...cassette. (b) pCAG-EGxxFP target plasmid and pX330-sgRNA plasmid (Mashiko et al. 2013). The pCAG-EGxxFP target...cloned directionally into the BbsI site. (c) The pCAG-EGxxFP target plasmid was co-transfected with pX330...Once amplified, you can insert this region into a pCAG-EGXXFP plasmid using standard cloning techniques...of transfecting HEK293T cells with your modified pCAG-EGXXFP plasmid along with the individual gRNAs in...Remember the primers you designed to generate your pCAG-EGXXFP plasmid? They are the perfect primer sets...

...including SpCas9, SaCas9, LbCas12a, enAsCas12a, SpCas9-NG, SaCas9-KKH, CP1028-SpCas9, and CP1041-SpCas9. The... weakly compatible with many Cas domains beyond SpCas9. In order to expand the gene-editing applications...

...initial tests, Cas13b from Prevotella sep. P5-125 (PspCas13b) yielded 62.9% average knockdown, and they chose...chose this enzyme for further studies. PspCas13b does not require a PFS, and it is sensitive to mismatches...They fused ADAR deaminase domains (ADARDD) to dPspCas13b, but observed low RNA editing. To increase A-...template has a cytosine mismatch at that position. PspCas13b-ADAR2DD(E488Q) displayed robust editing with various...showed that editing is mediated by ADAR2DD, not PspCas13b. Find the plasmids for the RESCUE RNA editors ...

...pCS2TAL3-RR Pawel Pelczar pCAG-T7-TALEN(Sangamo)-Destination series, pCAG-Golden-Gate-Esp3I-Destination...pCAG-Golden-Gate-Esp3I-Destination Takashi Yamamoto pcDNA-TAL-NC2, pCAGGS-TAL-NC2 Charles Gersbach pcDNA3.1-GoldenGate, pcDNA3.1...proteins with SpyTag/SpyCatcher and SnoopTag/SnoopCatcher The development of recombinant DNA technology...engineered a new peptide/protein pair, SnoopTag/SnoopCatcher, that can be used to irreversibly link desired...process by alternating the use of the SnoopTag/SnoopCatcher and SpyTag/SpyCatcher pairs. In order to improve...fusions through SpyTag/SpyCatcher and SnoopTag/SnoopCatcher linkages. Once all of the desired sequential...directional protein fusion, with a SpyCatcher-SnoopCatcher linker remaining between each protein unit, ...

...Eubacterium siraeum (EsCas13d) and Ruminococcus sp. (RspCas13d), while Konermann et al. characterized orthologs...WYL-domain-containing accessory protein WYL1 increases RspCas13d and EsCas13d cleavage activity The majority of... et al. from Arbor Biotechnologies found that RspCas13d accessory protein RspWYL1 increases both targeted...targeted and collateral RNA degradation by RspCas13d. RspWYL1 also increased EsCas13d activity, indicating...

...specific variants. HypaCas9, sSpCas9(1.1), SpCas9HF1, and evoCas9 are a few spCas9s which have been evolved...of Maya Kostman. The most commonly used Cas, spCas9, has gone through multiple rounds of optimization...

...Transfer Plasmid : 1 pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper: 2 RepCap Select what type of tissue...using 28 μg of total plasmid (Transfer, helper, and RepCap added together) per 15 cm dish or 2500 μg of plasmids...

...packaging plasmid The packaging plasmid aka the RepCap plasmid encodes two genes: the replication (rep... at the rep and cap genes. Figure 1. The RepCap plasmid and its products. Created with BioRender.com...Now you’ve got a basic understanding of the AAV RepCap plasmid! Check out this blog post to learn more...

...Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9 Day 10: Transcriptional regulation...The PAM Requirement and Expanding CRISPR Beyond SpCas9 Day 8: Repair mechanisms: CRISPR 101: Non-homologous...

...Once upon a time, not so long ago, spCas9 was the only Cas enzyme widely available and applied by researchers...getting the desired features and cutting (note: spCas9 isn’t always the winner!).   Fig. 3 – Comparison...The PAM Requirement and Expanding CRISPR Beyond SpCas9 More resources from the Innovative Genomics Institute...

...the fact that it is smaller than the traditional SpCas9 enzyme (about a third of the size) and does not...cleave RNA through the same mechanism as traditional spCas9. If you are looking to modify RNA, Cas9 just won...The PAM requirement and expanding CRISPR beyond spCas9 Cas proteins More resources on the Addgene Website...

...Yoon, S., & Kim, H. H. (2019). SpCas9 activity prediction by DeepSpCas9, a deep learning–based model with... and PE5 prime editing strategies) based on a DeepSpCas9 score ranking (Kim et al., 2019). PRIDICT saves...

...CRISPR, and specifically Cas9 from S. pyogenes (SpCas9), is truly an exceptional genome engineering tool... species, and has many applications. That said, SpCas9 is not the only game in town, and other Cas proteins... can circumvent the limitations associated with SpCas9. Cas13a (previously referred to as C2c2), has several...

...period of time. High fidelity Cas9s such as eSpCas9 and SpCas9-HF should be explored to reduce off-target...mutation in dystrophin exon 23. Long et al. used SpCas9 in conjunction with AAV9, whereas Nelson et al....

...capacity of a locus. When using S. pyogenes Cas9 (SpCas9), potential target sites are both [5’-20nt-NGG]...editing, there are usually few potential gRNAs. While SpCas9, with a PAM preference of NGG, is still the most...gRNAs that are likely to be active, not only for SpCas9 but also for some other Cas enzymes. It seems to...

...used for each gene. At the moment, 13 full genome SpCas9 knockout libraries designed by different academic...Library, for example related to strategies beyond SpCas9, or species not currently included in Green Listed...libraries. As of now I have focused on human and mouse SpCas9 knockout libraries, but there are of course great...

... makes it unuseable. With the large size of the SpCas9 nuclease (~4.2 kb) this is a particularly prominent...Institute to provide lentiviruses for delivery of SpCas9 and Cas9 nickase for genome editing. You can also...

...crRNAtracrRNACas9 crRNACpf1 Endonuclease size spCas9: ~4 kbsaCas9: ~3 kb ~3.9kb crRNA/gRNA length...sequence, tRNAs, multiple individual promoters).  spCas9 and its gRNAs are also larger than their Cpf1 counterparts...

...well characterized than the conventionally used SpCas9 (derived from the bacterium Streptococcus pyogenes...been shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative...

...protein The MLV Gag protein fused to a Flag-tagged SpCas9 is incorporated into Nanoblades, just like the ...these variants are fused to the MLV Gag in place of SpCas9. gRNA A gRNA targeting a DNA sequence of interest...