...accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library containing several... tag donor plasmids from the library can be used to generate functionally unique repair templates. A final...obtained as a universal donor plasmid. The CRISPaint toolkit includes several donor plasmids for protein ...Since the discovery of GFP over 50 years ago, the growing spectrum of fluorescent proteins (FPs) has ...repair pathway, a new user-defined DNA sequence (like GFP) can be inserted at the DSB. CRISPR has allowed scientists...individually generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it..., as well as the 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or ...

.... Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor plasmid contains...the introduced donor plasmid as a repair template. This results in the insertion of the GFP tag precisely...to endogenously tag a wide variety of genes with GFP. These plasmids are available through Addgene, and...is the addition of a useful coding sequence (i.e. GFP) into a gene while leaving other features of the ...we show an example gene targeted for tagging with GFP at the C terminus, resulting in one edited allele...allele, or gene copy. The coding sequence for GFP is transcribed and translated along with the gene, producing...shown in the example above, we inserted a linker and GFP tag at the end of the last exon of the gene to ensure...

...with ssDNA repair templates. Their work demonstrates that different repair template strategies activate...using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR uses a long ssDNA template to increase ...working with CRISPR than just designing a repair template that contains your desired edit, and I want to...Editing in Human Cells Hands-on tips for repair template design from guest bloggers Dominik Paquet and ...distance affects editing efficiency. Optimizing Donor DNA for Enhanced CRISPR Genome Editing Chris Richardson...of precise genome editing using oligonucleotide donors". Kan, Yinan, Brian Ruis, Taylor Takasugi, and ...PubMed Central PMCID: PMC5495063 Kan et al. use a GFP-BFP conversion system to assess precision genome ...

...drug resistance (Chen et al., 2013), fluorescence (GFP; Tzur et al., 2013), or rescue of mutant phenotypes...following a report that one can use linear repair templates (ie. PCR-derived dsDNA) with 30-60 basepair homology...homology arms to knock in large epitopes, such as GFP (Paix et al., 2014). A further development of this...ribonucleoprotein complex with linear DNA as repair templates which reduces time to generate edits to only four...optimize a range of editing parameters: oligo repair templates with homology arms of 35-80 bp, and DNA double-strand...CRISPR whether it be by improved gRNA or repair template design, delivery, screening, and more. Here are...some highlights from the past few years:  Repair template design The Meyer lab meticulously characterized...

...hiPSCs  EMX1  67% Mouse BM cells from GFP transgenic mouse  GFP  ~60-65% Human hepatocytes  Myd88  ~... of a fluorophore or tag. To utilize HDR, a donor template containing the desired sequence flank by homology...Nanoblades which carries a gRNA, Cas9, and a HDR donor template DNA can be generated by incubating Nanoblade...facilitated HDR with either a ssDNA or dsDNA donor template. In vivo editing of mouse zygotes and mice ...

...using ssDNA oligo as donor template, it's usually more efficient than plasmid donor. Typical design is ...the HR template, you usually would need to make mutations of the HR template to avoid this donor plasmid... HR template vector design guidelines? A few notes below are considerations for designing HR donor. Generally...my HR template an issue? Yes. If you have intact protospacer+PAM sequence within the HR template, it will...experiment, consider co-transfecting with GFP. This allows you to sort for GFP-positive cells and to enrich for...then select the best guide to co-transfect in HR donor. Alternatively, a new system described recently ...insertions or deletions), we typically use plasmid donor, with two homology arms on each side flanking your...

... power of ssDNA donor molecules for homology directed repair. Using long ssDNA donors, the Gurumurthy ... Single-stranded DNA repair templates Short single-stranded DNA templates have been shown to improve the...ameliorate this problem. Briefly, you design your donor template to include a T7 promoter upstream of the left...final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template. Although...from low efficiency when a standard dsDNA repair template is used. The original design for creating a conditional...targeting two intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand...vitro transcription, and use the IVT product as a template for reverse transcription to create your ssDNA...

