... encoding the gRNAs and Cas9. As with the Gersbach lab plasmids, multiple Cas9 variants are available:...subsequently inserted into a Cas9-containing destination vector based on pCAMBIA. Cas9 is plant-optimized, and...humanized Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric...options include humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator... then digested using BsmBI and ligated into Cas9 or dCas9-containing destination vectors. Destination ...the multiple gRNAs and Cas9 together into a single plasmid. Although only wt hCas9 entry vectors are supplied...target multiple sites! In this post, we'll cover Cas9 multiplexing options, but also check out our blog...

...needed by Cas9 for cleavage. In certain cases, Cpf1 may be better suited for genome editing than Cas9 - read...CRISPR system. Like Cas9, Cpf1 contains a RuvC-like endonuclease domain, but it lacks Cas9’s other HNH endonuclease...Potential advantages of Cpf1 over Cas9 Type II CRISPR systems based on Cas9 were thought to be the simplest...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...needed for Cas9 function. Since both Cpf1 and its guide RNAs are smaller than their SpCas9 counterparts...display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain a RuvC-like...

...several Cas9s that recognize NG PAMs. The Nureki lab generated SpCas9-NG, a rationally engineered Cas9 with...PAM-interacting domain of Cas9. After multiple rounds of engineering, they developed the SpRY Cas9 variant (Walton... at NGH PAMs (Zhang et al, 2021) NG Cas9 NG - SpG Cas9  NG  - SpRY nuclease  C to T Base Editor...notes SpCas9 NGG - Cas12a TTTV Great for AT-rich regions Cas9 EQR NGAG - xCas9 NG Less...of gRNA placement significantly over SpCas9 and more so than Cas9s with an NG PAM.   Cas PAM sequence...    Fig. 1: SpCas9 has sparse PAM coverage in the human genome. SpRY Cas9 has PAM sites covering...blog The PAM Requirement and Expanding Beyond Cas9 xCas9: Engineering a CRISPR Variant with PAM Flexibility...

...similarities and differences to Cas9. DNA targeting Cas enzymes Cas9 – The genome engineering workhorse...CRISPR’ without shortly thereafter hearing ‘Cas9’. The Cas9 nuclease is a genome engineering powerhouse...making Cas9 an adaptable choice for many genome engineering endeavors. For more information on Cas9 variants...same mechanism as traditional spCas9. If you are looking to modify RNA, Cas9 just won’t cut it – literally...We are all oh so familiar with the nuclease Cas9, but how does it stack up to the new Cas enzymes on ...proceed to generate a blunt DNA double strand break. Cas9 has been studied for genome engineering purposes...variants, check out our blog post on choosing a Cas9 enzyme. Fast facts – PAM requirement: NGG and an expanding...

...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...optogenetic control of CRISPR-Cas9. By combining AcrIIA4, a Streptococcus pyogenes Cas9 inhibitor, with the LOV2...Anti-CRISPR proteins for optogenetic control of CRISPR-Cas9 Anti-CRISPR proteins that inhibit type II CRISPR...

...Comparing the Cas9 and Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 ... guide page for a review of Cas9.  For a brief comparison of Cpf1 vs. Cas9, see the table below. Table...Editing Which Cas9 Do I Choose for My CRISPR Experiment A Match Made in Heaven: CRISPR/Cas9 and AAV Resources...promoters).  spCas9 and its gRNAs are also larger than their Cpf1 counterparts. Table 2. Cas9 Multiplexing... system for editing multiple targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing....cleaves 5' of the protospacer Multiplexing CRISPR-Cas9 options before Cpf1 crRNA array Prior to this new...multiplex CRISPR gene editing methods relied solely on Cas9. Overall, these approaches have two main drawbacks...

...chromosome encodes a tracrRNA (in some systems including Cas9), Cas proteins, and a CRISPR array. The CRISPR array...bacteria, these crRNAs are bound by Cas proteins (Cas9 shown here). The repeat sequence base pairs with...“single-guide RNA” that performs both functions. Cas9 cuts both the target and nontarget DNA strands upstream...CRISPR-associated.” The most famous of these proteins is Cas9 nuclease, but there are many other Cas proteins ... strand: The paired strand of foreign DNA cut by Cas9, whose sequence is not checked. Assuming full complementarity...these sequences. Pro tip! Various labs have created Cas9 enzymes with looser PAM requirements, which are ...sequence is the RNA guide, like a wanted poster that Cas9 uses to track down its suspect—the target DNA. tracrRNA...

