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Sandra Grady

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Aug 3, 2024, 5:21:28 PM8/3/24

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When you open the editor on a small screen, the options condense into menus or second line menus that will remain open until you select the menu again or select a different option menu, for easy access:

You can also add a link to text you've already entered by selecting the section of text that you want to turn into a link. The link text is automatically added based on the text that you've selected in the editor.

The Content Market is your gateway to valuable learning materials from trusted content providers. You can also access tools and links your administrator has made available across your institution. You can add content from the Content Market directly to the editor in your course's assignments, tests, and documents.

Select a tool to launch and browse for content to add. Or, select the plus sign on the Institution Tool's card to add the entire tool in the editor. When you add a tool or a piece of content to the editor, it appears as a link.

You can choose an aspect ratio for the image. You can keep the original aspect ratio, or convert it to a landscape or portrait display. The white grid lines show you what part of the original image will be displayed on screens.

This tutorial demonstrates how to use the Content Editor in the Blackboard Ultra course view. The editor appears wherever you can format text, such as in assignments, tests, and discussions. You can add bullet and numbered lists, and bold and italicized text. Use the Text Style menu to add headings. You can also launch the math editor to embed mathematical formulas in your text. The WIRIS editor opens in a new window.

Create or edit table. Note: Cells cannot be merged, nor can the height or width or rows and columns be changed. This is meant for screen readers to have an easier time parsing the content and ensures that tables appear properly on both mobile and desktop screens.

Links: Link to an external site by entering the site's URL. The text editor does not recognize links unless you select this tool. Note: To ensure your link is accessible, you should input Link text that describes where the link will lead.

Attach Files: Attach files from your computer (i.e., documents, PDFs, PowerPoints, or images). Once uploaded, you can select whether items are available for download only, view only, or both. Allowing students to view files lets them read the files from the page, without needing to download them.

The text editor does not have some functions that you might be used to. One example is highlighting. You are not able to highlight text in your course. This change is made to keep all content accessible.

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In earlier studies, we and others reported that FnCas9 has a very high intrinsic specificity, resulting in dissociation from off-targets presented in vitro4,19. In contrast, SpCas9 and its high-fidelity variants remain bound to off-target sites in a cleavage incompetent fashion, a property that might cause non-specific off-targeting outcomes from such regions20,21,22. Where nuclease activity is not utilised, such as in the realm of base editing, constraints introduced by the targeting window of the base editors, concerning the nearest PAM, have necessitated engineering Cas9 proteins with altered PAM specificities to access nucleobase targets on a case-to-case basis23. Taken together, there is an unmet need for Cas effectors that can combine high activity and specificity on one hand, and flexibility in base editing on the other, especially when a favorable PAM is not present in the vicinity of the editing window.

In this study, we have rationally engineered FnCas9 by modifying its WED-PI domain and phosphate-lock loop (PLL) to develop enhanced (en) FnCas9 variants. We have identified and characterized three kinetically enhanced, PAM-flexible enFnCas9 variants (en1, en15 and en31) without altering the intrinsic DNA interrogation specificity of FnCas9, as reported earlier by our group4. enFnCas9 variants expand the range of single-nucleotide variant (SNV) detection by FnCas9-based CRISPR diagnostics (CRISPRDx) platforms. PAM-flexible robust cellular editing, improved HDR-mediated knock-in rate, and single nucleobase specificity underscores the superior performance of enFnCas9 variants in human cells. Furthermore, the compatibility of enFnCas9 variant, en31 with extended (x-) or super-extended (sx-)-gRNAs, enables tuning of adenine base editing window for adenine base editors (ABEs) by en31-ABEmax8.17 without the requirement of PAM engineering to target intended bases. This attribute expands the range of pathogenic single-nucleotide polymorphisms (SNPs) targeting by en31-ABE. Finally, we demonstrate the precise correction of a Leber congenital amaurosis, type 2 (LCA2) disease-associated point mutation in the RPE65 gene which results in the generation of a premature stop codon in the disease condition. We show restoration of the full-length protein expression in patient-specific iPSC-derived retinal pigmented epithelium using the en31-ABEmax8.17. Overall, we show engineered enFnCas9 variants with broad applications in therapeutics and diagnostics.

