Allele-specific CRISPR-Cas9 editing inactivates a single nucleotide variant associated with collagen VI muscular dystrophy.

The application of allele-specific gene editing tools can expand the therapeutic options for dominant genetic conditions, either via gene correction or via allelic gene inactivation in situations where haploinsufficiency is tolerated. Here, we used allele-targeted CRISPR-Cas9 guide RNAs (gRNAs) to introduce inactivating frameshifting indels at an SNV in the COL6A1 gene (c.868G>A; G290R), a variant that acts as dominant negative and that is associated with a severe form of congenital muscular dystrophy. We expressed SpCas9 along with allele-targeted gRNAs, without providing a repair template, in primary fibroblasts derived from four patients and one control subject. Amplicon deep sequencing for two gRNAs tested showed that single-nucleotide deletions accounted for the majority of indels introduced. While activity of the two gRNAs was greater at the G290R allele, both gRNAs were also active at the wild-type allele. To enhance allele selectivity, we introduced deliberate additional mismatches to one gRNA. One of these optimized gRNAs showed minimal activity at the WT allele, while generating productive edits and improving collagen VI matrix in cultured patient fibroblasts. This study strengthens the potential of gene editing to treat dominant-negative disorders, but also underscores the challenges in achieving allele selectivity with gRNAs.

Allele-specific CRISPR/Cas9 editing inactivates a single nucleotide variant associated with collagen VI muscular dystrophy.

The application of allele-specific gene editing tools can expand the therapeutic options for dominant genetic conditions, either via gene correction or via allelic gene inactivation in situations where haploinsufficiency is tolerated. Here, we used allele-targeted CRISPR/Cas9 guide RNAs (gRNAs) to introduce inactivating frameshifting indels at a single nucleotide variant in the COL6A1 gene (c.868G>A; G290R), a variant that acts as dominant negative and that is associated with a severe form of congenital muscular dystrophy. We expressed spCas9 along with allele-targeted gRNAs, without providing a repair template, in primary fibroblasts derived from four patients and one control subject. Amplicon deep-sequencing for two gRNAs tested showed that single nucleotide deletions accounted for the majority of indels introduced. While activity of the two gRNAs was greater at the G290R allele, both gRNAs were also active at the wild-type allele. To enhance allele-selectivity, we introduced deliberate additional mismatches to one gRNA. One of these optimized gRNAs showed minimal activity at the WT allele, while generating productive edits and improving collagen VI matrix in cultured patient fibroblasts. This study strengthens the potential of gene editing to treat dominant-negative disorders, but also underscores the challenges in achieving allele selectivity with gRNAs.

Designing Guide-RNA for Generating Premature Stop Codons for Gene Knockout Using CRISPR-BETS.

Cytosine base editors (CBEs) accurately modify target sites by mediating a C to T change (or a G to A change on the opposite strand). This allows us to install premature stop codons for gene knockout. However, highly specific sgRNAs (single-guide RNAs) are necessary for the CRISPR-Cas nuclease to work efficiently. In this study, we introduce a method of designing highly specific gRNA to generate premature stop codons and knock out a gene using CRISPR-BETS software.

CRISPR-BETS: a base-editing design tool for generating stop codons.

Cytosine base editors (CBEs) can install a predefined stop codon at the target site, representing a more predictable and neater method for creating genetic knockouts without altering the genome size. Due to the enhanced predictability of the editing outcomes, it is also more efficient to obtain homozygous mutants in the first generation. With the recent advancement of CBEs on improved editing activity, purify and specificity in plants and animals, base editing has become a more appealing technology for generating knockouts. However, there is a lack of design tools that can aid the adoption of CBEs for achieving such a purpose, especially in plants. Here, we developed a user-friendly design tool named CRISPR-BETS (base editing to stop), which helps with guide RNA (gRNA) design for introducing stop codons in the protein-coding genes of interest. We demonstrated in rice and tomato that CRISPR-BETS is easy-to-use, and its generated gRNAs are highly specific and efficient for generating stop codons and obtaining homozygous knockout lines. While we tailored the tool for the plant research community, CRISPR-BETS can also serve non-plant species.

Enhanced Cas12a multi-gene regulation using a CRISPR array separator.

The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

PnB Designer: a web application to design prime and base editor guide RNAs for animals and plants.

BACKGROUND: The rapid expansion of the CRISPR toolbox through tagging effector domains to either enzymatically inactive Cas9 (dCas9) or Cas9 nickase (nCas9) has led to several promising new gene editing strategies. Recent additions include CRISPR cytosine or adenine base editors (CBEs and ABEs) and the CRISPR prime editors (PEs), in which a deaminase or reverse transcriptase are fused to nCas9, respectively. These tools hold great promise to model and correct disease-causing mutations in animal and plant models. But so far, no widely-available tools exist to automate the design of both BE and PE reagents. RESULTS: We developed PnB Designer, a web-based application for the design of pegRNAs for PEs and guide RNAs for BEs. PnB Designer makes it easy to design targeting guide RNAs for single or multiple targets on a variant or reference genome from organisms spanning multiple kingdoms. With PnB Designer, we designed pegRNAs to model all known disease causing mutations available in ClinVar. Additionally, PnB Designer can be used to design guide RNAs to install or revert a SNV, scanning the genome with one CBE and seven different ABE PAM variants and returning the best BE to use. PnB Designer is publicly accessible at http://fgcz-shiny.uzh.ch/PnBDesigner/ CONCLUSION: With PnB Designer we created a user-friendly design tool for CRISPR PE and BE reagents, which should simplify choosing editing strategy and avoiding design complications.