...express Cas9 and a gRNA from the same plasmid. LentiCas9-blast virus can be used to generate Cas9-expressing...While various Cas9 mutants have been engineered to perform unique editing tasks, classic Cas9 nuclease, which...gRNAs in a monoclonal Cas9 background because you’ll have more consistent levels of Cas9 expression between...this plasmid Cas9 expression is typically cytotoxic, which makes generation of high titer Cas9 lentiviruses... expressed from lentiCas9-Blast. Purpose of LentiCas9-blast LentiCas9-blast is a Cas9 expression vector...Secondly, you can use a LentiCas9-Blast viral prep to create a single stable Cas9-expressing cell line and...growth rate variability when using lentiCas9-blast to generate Cas9-expressing cells. To ensure that this...

...contains Cas9. The Madison lab also created an inducible SRIRACCHA system (iSRIRACCHA) where Cas9 expression... labor intensive and quite time consuming.   As Cas9/gRNA activity is crucial for successful CRISPR editing...the scientific community for efficient real-time Cas9 activity assays. Recently scientists have created...fluorescent reporter assays to quantify targeted Cas9 cleavage, and repair through homology directed repair...containing the reporter gene that was cleaved by Cas9 and repaired via HDR using the supplied donor plasmid...expressing cells provides a real-time read out of Cas9 activity.  By using the RFP:GFP ratio, edited cells...Thus SRIRACCHA enables scientists to both quantify Cas9 activity and sgRNA efficiency as well as enrich ...

...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... engineering experiments, consider that wildtype Cas9 with optimized chimeric gRNA has high efficiency...effect. The double nickase system is based on the Cas9 D10A nickase described in Figure 4 of the Cong, ...can express two different chimeric gRNAs with the Cas9 nickase which will together introduce cleavage of...arms that were 800bp long. Tips for using CRISPR-Cas9 at the lab bench Q4: After the introduction of a...CRISPR guide for background information on CRISPR/Cas9 systems Peruse the most recent genome editing review...

... 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 RNP activity or longevity. GeoCas9 is active at high...used to make eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally occurring Cas9 diversity ...bacteria, such as Streptococcus pyogene’s SpCas9. These Cas9 proteins don’t work well at high temperatures...discovery of GeoCas9 by the Doudna lab has opened up the field of thermophilic bacteria to CRISPR/Cas9 genome...discovered a Cas9 protein from the thermophilic bacteria Geobacillus stearothermophilus (GeoCas9). G. stearothermophilus...human plasma. The discovery of GeoCas9 clears the way for CRISPR/Cas9 genome editing in facultative and... Cas9 is the genome editing tool of choice for a number of model organisms: mammalian cells, yeast, drosophila...

...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...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... “gDNAs”) rather than the RNA guides employed by Cas9 or Cpf1. These short, 24 base DNA guides may be ...

...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... could be rescued by coinjection into zygotes of Cas9 mRNA and a single-guide RNA targeting the mutant...Hubrecht Institute in The Netherlands used the CRISPR/Cas9 genome editing system to correct the cystic fibrosis...cystic fibrosis (8).   The difference with CRISPR/Cas9 in comparison to other genome editing techniques...

...CRISPR/Cas9 over previous genome editing techniques is its simplicity and versatility. CRISPR/Cas9 consists...in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be ...of two components: the non-specific endonuclease (Cas9) and a single stranded guide RNA (gRNA). The ~20...the user, and it can be easily modified to target Cas9 to virtually any genomic locus, provided the target...adjacent motif (PAM) sequence. Co-delivery of wild-type Cas9 and a gRNA generates a double-strand break in the...genomic target. The goal of a CRISPR screen is to use Cas9 and a pool of gRNAs to identify genes that are essential...lentivirus to deliver a pooled population of gRNAs to Cas9 expressing cells. Pooled lentiviral CRISPR libraries...

