...73178 Human sgRNA library Brunello in lentiGuide-Puro Human sgRNA library in backbone lentiGuide-Puro targeting...73633 Mouse sgRNA library Brie in lentiGuide-Puro Mouse sgRNA library in backbone lentiGuide-Puro containing...Human CRISPRa sgRNA library Calabrese in backbone XPR_502 (P65 HSF), Set A Human CRISPRa sgRNA activation...containing 56,762 unique sgRNAs. This backbone does not contain dCas9. 56,762 unique sgRNAs targeting 18,885 ...GFP Zhang 61425 lenti dCAS-VP64_Blast dCAS9 (D10A, N863A) Blasticidin 3rd generation lenti vector encoding...2A-Blast-WPRE) Zhang 61426 lenti MS2-P65-HSF1_Hygro MS2-P65-HSF1 Hygromycin lenti vector encoding the MS2...gRNA pooled library in lentiCRISPRv2 Human sgRNA library in backbone lentiCRISPRv2 targeting 19,114 genes...

...sequential infection first of Cas9, then with the sgRNAs, and can result in a higher titer due to the smaller...study genes all across the genome? Two new human lentiviral CRISPR library systems described in companion... article describes the development of a mouse lentiviral CRISPR library.  “This enables you to do customized...technology.” GeCKO library for genomic screens Using lentivirus as the delivery vehicle, Neville Sanjana, also...for select gene groups The 6 available enriched lentiviral CRISPR libraries developed by MIT's Tim Wang ...unique gRNAs. Their gRNA library is delivered via lentiviral infection into target cells that have previously...as well as non-coding genetic elements.” These lentiviral libraries and their demonstrated application ...

...S. pyogenes Puro Zhang lenti sgRNA(MS2)_zeo backbone 61427 Mammalian/Lentiviral BsmBI none S. pyogenes... pLCKO 73311 Mammalian/Lentiviral hU6 none S. pyogenes Puro Moffat lenti sgRNA(MS2)_puro backbone 73795...pyogenes URA3 Davis lenti sgRNA(MS2)_puro optimized backbone 73797 Mammalian/Lentiviral U6 none S. pyogenes...pyogenes Joung sgRNA with U6 promoter 48962 Worm HindIII none S. pyogenes de Bono sgRNA with rpr-1 promoter...dCas9-VP64 along with 1-4 sgRNAs expressed from independent promoters. sgRNAs are cloned into separate ... and allow for expression of more sgRNAs in a single vector. sgRNAs are expressed as polycistronic glycine...transcription BsaI none S. pyogenes Mark pLenti-sgRNA-Lib 53121 Mammalian/Lentiviral BsmBI none S. pyogenes ccdB Wei...

...CRISPRko sgRNA library The Brunello library consists of 77,441 sgRNAs with an average of 4 sgRNAs per gene...contains two sets of sgRNAs; A, the top 3 ranked sgRNAs, and B, the next 3 ranked sgRNAs. Dolcetto was able...containing only 3 sgRNAs (setA) also outperformed CRISPRi libraries with 10 sgRNAs in the sgRNA metrics, confirming... CRISPRko sgRNA library as pooled library or lentiviral prep! Dolcetto: human CRISPRi sgRNA library While...2014 to predict sgRNA activity. In this study scientists examined approximately 1,900 sgRNAs and identified...subsampling analysis of the sgRNAs, Brunello even with only a single sgRNA outperformed another CRISPR...CRISPR library with 6 sgRNAS, highlighting the benefit of sgRNA design in CRISPR libraries. Thus, the Brunello...

...et al., 2019). This dual lentivirus system pairs ubiquitous expression of sgRNA(s) and mCherry (vector .... Figure 2: Dual lentivirus system with ubiquitous expression of sgRNA (top) and neuron-specific...transcription at genomic sites targeted by specific sgRNAs. Its RNA-guided nature makes CRISPRa much simpler...all the protein and RNA components, and several sgRNAs must be screened to identify effective ones. Simplified...constrained by AAV packaging limits (i.e., how many sgRNA expression cassettes will fit). Cre-dependent expression...expression of the SPH activator paired with AAV sgRNA delivery make the SPH mouse an attractive tool to...Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can be found here. Neuron-optimized viral vectors...

... activator (CRISPRa) sgRNA library uses the sunCas9 system and contains 10 sgRNAs for each transcription...nucleolytically inactive Cas9-VP64 fusion; 2. A sgRNA incorporating two MS2 RNA aptamers at the tetraloop...gain-of-function screening when combined with a compact pooled sgRNA library. Our SAM-mediated screens exhibited a high...transcriptional repressor (CRISPRi) library contains 10 sgRNAs for each transcription start site in those 15,977...further optimize those now. They are also developing sgRNAs that will enable even more fine-tuning of gene ...conducted earlier by the Zhang lab for GeCKO, a lentiviral CRISPR library enabling genome-scale, knockout...that you review the steps for how to use pooled lentiviral CRISPR libraries and keep the following in mind...

