![graphpad prism 8 step-by-step example graphpad prism 8 step-by-step example](https://cdn.graphpad.com/faq/802/images/802c.png)
(iv) One hundred microliters of the mixture was spread on carbenicillin (100 μg/ml)-containing agar plates.(iii) Of the NEB Stable outgrowth medium, 450 μl was added into the mixture and the tube was rotated at 37☌ for 1 h.
![graphpad prism 8 step-by-step example graphpad prism 8 step-by-step example](https://greencracks.com/wp-content/uploads/2019/07/GraphPad-Prism-crack.png)
(ii) The mixture was placed on ice for 30 min and heat-shocked at 42☌ for 30 s before being placed on ice for 5 min.Eppendorf tubes were flicked five times to mix the cells with the plasmid DNA. (i) Ten microliters of the ligation reaction or 2 μl of the assembly reaction was added into 50 μl of NEB Stable Competent E.In addition, short fragments of CasRx sgRNA expression cassette (gBlocks TM), CasRx pre-sgRNA expression plasmid, and all-in-one CasRx AAV plasmid were designed and illustrated using Benchling 3. The other two sgRNA/pre-sgRNAs were manually designed to target the different regions in VEGFA mRNA, avoiding the stem–loop.
#GRAPHPAD PRISM 8 STEP BY STEP EXAMPLE TRIAL#
The first VEGFA sgRNA/pre-sgRNA was designed based on the targeting sequence of bevasiranib, the first VEGFA-targeted siRNA to reach phase III clinical trial ( Garba and Mousa, 2010). Three sgRNA/pre-sgRNAs targeting human VEGFA mRNA were designed for the study. Since the selection parameters of the sgRNA target regions are similar to RNA interference, small interfering RNA (siRNA) designed web tools can be used for sgRNA selection. The secondary structure of the target mRNA can be predicted using the RNAfold web server 2 or other online tools. Two forms of CasRx sgRNAs, sgRNAs (22 nt) and pre-sgRNAs (30 nt), that do not target the stem–loop region of the target mRNA were selected for an increased targeting rate. The transcript sequence of the target gene (human VEGFA) was obtained from NCBI 1 ( Figure 1A and Supplementary Figure 1).
![graphpad prism 8 step-by-step example graphpad prism 8 step-by-step example](https://i.ytimg.com/vi/OTt4aN8Zcqk/maxresdefault.jpg)
#GRAPHPAD PRISM 8 STEP BY STEP EXAMPLE MANUAL#
An instructional manual for RNA editing in vitro with CasRx using variants of guide RNAs is described. For application in development of therapeutics, we generated all-in-one AAV constructs consisting of CasRx and a single pre-sgRNA or multiple pre-sgRNAs (array) to examine the RNA knockdown efficiency of the system. We examined the RNA knockdown efficiencies of the CRISPR/CasRx system using different forms of single guide RNA (sgRNA) by targeting VEGFA mRNA. Here, we describe three different methods to perform efficient CRISPR/CasRx-mediated RNA knockdown of vascular endothelial growth factor A (VEGFA). These studies indicate the therapeutic potential for the CRISPR/CasRx system. Delivery of the CRISPR/CasRx system successfully suppressed the mRNA of vascular endothelial growth factor (VEGF), the key factor for pathogenic ocular angiogenesis, and also subsequently showed a reduction in the area of choroidal neovascularization (CNV), the hallmark of nAMD ( Zhou et al., 2020). Notably, the therapeutic potential of CRISPR/CasRx was demonstrated in mouse models of neovascular age-related macular degeneration (nAMD) using AAV vectors.
![graphpad prism 8 step-by-step example graphpad prism 8 step-by-step example](https://graphstats.net/wp-content/uploads/2020/11/prism-bubbles2-w1210-800.png)
Subsequent studies with CasRx have shown efficient messenger RNA (mRNA) knockdown in various animal models and transgenic expression in plants ( Mahas et al., 2019 Kushawah et al., 2020). Importantly, Cas13d nucleases can process CRISPR arrays, allowing for multiplex targeting ( Konermann et al., 2018). RNA targeting by CasRx also fared better than short hairpin RNA (shRNA) interference. Within the Cas13d family, CasRx (also known as RfxCas13d), from Ruminococcus flavifaciens, possesses the highest RNA cleavage activity and specificity in human cells. In 2018, the smallest of RNA-targeting Cas nucleases, Cas13d, was described. CRISPR/Cas13 was also reported safer than existing CRISPR/Cas systems due to the lack of genomic alterations. Recently, scientists characterized Cas13 enzymes and demonstrated programmable RNA editing superior in efficiency and specificity compared to existing RNA-targeting approaches ( Abudayyeh et al., 2017 Cox et al., 2017 Liu et al., 2017 Konermann et al., 2018). Unlike DNA manipulation using the CRISPR/Cas system, there is a significant lack of literature for CRISPR/Cas-based RNA modification. This warrants the exploitation of smaller-sized enzymes. However, the large size of SpCas9 makes packaging into low-capacity vectors such as adeno-associated virus (AAV) for in vivo delivery or other therapeutic applications challenging. CRISPR/Cas-type II RNA-guided nucleases, including Streptococcus pyogenes Cas9 (SpCas9), have been extensively explored and demonstrated to possess excellent DNA editing capabilities ( Jinek et al., 2012). The discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has provided the opportunity for scientists to make precise changes in the genome.