... the genomic site and the donor vector. The PITCh donor plasmid with an EGFP-2A-PuroR cassette, flanked... and donor vectors as well as pX330S-2-PITCh, expressing a generic gRNA targeting the PITCh donor plasmid...following articles provide PCR templates for amplification of the tag (eg GFP, Flag, YFP, etc) and selection...Plasmids Protein Species Tag Donor Plasmid gRNA plasmid gRNA plasmid FLAG pFETCh_Donor CEBPB Human FLAG pFETCh_CEBPB...FBL-specific gRNA pCRIS-PITChv2-FBL - PITCh donor vector for EGFP-2A-PuroR knock-in into human FBL locus Jorgensen...created using CRISPR and the donor plasmids containing homology arms and EGFP are available at Addgene. ...101 post for more information about HDR/repair template design. How to use CRISPR to tag your gene of ...

... describes how you can better design your donor DNA template to achieve more efficient genome editing ...Favorite Fluorescent Protein Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts ...post and the associated publication. Optimizing Donor DNA for Enhanced CRISPR Genome Editing CRISPR has...

...Adenoviral CRISPR vectors (left) or CRISPR vectors and donor DNA vectors (right) are transduced to target cells...non-homologous end joining (NHEJ). In targeted DNA knock-in, donor DNA is copied into the cut site through error-free...genes. For this you would need to provide a DNA template to repair the double strand break via homology...Cas9/sgRNA and one vector for your specific DNA template. Using this two vectors system, Manuel Gonçalves...RNA-guided nucleases or TALENs together with AdV donor DNA leads to a vast majority of AdV-modified human...structures presumably reduce the likelihood that donor DNA will interact with sporadic double-stranded ... via simple switching of the relevant gRNAs and donor DNA Adenoviral CRISPR/Cas9 genome editing tools ...

... create a fusion with a reporter protein such as GFP? All of these outcomes can be most effectively achieved...want to include a homology directed repair (HDR) template to create knock-ins, point mutations, or to add...add a tag, you will also need to deliver a donor plasmid or single-stranded DNA oligonucleotide, so you...typically used for gRNA. May contain reporter gene (e.g. GFP) to identify and enrich positive cells or a selection...transfer vectors. May contain reporter gene (e.g. GFP) to identify and enrich positive cells. Likely contains...repair by non-homologous end joining (NHEJ) or HDR. EGFP validation of sgRNAs: Can evaluate component delivery...efficacy by cloning in genome target sequence into EGFP reporter. Target DNA reporter system: Can evaluate...

...genomic edits by copying from a DNA donor template . The donor template contains the desired sequence and...linear dsDNA templates work better for large edits or insertions. When designing the donor template, incorporating...out integration of donor DNA. Both types of CASTs have been found to achieve donor DNA integration for...pathway. HDR relies on copying DNA from a matching template to accurately repair or fill in the missing sequence...the break ends without the need for a homologous template. Both NHEJ and HDR have their uses, but we’ll ...cell along with the gRNA(s) and Cas enzyme. The donor DNA contains the desired edit as well as additional...homology arms. Depending on the application, the donor DNA may be a single-stranded oligonucleotide, double-stranded...

...activator with 2A GFP 61423 : Expresses the MS2-P65-HSF1 activator helper complex with 2A GFP 61424 : sgRNA...(SapI)_hSyn-GFP-KASH-bGH (SpGuide acceptor). This is a AAV plasmid for sgRNA cloning. GFP-KASH fusion ...contains a Kras G12D homology directed repair donor template. #60225 - AAV:ITR-U6-sgRNA(LacZ)-pEFS-Rluc-...PX552; U6 promoter-driven; for sgRNA cloning; has GFP-KASH for FACS sorting SaCas9 + single guide RNA: ...luciferase-2A-Cre recombinase, and Kras G12D HDR template 60225 : control for AAV-KPL; sgRNA targeted to... recombinase-2A-EGFP-KASH 60231 : sgRNA cloning backbone with Cre recombinase-2A-EGFP-KASH Detailed backbone...recombinase-2A-EGFP-KASH and an sgRNA. #60231 - AAV:ITR-U6-sgRNA(backbone)-hSyn-Cre-2A-EGFP-KASH-WPRE-shortPA-ITR...