...The team designed sgRNAs that include an internal barcode and separated multiple sgRNAs with tRNAs, which...uses two sgRNAs with internal barcodes (iBARs), multiplexed using tRNAs, within the lentiviral 3’ long ...is duplicated during lentiviral integration, producing a second copy of the sgRNAs. Image reused from Walton... polymerase III transcript, the sgRNAs were encoded on the lentiviral minus strand in the 3’ LTR. These...license. In this iteration, the size of the lentiviral insert was minimized to reduce recombination ...

...Veening is now available with one sgRNA per operon or gene. The sgRNA pool can also target core operons... cell lines. The Turner Lab Human messenger-RBP sgRNA Library targets human mRNA binding proteins with...operons in other pneumococcal strains. The BARBEKO sgRNA library uses CRISPR cytosine base editors to disrupt... 10 guide RNAs per gene. The lab modified the lentiviral backbone to encode the CD90.1 selection marker...

...fused to an sgRNA used to direct Cas9 to a specific genomic locus. When fused to the sgRNA and co-expressed...dCas9 – the dCas9, sgRNA/RNA cargo complex localizes to the sequence targeted by the sgRNA. This allows scientists...machinery that brings the sgRNA/cargo RNA to the appropriate DNA locus. The sgRNA/RNA fusions retained their...construct. The sgRNA targeting Gaussia luciferase can be easily replaced with an sgRNA targeting your ...specific recruitment of multiple copies of VP64 to the sgRNA-targeted gene, resulting in increased activation...SV40, SFFV) or inducible (CMV-TO) cDNA expression, sgRNA (U6) expression, shRNA/shRNA-miR30 constitutive ...and shRNAs with a reporter gene, or (4) multiple sgRNAs with Cas9 for genome engineering. The versatility...

...described here. #60224 - AAV:ITR-U6-sgRNA(Kras)-U6-sgRNA(p53)-U6-sgRNA(Lkb1)-pEFS-Rluc-2A-Cre-shortPA-KrasG12D_HDRdonor-ITR...CMV-driven SaCas9; U6-driven sgRNA 61593 : PX602; TBG-driven SaCas9; U6-driven sgRNA 61592 : PX600; CMV-driven... SaCas9 sgRNA can be co-transfected as PCR amplicons using a protocol similar to SpCas9 sgRNA PCR (Ran... pAAV-U6::sgRNA(SapI)_hSyn-GFP-KASH-bGH (SpGuide acceptor). This is a AAV plasmid for sgRNA cloning. GFP-KASH...implemented by delivering Cre and sgRNA to a Cre-dependent mouse or sgRNA to a constitutively Cas9-expressing...control for AAV-KPL; sgRNA targeted to LacZ, plus luciferase-2A-Cre recombinase 60226 : sgRNA cloning backbone...genome editing 60227 : sgRNA targeted to NeuN, plus Cre recombinase 60228 : sgRNA targeted to LacZ, plus...

...targeting requires two sgRNAS: one that targets the knock-in site and a universal sgRNA that targets the donor... the genomic loci of interest. As the universal sgRNA is not predicted to target sites in the human, pig...CRISPR-Cpf1(Cas12a) Double Knockout Library is a lentiviral library targeting 26 genes chosen from an analysis...

...imaging 51024 pSLQ1651-sgTelomere(F+E) Lentiviral vector with sgRNA targeting human telomeres 46913 pMSCV-LTR-dCas9...N 51025 pSLQ1661-sgMUC4-E3(F+E) Lentiviral vector that contains sgRNA targeting repetitive sequence of...dCas9) protein and a customized s ingle g uide RNA (sgRNA) designed with a 20-basepair complementary region...any gene of interest. Co-expression of dCas9 and sgRNA can efficiently block transcription (in bacteria...determined jointly by 20-bp matching region on the sgRNA and a short DNA motif (protospacer adjacent motif...plasmid ( pgRNA_bacteria ) contains a constitutive sgRNA expression cassette on a ColE1 vector with ampicillin...pgRNA_humanized ) contains a murine U6 promoter controlled sgRNA cassette, wherein the GN19 can be custom designed...

...or HDR. EGFP validation of sgRNAs: Can evaluate component delivery and sgRNA efficacy by cloning in genome...you will need to deliver. Minimally, one or more sgRNAs and Cas9 are required for any application. If you...efficiency. Lentiviral transduction Cas9 and gRNA can be present in a single lentiviral transfer vector...plasmids provide the necessary components to make lentiviral particles. Stable, tunable expression of Cas9...Difficulty Very easy Very hard Viral delivery   Lentivirus Retrovirus AAV Cells Dividing or nondividing...: Traffic Light Reporter System: Can evaluate lentiviral component delivery as well as genome repair by...