FnCas9 is evolutionarily divergent to SpCas9 and harbors structural dissimilarities such as unique interactions between the RuvC and REC3 domains, and the PI and WED domains with the latter sharing contacts with the REC1 and REC2 domains1,3,4. However, PAM recognition is conserved among Cas9 orthologs, which trigger directional target DNA unwinding, R-loop formation and expansion, which eventually lead to reorientation of the HNH endonuclease domain to DNA cutting and concomitant RuvC activation leading to concerted DNA cleavage27. Recent mechanistic studies showed that the directional PAM-duplex DNA unwinding serves as the rate-limiting checkpoint of Cas9 action and a conformational switch discriminates Cas9 DNA binding and cleavage events21,28,29,30,31,32. Moreover, the loss of nucleobase-specific interaction between the target DNA and Cas9 was reported to be rescued by base non-specific Cas9 interactions3,33. Thus, we reasoned that stabilizing FnCas9:DNA duplex binding by introducing base non-specific interactions between PAM duplex and the protein might improve FnCas9 nuclease activity without altering its intrinsic specificity. Additionally, to investigate the optimal spacer length in DNA cleavage activity of FnCas9, we performed an in vitro cleavage assay using a previously reported target DNA harboring a stretch of guanines with FnCas9 RNP containing gRNAs of variable lengths ranging from 20 to 24 nucleotides (g20-g24, hereafter referred as extended-gRNA, x-gRNA)34. Interestingly, we observed the lowest activity with the canonical g20, while x-gRNAs exhibited an enhanced DNA cleavage rate, with g21 inducing the fastest rate of cleavage (Supplementary Fig. 3a). We have used g21 in all subsequent assays unless otherwise stated.

Intrigued by the improved kinetic activity of the variants, we tested the cleavage efficiency of two of the enFnCas9 variants (en15 and en31) using super-extended (sx)- gRNAs with spacer lengths ranging from 26 to 28 nucleotides (g26-g28, hereafter referred to as sx-gRNA). We confirmed similar cleavage efficiencies as g21, suggesting the compatibility of enFnCas9 variants with sx-gRNAs (Supplementary Fig. 3b). This could be attributed to the elongated REC3 domain, which interacts with the nucleobases away from the PAM, as visualized in its crystal structure3. To our knowledge, similar observations have not been reported so far for other Cas9 proteins until recently, during the revision of this work35. We speculated that this feature might offer further enhancement of specificity and nucleobase accessibility away from PAM as shown later.

The remarkable intrinsic specificity of FnCas9 to single nucleotide mismatches in the target has applications both in disease diagnostics and disease correction4,40,41. At the level of diagnostics, FnCas9 has recently been utilised for paper strip-based robust diagnostics of nucleic acid targets through the FnCas9 Editor Linked Uniform Detection Assay (FELUDA)40 and Rapid Variant Assay (RAY) platforms41. In contrast to collateral cleavage-based platforms employed by Type V effectors (such as Cas12a42or Cas12f43) or Type VI effectors (such as Cas1344), FELUDA and RAY uses the specificity of direct FnCas9:DNA binding as a lateral-flow readout through a combination of FAM-labeled FnCas9:sgRNA complex and paper-strip chemistry40,41 (Fig. 2a). Importantly, FnCas9 showed comparable resolution of single-nucleotide variant (SNV) diagnosis (4.4-fold) as AaCas12b (4.6-fold) and Cas14a1 (5.1-fold) both of which belong to type V DNA targeting Cas systems and have been reported to have higher intrinsic specificity than SpCas910,11,15,16,45 (Supplementary Fig. 6a). Taken together, this underscores the utility of these variants as a diagnostic platform.

Taken together, enFnCas9 variants have a very high specificity of mismatch discrimination similar to Cas12a or Cas12f/Cas14a but due to their wider PAM accessibility, these can potentially target more genomic sites and pathogenic SNVs for detection.

The safety of therapeutic genome editing is determined by off-target interrogation of CRISPR effectors. Although Cas12a and Cas12f have higher specificity than SpCas9, their therapeutic success relies on minimum ssDNA cleavage inside the cell such as those formed during replication, homology-directed repair, or transcription42,46. Interestingly, Cas12a has been reported to nick off-target DNA substrates with up to four mismatches depending upon the crRNA sequences employed47. On the contrary, enFnCas9 does not produce trans-cleavage products, and its high specificity, both at the level of DNA interrogation and cleavage, might be beneficial for safe nuclease-mediated genome editing. Although construction of high-fidelity SpCas9 proteins have improved its overall specificity, this is also accompanied by lower editing efficiencies5,48,49. We selected two such proteins (SpCas9-HF1 and eSpCas9) due to their balanced activity and specificity as reported in literature5,48,49, and compared their cellular editing rates (insertion/deletions, indels) with one of the enFnCas9 variants, en1. To assess the cellular genome editing potential of enFnCas9 variants, we constructed mammalian expression vectors which encode both the sgRNA and Cas9. Cas9 was fused to EGFP via a T2A linker, all under the control of an identical promoter, chicken β-actin (Fig. 3a). This design ensures uniform protein synthesis within cells and self-cleavage of the EGFP from the Cas9 by T2A. GFP-based fluorescence-activated cell sorting (FACS) was employed to ensure assay uniformity, followed by amplicon sequencing (Supplementary Fig. 1a, b).