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CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs.

ABSTRACT: Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.

SUBMITTER: Coelho MA

PROVIDER: S-EPMC7431537 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs.

Coelho Matthew A MA De Braekeleer Etienne E Firth Mike M Bista Michal M Lukasiak Sebastian S Cuomo Maria Emanuela ME Taylor Benjamin J M BJM

Nature communications 20200817 1

Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR Guide RNA Assisted Reduction of Damage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-targe ...[more]

PMID: 32807781

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Project description:CRISPR-Cas systems are a diverse family of RNA-protein complexes in bacteria that target foreign DNA sequences for cleavage. Derivatives of these complexes have been engineered to cleave specific target sequences depending on the sequence of a CRISPR-derived guide RNA (gRNA) and the source of the Cas9 protein. Important considerations for the design of gRNAs are to maximize aimed activity at the desired target site while minimizing off-target cleavage. Because of the rapid advances in the understanding of existing CRISPR-Cas9-derived RNA-guided nucleases and the development of novel RNA-guided nuclease systems, it is critical to have computational tools that can accommodate a wide range of different parameters for the design of target-specific RNA-guided nuclease systems. We have developed CRISPRseek, a highly flexible, open source software package to identify gRNAs that target a given input sequence while minimizing off-target cleavage at other sites within any selected genome. CRISPRseek will identify potential gRNAs that target a sequence of interest for CRISPR-Cas9 systems from different bacterial species and generate a cleavage score for potential off-target sequences utilizing published or user-supplied weight matrices with position-specific mismatch penalty scores. Identified gRNAs may be further filtered to only include those that occur in paired orientations for increased specificity and/or those that overlap restriction enzyme sites. For applications where gRNAs are desired to discriminate between two related sequences, CRISPRseek can rank gRNAs based on the difference between predicted cleavage scores in each input sequence. CRISPRseek is implemented as a Bioconductor package within the R statistical programming environment, allowing it to be incorporated into computational pipelines to automate the design of gRNAs for target sequences identified in a wide variety of genome-wide analyses. CRISPRseek is available under the GNU General Public Licence v3.0 at http://www.bioconductor.org.

Project description:MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.

Dataset Information

CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs.

Publications

CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs.

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