... Sabatini and Lander gRNA cloning into pLX-sgRNA pLX-sgRNA PDF 125.9 KB Sontheimer gRNA design and cloning...Cas9 D10A (pX335) ; PDF 248.6 KB Zhang sgRNA cloning lentiCRISPR v2 packaging plasmids: pVSVg , psPAX2 ...as an optional input to assess HDR frequency. An sgRNA sequence (without PAM sequence) can be provided,... can identify positively or negatively selected sgRNAs, genes, or pathways. CrispyCrunch CrispyCrunch ...CRISPR in 180+ genomes. PubMed PMID: 27380939 . sgRNA Scorer 2.0 From the Church Lab : a tool that identifies... epression, and a ctivation From the Qi Lab , a sgRNA design tool for genome editing, as well as gene ...Cas9 target online predictor Identifies candidate sgRNA target sites by off-target quality. Validated for...

...assemble multiple unique sgRNAs into a multi-TU targeting construct (Cas9, sgRNA, reporter, etc.). This ...for the multiplexing of sgRNAs against unique genomic targets or multiple sgRNAs against the same target...target to ensure efficient cutting. Beyond sgRNAs, MoClo can also be used to test a variety of Cas enzymes...Fragmid system was used to assess how different lentiviral plasmid backbone elements impact viral titer ...expand the Fragmid toolkit to facilitate other lentivirus- and AAV-based studies. Unified MoClo Systems...

...complexed with sgRNA. Direct comparison of Cas9 RNPs prepared with an in vitro-transcribed (IVT) sgRNA versus ...cumbersome in vitro synthesis of single guide RNAs (sgRNAs), are time-consuming, have been typically optimised... the in vitro-transcribed single guide RNA (IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not...construct plasmid vectors or synthesize/purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2...our CRISPR guide pages Find ready-to-use Cas9 lentivirus ...

...endosomal escape. Combining Cas9-PAGE with lentiviral sgRNA expression yielded editing of 80–90% in human...limited (likely due to its long, negatively charged sgRNA), RNP delivery was much better with Cas12, which...Workflow to assess Cas9-PAGE editing with viral sgRNA targeting mCherry reporter (mChe), with co-incubation...

...46,354 BARBEKO sgRNA Library 174163 Knockout, Base Editing Human Wei 3rd 3 53,502 sgRNA Pool for Streptococcus...131625 Inhibition E. coli Bikard N/A ∼5 21,417 Bison sgRNA Library 169942 Knockout Human Ebert 3rd 4 2,852 ...Fly Perrimon 3rd 8 6 6 7,956 17,827 59,406 Dual sgRNA CRISPRi Libraries 187246 — 187248 1000000192 Inhibition...Inhibition Human Weissman 3rd 6 Varies Dual sgRNA CRISPRa Libraries 187249 — 187251 1000000193 Activation...3rd 2 37,722 Green monkey (Chlorocebus sabaeus) sgRNA library 178284 Knockout Green Monkey Doench and ...Knockout Human Parts 3rd 3 59,576 Human Whole Genome sgRNA iBAR Library 140633 Knockout Human Wei 3rd 3 58,... Mouse Knouse 3rd ~5 71,878 Mouse messenger-RBP sgRNA Library 169082 Knockout Mouse Turner N/A 10 13,500...

...This ‘Caspex’ construct is guided by a pre-defined sgRNA sequence to a specific genomic locus and labels ... and targeted to a sequence of interest with an sgRNA expressed from a separate plasmid. When cells are...specific DNA sequence in only 1 min. Using these lentiviral plasmids, catalytically inactive Cas9 (dSpyCas9...

...Cas9 from Streptococcus pyogenes with an invariant sgRNA scaffold and cloning sites for inserting the guide...pyogenes CRISPR chimeric RNA element with customizable sgRNA from the U6 promoter and puromycin resistance from...pSpCas9(BB)-2A-GFP (PX458). lentiCRISPR v2 - This 3rd generation lentiviral transfer vector also from the...promoter. It’s a 3rd generation lentiviral backbone that must be used with lentiCas9-Blast or with cell lines...the last 15 years - the majority of these being lentiviral or CRISPR tools. How many times have these plasmids...Addgene psPAX2  - psPAX2 is a 2nd generation lentiviral packaging plasmid from the Didier Trono lab that...genes. It can be used with 2nd or 3rd generation lentiviral vectors and an envelope expressing plasmid, such...