...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...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...Again, they were in for a surprise! Whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1...

...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...

... 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...plasmids from this paper at Addgene. 2. Kabadi, Ami M., et al. “Multiplex CRISPR/Cas9-based genome engineering...

...or see Addgene's CRISPR guide page for a review of Cas9.  For a brief comparison of Cpf1 vs. Cas9, see ...Comparing the Cas9 and Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 ...method, other multiplex CRISPR gene editing methods relied solely on Cas9. Overall, these approaches have...promoters).  spCas9 and its gRNAs are also larger than their Cpf1 counterparts. Table 2. Cas9 Multiplexing...Resources on the Addgene Blog Cpf1: A New Tool for CRISPR Genome Editing Which Cas9 Do I Choose for My... A Match Made in Heaven: CRISPR/Cas9 and AAV Resources on Addgene.org Browse Our CRISPR Multiplexing... system for editing multiple targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing....

...stably integrate a reporter gene that contains a puromycin resistance gene followed by the target site...contains Cas9. The Madison lab also created an inducible SRIRACCHA system (iSRIRACCHA) where Cas9 expression...that targets your desired gene (whose sequence is also found in the reporter gene). A control RFP expression...while cells containing the reporter gene that was cleaved by Cas9 and repaired via HDR using the supplied...plasmids at Addgene GEMCherry2, an improved monomeric fluorescent protein for assessing CRISPR/Cas9 activity... 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...

...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...genome where it will proceed to generate a blunt DNA double strand break. Cas9 has been studied for genome...Cpf1, Cas12’s primary difference from Cas9 is that it generates staggered breaks, also known as sticky...same mechanism as traditional spCas9. If you are looking to modify RNA, Cas9 just won’t cut it – literally..., unlike Cas9, it targets RNA instead of DNA. This makes it particularly useful in generating transient...

...express Cas9 and a gRNA from the same plasmid. LentiCas9-blast virus can be used to generate Cas9-expressing...this plasmid Cas9 expression is typically cytotoxic, which makes generation of high titer Cas9 lentiviruses...While various Cas9 mutants have been engineered to perform unique editing tasks, classic Cas9 nuclease, which...libraries) and individual gene knockouts (using gRNA lentiviruses). Separating Cas9 and the gRNA into two ...gRNAs in a monoclonal Cas9 background because you’ll have more consistent levels of Cas9 expression between... expressed from lentiCas9-Blast. Purpose of LentiCas9-blast LentiCas9-blast is a Cas9 expression vector...growth rate variability when using lentiCas9-blast to generate Cas9-expressing cells. To ensure that this...

...have created Cas9 enzymes with looser PAM requirements, which are available from Addgene!   RNA crRNA:..., simplifying the CRISPR-Cas9 system for laboratory applications. At Addgene, we sometimes refer to the... Sheet  Resources on the Addgene blog A Needle in a Base-Stack: Cas9 Structural Biology...elegant bacterial immune system and an efficient gene editing tool… but boy does it have a lot of parts...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...

...Publication at Addgene Cas9 and NgAgo head to head NgAgo has a number of unique features compared to Cas9. These...Resources on the Addgene Blog Read About Cas9 Homolog Cpf1 Check Out High Fidelty Cas9 Mutants Browse ...lines, with an efficiency similar to that of Cas9. Like Cas9, NgAgo is also able to mediate homology-directed... certain guide sequences - using 5 guides/gene for 10 genes, they did not observe differences in cleavage... BELOW Biologists are going gaga over the newest gene-editing protein - a DNA-cleaving Argonaute from ... why you might choose NgAgo over CRISPR proteins Cas9 or Cpf1 - keep reading to learn more! Background...These include: More targeting flexibility Unlike Cas9 and the other CRISPR enzymes, NgAgo does not require...

...researching a way to treat AD using CRISPR/Cas9. They are targeting the genes responsible for both early onset ...mutation in a gene that causes cataracts could be rescued by coinjection into zygotes of Cas9 mRNA and a ...5. Rohn et al. 2018. “The Potential of CRISPR/Cas9 Gene Editing as a Treatment Strategy for Alzheimer’...Lucie is an Addgene co-op from Northeastern University.  There can be no doubt that CRISPR/Cas9 technology...applications.” Li’s team noted that CRISPR/Cas9 has been used to generate mutations in a wide range of organisms...glance at genome editing with CRISPR-Cas9 technology.” Current genetics. https://doi.org/10.1007/s00294-019...2020. . “Advances in CRISPR/Cas-based Gene Therapy in Human Genetic Diseases.” Theranostics. https://doi.org...