...both the GFP and the RFP proteins in the nucleus. During the first round of translation, the GFP proteins...Compartment mCherry-Rab5 BFP-Rab5 GFP-Rab5B Vacuolar & Budding Compartment GFP-Rab4B Mitochondria mito-BFP...GTPase in division) GFP-Mff (outer membrane protein) Late Endosome mCherry-Rab7A GFP-Rab7A     ...directed repair templates (see the supplementary info from the paper for details on repair template design). ...protein Knock-off, is made up of three components: a GFP protein that binds to the coding region of a reporter...reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) a RFP protein that binds to the 3’ UTR of the reporter...these dCas9s with one of three fluorescent proteins (GFP, BFP and mCherry), the authors were able to visualize...

...genomic edits by copying from a DNA donor template . The donor template contains the desired sequence and...linear dsDNA templates work better for large edits or insertions. When designing the donor template, incorporating...out integration of donor DNA. Both types of CASTs have been found to achieve donor DNA integration for...pathway. HDR relies on copying DNA from a matching template to accurately repair or fill in the missing sequence...the break ends without the need for a homologous template. Both NHEJ and HDR have their uses, but we’ll ...cell along with the gRNA(s) and Cas enzyme. The donor DNA contains the desired edit as well as additional...homology arms. Depending on the application, the donor DNA may be a single-stranded oligonucleotide, double-stranded...

...Lin and Yasuda groups use both mCyFP1 and EGFP as FRET donors in experiments simultaneously measuring the...-positive assay) as well as stabilization (Nanog-GFP reporter activation and silencing of factor-linked...makes mCyRFP1 a powerful tool for use as a donor in FRET (fluorescence resonance energy transfer) and ...that can be combined with your gRNA and repair templates of choice though modular cloning steps. The Voytas...along with EGFP while retaining an emission spectrum that is easily separable from that of EGFP. This makes...spectra (N- and C-terminal tags for mTagBFP, TagRFPt, EGFP, mVenus, mCerulean3, mKOFP2) and empty vectors that...and combined it with a variety of reporters (i.e. eGFP) and effectors (i.e. DREADDs). The team demonstrated...

..., Hess et al. successfully evolved wild type GFP to EGFP using CRISPR-X and subsequent FACS sorting. They...with unique tissue-specific promoters expressing GFP, tetracycline response elements, and shRNAs many ... of DsRed connected to a pH-sensitive variant of GFP (SEP) by a 9 amino-acid linker (see Figure 1). The...However, the laborious construction of repair templates and guide RNA constructs, as well as the difficult...encode both a guide RNA transcript and a repair template for an individual tagging event. Commonly used...gene are supplied to the reaction from a prebuilt donor plasmid library, available from Addgene. Site-specific...available). Second, they co-transfected short-arm donor vectors containing mAID and either Neo or Hygro ...

... pCAG-SpCas9-GFP-U6-gRNA 79144 Mammalian hU6 yes, cut S. pyogenes EGFP Zou pCAG-eCas9-GFP-U6-gRNA 79145...Cerulean Church LRG (Lenti_sgRNA_EFS_GFP) 65656 Mammalian/Lentiviral LIC none S. pyogenes GFP Vakoc U6>sgRNA...cut Lb Cpf1 Neo Welker AIO-GFP 74119 Mammalian U6x2 yes, nick S. pyogenes EGFP Jackson AIO-mCherry 74120...S. pyogenes Kamoun pUC119-gRNA 52255 Plant PCR template none S. pyogenes Sheen pRGEB31 51295 Plant BsaI...pyogenes Zhang pL-CRISPR.EFS.GFP 57818 Mammalian/Lentiviral BsmBI yes, cut S. pyogenes EGFP Ebert pLKO.1-puro...pyogenes Zhang pLKO5.sgRNA.EFS.GFP 57822 Mammalian/Lentiviral BsmBI none S. pyogenes EGFP Ebert pSECC 60820...tagRFP657 Ebert pL-CRISPR.SFFV.GFP 57827 Mammalian/Lentiviral BsmBI yes, cut S. pyogenes EGFP Ebert pLKO5.sgRNA.EFS.tRFP...