, as shown in the figure...modular nature of Cas9, Nihongaki et al. showed that it was possible to swap out the Cas9 domains in their...light-activated split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while transcription...

...DNA sequence in only 1 min. Using these lentiviral plasmids, catalytically inactive Cas9 (dSpyCas9) fused...transcription of RNA such as long-read cDNA sequencing and RNA sequencing of complex heterogeneous RNA samples...Caspex’ construct is guided by a pre-defined sgRNA sequence to a specific genomic locus and labels the ...inducible CMV promoter and targeted to a sequence of interest with an sgRNA expressed from a separate plasmid...functional element (tyrosine sulfation consensus sequence, fluorophore, or APEX2), allowing for quantitative...Find the Tet-inducible caspex plasmid here. Myers SA et al. Nature Methods. 2018. PMID: 29735997 bioRxiv...genomic loci. He made use of catalytically inactive dCas9 by fusing it to an engineered peroxidase, (APEX)...

...stabilizing the 3’ end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed...reconstituting coding sequences for a range of applications, including CRISPR activation by dCas9-VPR, prime editing...Blainey’s lab has developed a multiplexing approach to Cas9-based pooled CRISPR screens called CROPseq-multi...platform is compatible with enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene...Anzalone et al., 2019, including modifications to the Cas9 or reverse transcriptase enzymes, inhibition of ... improve the specificity of ssAAV, a targeting sequence of miR124T was inserted into the vector to silence...alone or in combination with miR124T targeting sequences, provides a simple yet valuable tool for microglia...

...determined by both the gRNA sequence and the Cas9 enzyme. Ideally, a gRNA targeting sequence will have perfect... an sgRNA. gRNA scaffold sequence The sequence within the gRNA that is responsible for Cas9 binding, it...spacer/targeting sequence that is used to guide Cas9 to target DNA gRNA targeting sequence The 20 nucleotides...any genomic sequence specified by a particular gRNA. Epitope tag(s) are fused to dCas9 or gRNA for efficient... and PAM Sequences Species/Variant of Cas9 PAM Sequence* Streptococcus pyogenes (SP); SpCas9 3' NGG SpCas9...(PAM) The PAM sequence (NGG) serves as a binding signal for Cas9, but the exact sequence depends on which...proteins and PAM sequences . Once you’ve selected your target, you’ll express your gRNA and Cas9 protein, which...

...determined by both the gRNA sequence and the Cas9 enzyme. Ideally, a gRNA targeting sequence will have perfect... an sgRNA. gRNA scaffold sequence The sequence within the gRNA that is responsible for Cas9 binding, it...spacer/targeting sequence that is used to guide Cas9 to target DNA gRNA targeting sequence The 20 nucleotides...any genomic sequence specified by a particular gRNA. Epitope tag(s) are fused to dCas9 or gRNA for efficient... and PAM Sequences Species/Variant of Cas9 PAM Sequence* Streptococcus pyogenes (SP); SpCas9 3' NGG SpCas9...(PAM) The PAM sequence (NGG) serves as a binding signal for Cas9, but the exact sequence depends on which...proteins and PAM sequences . Once you’ve selected your target, you’ll express your gRNA and Cas9 protein, which...