...trying to target every gene in the genome, or a specific class of genes? Addgene currently carries several...versatility. CRISPR/Cas9 consists of two components: the non-specific endonuclease (Cas9) and a single stranded...within the target gene. CRISPR can also be used to activate or repress target genes without permanently... of a CRISPR screen is to use Cas9 and a pool of gRNAs to identify genes that are essential for a given...repress target genes by combining a gRNA library with the aforementioned derivatives of Cas9. Several CRISPR... in a different gene. Libraries may be available in a 1-plasmid system, in which Cas9 is included on the... gRNAs targeting the same gene can be strong evidence that a particular gene is actually important for...

...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...proteins for optogenetic control of CRISPR-Cas9. By combining AcrIIA4, a Streptococcus pyogenes Cas9 inhibitor...plasmids Anti-CRISPR proteins for optogenetic control of CRISPR-Cas9 Anti-CRISPR proteins that inhibit... a few of the new CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR...adapted for detecting SNPs. Learn more from this Addgene blog post Read the paper in Science Find the...editing is now possible. Learn more from this Addgene blog post Read the paper in Nature Methods Find...

...selection-based gene tagging approach Multiple features of C. elegans have made CRISPR/Cas9 so successful...is inserted at the 5’ end of a gene, the SEC separates the targeted gene from its promoter, creating a ...the targeted gene. After initial testing, Dickinson et al. tested this system across 7 genes in parallel...animals per gene, they obtained the desired insertions on the first attempt for 6/7 genes. The data are...efficient selection-based CRISPR/Cas9 method to generate knock-ins on any genetic background. Their new method...publication at Addgene. Additional Resources on the Addgene Blog An “elegans” Approach to Better Cas9 Editing...plasmids from this publication at Addgene. Other strategies for CRISPR/Cas9 editing in C. elegans: Engineering...

...the Cas9 nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does...answered by Le Cong Read Addgene's CRISPR guide for background information on CRISPR/Cas9 systems Peruse the...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...

...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...editing and mouse-model generation. Key components of Nanoblades Nanoblades are a Cas9 RNP delivery vehicle...centrifugation. Gag-Cas9 fusion protein The MLV Gag protein fused to a Flag-tagged SpCas9 is incorporated ...alternative method for generating transgenic mice strains and for non-invasive in vivo gene editing. The bottom... leukemia virus (MLV)-like particles loaded with Cas9-gRNA ribonucleoproteins (RNPs). According to the...

...your application: Wildtype Cas9 or Cas9 nickase are appropriate for generating knockouts or knock-ins, or...used for generating transgenic embryos. Cas9-gRNA ribonucleoprotein complexes Purified Cas9 protein ...permanently knock out or knock-in a gene? Do you want to activate or repress gene expression? Are you trying to...in vivo viral delivery. Cas9 mRNA and gRNA Plasmids containing gRNA and Cas9 are used in in vitro transcription...catalytically dead dCas9 can be used in conjunction with activator or repressor domains to control gene expression...reporter gene (e.g. GFP) to identify and enrich positive cells or a selection marker to generate stable ...transcription reactions to generate mature Cas9 mRNA and gRNA. RNA is delivered to target cells using microinjection...

... 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 RNP activity or longevity. GeoCas9 is active at high...loving) bacteria, such as Streptococcus pyogene’s SpCas9. These Cas9 proteins don’t work well at high temperatures...used to make eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally occurring Cas9 diversity ...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...

...used for genome editing, activating gene expression, repressing gene expression, and much more. But there...engineering viruses. For example, an Acr gene was used to replace genes in the difficult-to-engineer Sulfolobus...Pawluk et al., 2016). While Streptococcus pyogenes Cas9 (SpyCas9) is the most used and well studied CRISPR... the Addgene Blog First time CRISPR user? Check out this post. Learn how to Choose the Best Cas9 Variant... 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...

...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 ...

...systems, expression of S. pyogenes Cas9 or Cas9 variants (hereafter referred to as Cas9) and a single stranded...Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922. PLoS ONE ... to activate or repress target genes in plant cells, respectively. Cas9 expression is commonly driven ... as mRNAs for gene expression). Examples of commonly used RNA pol II promoters for Cas9 expression include... target genes in plants and many of the aforementioned empty gRNA backbones also contain Cas9, which enables...Nuclease dead Cas9 (dCas9) based activators (such as dCas9-VP64) or repressors (dCas9-KRAB or dCas9-SRDX) can...knockout, activate or repress target genes in plants using the same general experimental design principles ...