...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...3xFLAG-tag fusion proteins. The repair template and Cas9-sgRNA plasmid is then injected into the germline...species or in cell culture. In addition to CRISPR/Cas9 applications, SEC selection could replace other,...system represents another leap forward for the CRISPR/Cas9 revolution!   References Streamlined Genome Engineering...

...releasing Cas9. Nanoblades can also deliver Cas9 variants, such as a transcriptional activator dCas9-VPR, ...Nanoblades by association with the the Cas9 portion of the Gag-Cas9 fusion protein. A single Nanoblade particle...along with the gRNA and Cas9. An “all-in-one” Nanoblades which carries a gRNA, Cas9, and a HDR donor template...centrifugation. Gag-Cas9 fusion protein The MLV Gag protein fused to a Flag-tagged SpCas9 is incorporated ... leukemia virus (MLV)-like particles loaded with Cas9-gRNA ribonucleoproteins (RNPs). According to the...generation. Key components of Nanoblades Nanoblades are a Cas9 RNP delivery vehicle that’s based on mouse leukemia...just like the unmodified MLV Gag protein. After Gag-Cas9 is incorporated, the MLV protease, supplied as part...

... CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate for generating knockouts...in vivo viral delivery. Cas9 mRNA and gRNA Plasmids containing gRNA and Cas9 are used in in vitro transcription...generating transgenic embryos. Cas9-gRNA ribonucleoprotein complexes Purified Cas9 protein and in vitro transcribed...need to deliver. Minimally, one or more sgRNAs and Cas9 are required for any application. If you want to...Application Mammalian expression vector Promoter driving Cas9 expression can be constitutive or inducible. U6 ...stable cell lines. Transient or stable expression of Cas9 and/or gRNA in a mammalian cell line that can be...transfected at high efficiency. Lentiviral transduction Cas9 and gRNA can be present in a single lentiviral transfer...

...single base editing by APOBEC-Cas9 Though the ACE reporter quantifies APOBEC-Cas9 efficiency in real-time, ...and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells. Nucleic Acids Research... a APOBEC-Cas9n-UGI complex. So how exactly does this work? Figure 1: APOBEC- and Cas9-mediated ...genome, but they’re inefficient compared to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due...can help you do just that. Monitoring APOBEC and Cas9-mediated editing in real time The Harris lab created...APOBEC (a family of cytidine deaminase proteins) and Cas9-mediated editing in real time (St. Martin et al....quantify the on-target DNA editing efficiency of APOBEC-Cas9 editisome complexes in real-time (St. Martin et ...

...et al., 2016). While Streptococcus pyogenes Cas9 (SpyCas9) is the most used and well studied CRISPR system... study, the labs found that anti-CRISPR prevents Cas9 activity in HEK293 by reducing editing from ~30%...interact with CRISPR-Cas systems such as Cascade-Cas3, Cas9, Cas12, and Cas13 (Marino et al., 2020). Some Acr...ways: Inhibit DNA binding Example:  AcrIIA4 blocks Cas9’s interaction with the PAM site (Dong et al., 2017... crRNA loadingExample: AcrIIC2 interactions with Cas9 interferes and prevents the correct assembly of ... AcrIIC1 binds to the HNH endonuclease domain of Cas9 and prevents target DNA cleavage (Harrington et ... Doudna lab found that at least 50% of on-target Cas9 edits happens within the first six hours. When added...

.... Repair of Cas9-induced breaks by NHEJ As illustrated below, NHEJ-mediated repair of Cas9-generated breaks...Cas system for genome engineering is the fact that Cas9 can be easily programmed to make a DNA double strand...discuss NHEJ, and how it impacts what happens to Cas9-mediated DSBs in the genome. Non-homologous end...consequently the principal means by which CRISPR/Cas9-introduced breaks are repaired. The following factors...introduce indels. Given the end structure of the Cas9 DSB (blunt or near-blunt ends without nucleotide...population of cells constitutively expressing a targeted Cas9 should possess indels in the majority of their chromosomes...Another factor expected to impact repair is that the Cas9 protein doesn’t immediately release from the broken...