CrisPam: SNP-Derived PAM Analysis Tool for Allele-Specific Targeting of Genetic Variants Using CRISPR-Cas Systems.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a promising novel technology that holds the potential of treating genetic diseases. Safety and specificity of the treatment are to be further studied and developed prior to implementation of the technology into the clinic. The guide-RNA (gRNA) allows precise position-specific DNA targeting, although it may tolerate small changes such as point mutations. The permissive nature of the CRISPR-Cas system makes allele-specific targeting a challenging goal. Hence, an allele-specific targeting approach is in need for future treatments of heterozygous patients suffering from diseases caused by dominant negative mutations. The single-nucleotide polymorphism (SNP)-derived protospacer adjacent motif (PAM) approach allows highly allele-specific DNA cleavage due to the existence of a novel PAM sequence only at the target allele. Here, we present CrisPam, a computational tool that detects PAMs within the variant allele for allele-specific targeting by CRISPR-Cas systems. The algorithm scans the sequences and attempts to identify the generation of multiple PAMs for a given reference sequence and its variations. A successful result is such that at least a single PAM is generated by the variation nucleotide. Since the PAM is present within the variant allele only, the Cas enzyme will bind the variant allele exclusively. Analyzing a dataset of human pathogenic point mutations revealed that 90% of the analyzed mutations generated at least a single PAM. Thus, the SNP-derived PAM approach is ideal for targeting most of the point mutations in an allele-specific manner. CrisPam simplifies the gRNAs design process to specifically target the allele of interest and scans a wide range of 26 unique PAMs derived from 23 Cas enzymes. CrisPam is freely available at https://www.danioffenlab.com/crispam.

In silico Method in CRISPR/Cas System: An Expedite and Powerful Booster.

The CRISPR/Cas system has stood in the center of attention in the last few years as a revolutionary gene editing tool with a wide application to investigate gene functions. However, the labor-intensive workflow requires a sophisticated pre-experimental and post-experimental analysis, thus becoming one of the hindrances for the further popularization of practical applications. Recently, the increasing emergence and advancement of the in silico methods play a formidable role to support and boost experimental work. However, various tools based on distinctive design principles and frameworks harbor unique characteristics that are likely to confuse users about how to choose the most appropriate one for their purpose. In this review, we will present a comprehensive overview and comparisons on the in silico methods from the aspects of CRISPR/Cas system identification, guide RNA design, and post-experimental assistance. Furthermore, we establish the hypotheses in light of the new trends around the technical optimization and hope to provide significant clues for future tools development.

Cas9 Cuts and Consequences; Detecting, Predicting, and Mitigating CRISPR/Cas9 On- and Off-Target Damage: Techniques for Detecting, Predicting, and Mitigating the On- and off-target Effects of Cas9 Editing.

Large deletions and genomic re-arrangements are increasingly recognized as common products of double-strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats - CRISPR associated protein 9 (CRISPR/Cas9) on-target sites. Together with well-known off-target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9-editing is necessary to ensure validity of observed phenotypes in Cas9-edited cell-lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide-RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off-targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on- and off-target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.

Computational approaches for effective CRISPR guide RNA design and evaluation.

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/ CRISPR-associated (Cas) system has emerged as the main technology for gene editing. Successful editing by CRISPR requires an appropriate Cas protein and guide RNA. However, low cleavage efficiency and off-target effects hamper the development and application of CRISPR/Cas systems. To predict cleavage efficiency and specificity, numerous computational approaches have been developed for scoring guide RNAs. Most scores are empirical or trained by experimental datasets, and scores are implemented using various computational methods. Herein, we discuss these approaches, focusing mainly on the features or computational methods they utilise. Furthermore, we summarise these tools and give some suggestions for their usage. We also recommend three versatile web-based tools with user-friendly interfaces and preferable functions. The review provides a comprehensive and up-to-date overview of computational approaches for guide RNA design that could help users to select the optimal tools for their research.

CRISPR Explorer: A fast and intuitive tool for designing guide RNA for genome editing.

The RNA-guided CRISPR-Cas9 (clustered, regularly interspaced, short palindromic repeat-CRISPR-associated 9) system has become a revolutionary technology for targeted genome engineering. The critical step of this technology requires the design of a highly specific and efficient guide RNA (gRNA) that will guide the Cas9 nuclease to the complementary DNA target sequence. CRISPR-Explorer is a new and user-friendly web server for selecting optimal CRISPR sites. It implements the latest scoring schemes of gRNA specificity and efficiency based on published empirical studies. The gRNA design results are generated instantly, thus removing wait times. The user can visualize the high-quality gRNAs with detailed design information through an interactive genome browser. Furthermore, the user can define and specify the parameters for gRNA selection in the Batch Design mode, which recognizes various input formats. CRISPR Explorer is freely accessible at: http://crisprexplorer.org.