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Regenerating Urethral Striated Muscle by CRISPRi/dCas9-KRAB-Mediated Myostatin Silencing for Obesity-Associated Stress Urinary Incontinence.
Overweight females are prone to obesity-associated stress urinary incontinence (OA-SUI), and there are no definitive medical therapies for this common urologic condition. This study was designed to test the hypothesis that regenerative therapy to restore urethral striated muscle (stM) and pelvic floor muscles might represent a valuable therapeutic approach. For the in vitro experiment, single-guide RNAs targeting myostatin (MSTN) were used for CRISPRi/dCas9-Kruppel associated box (KRAB)-mediated gene silencing. For the in vivo experiment, a total of 14 female lean ZUC-Leprfa 186 and 14 fatty ZUC-Leprfa 185 rats were used as control and CRISPRi-MSTN treated groups, respectively. The results indicated that lentivirus-mediated expression of MSTN CRISPRi/dCas9-KRAB caused sustained downregulation of MSTN in rat L6 myoblast cells and significantly enhanced myogenesis in vitro. In vivo, the urethral sphincter injection of lentiviral-MSTN sgRNA and lentiviral-dCas9-KRAB significantly increased the leak point pressure, the thickness of the stM layer, the ratio of stM to smooth muscle, and the number of neuromuscular junctions. Downregulation of MSTN with CRISPRi/dCas9-KRAB-mediated gene silencing significantly enhanced myogenesis in vitro and in vivo. It also improved urethral continence in the OA-SUI rat model.
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Improving FnCas12a Genome Editing by Exonuclease Fusion.
Among current reported Cas12a orthologs, Francisella novicida Cas12a (FnCas12a) is less restricted by protospacer adjacent motif (PAM). However, the activity of FnCas12a nuclease is relatively low or undetectable in human cells, limiting its application as desirable genome engineering tools. Here, we describe TEXT (Tethering EXonuclease T5 with FnCas12a)-a fusion strategy that significantly increased the knockout efficiency of FnCas12a in human cells at multiple genomic loci in three different cell lines. TEXT results in higher insertion and deletion efficiency than FnCas12a under different spacer lengths from 18 nt to 23 nt. Deep sequencing shows that TEXT substantially increased the deletion frequency and deletion size at the targeted locus. Compared to other Cas12a orthologs, including AsCas12a and LbCas12a, TEXT achieves the highest on-targeting efficiency and shows minimal off-targeting effects at all tested sites. TEXT enhances the activity of FnCas12a nuclease and expands its targeting scope and efficiency in human cell genome engineering.
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Application of a cost-effective DNA extraction protocol for screening transgenic and CRISPR-edited primary goat cells.
The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells-GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A-0%, protocol B-93%, protocol C-13.33%, P <0.05). In a proof-of-principle experiment, the TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.
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CRISPR-Based Regulation for High-Throughput Screening.
CRISPR technology has revolutionized genome editing by enabling precise, permanent modifications to genetic material. To circumvent the irreversible alterations associated with traditional CRISPR methods and facilitate research on both essential and nonessential genes, CRISPR interference or inhibition (CRISPRi) and CRISPR activation (CRISPRa) were developed. The gene-silencing approach leverages an inactivated Cas effector protein paired with guide RNA to obstruct transcription initiation or elongation, while the gene-activation approach exploits the programmability of CRISPR to activate gene expression. Recent advances in CRISPRi technology, in combination with other technologies (e.g., biosensing, sequencing), have significantly expanded its applications, allowing for genome-wide high-throughput screening (HTS) to identify genetic determinants of phenotypes. These screening strategies have been applied in biomedicine, industry, and basic research. This review explores the CRISPR regulation mechanisms, offers an overview of the workflow for genome-wide CRISPR-based regulation for screens, and highlights its superior suitability for HTS across biomedical and industrial applications. Finally, we discuss the limitations of current CRISPRi/a HTS screening methods and envision future directions in CRISPR-mediated HTS research, considering its potential for broader application across diverse fields.
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CRISPR Gene Therapy of the Eye: Targeted Knockout of Vegfa in Mouse Retina by Lentiviral Delivery.
Genome editing and knockout by virus-based delivery of CRISPR/Cas9 may provide a new option to cure inherited and acquired ocular diseases. Here we describe development and application of lentivirus-based delivery vectors enabling knockout of the Vegfa gene. We show that Streptococcus pyogenes (Sp) Cas9 and single-guide RNAs (sgRNAs) delivered by such vectors selectively can ablate the vascular endothelial growth factor A (Vegfa) gene in mouse retina following a single administration. These findings may contribute to the development of a new therapeutic path in the treatment of ocular diseases including exudative age-related macular degeneration (AMD).
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Multiple Applications of a Transient CRISPR-Cas9 Coupled with Electroporation (TRACE) System in the Cryptococcus neoformans Species Complex.
Cryptococcus neoformans is a fungal pathogen that claims hundreds of thousands of lives annually. Targeted genetic manipulation through biolistic transformation in C. neoformans drove the investigation of this clinically important pathogen at the molecular level. Although costly and inefficient, biolistic transformation remains the major method for editing the Cryptococcus genome as foreign DNAs introduced by other methods such as electroporation are predominantly not integrated into the genome. Although the majority of DNAs introduced by biolistic transformation are stably inherited, the transformation efficiency and the homologous integration rate (∼1-10%) are low. Here, we developed a Transient CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with Electroporation (TRACE) system for targeted genetic manipulations in the C. neoformans species complex. This method took advantages of efficient genome integration due to double-strand breaks created at specific sites by the transient CRISPR-Cas9 system and the high transformation efficiency of electroporation. We demonstrated that TRACE can efficiently generate precise single-gene deletion mutants using the ADE2 locus as an example. This system can also effectively delete multiple genes in a single transformation, as evident by the successful generation of quadruple mfα1Δ2Δ3Δ4Δ mutants. In addition to generating gene deletion mutants, we complemented the ade2Δ mutant by integrating a wild-type ADE2 allele at the "safe haven" region (SH2) via homologous recombination using TRACE. Interestingly, introduced DNAs can be inserted at a designated genetic site without any homologous sequences, opening up numerous other applications. We expect that TRACE, an efficient, versatile, and cost-effective gene editing approach, will greatly accelerate research in this field.
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Protocol for NT-CRISPR: A Method for Efficient Genome Engineering in Vibrio natriegens.
Vibrio natriegens is a gram-negative bacterium, which has received increasing attention due to its very fast growth with a doubling time of under 10 min under optimal conditions. To enable a wide range of projects spanning from basic research to biotechnological applications, we developed NT-CRISPR as a new method for genome engineering. This book chapter provides a step-by-step protocol for the use of this previously published tool. NT-CRISPR combines natural transformation with counterselection through CRISPR-Cas9. Thereby, genomic regions can be deleted, foreign sequences can be integrated, and point mutations can be introduced. Furthermore, up to three simultaneous modifications are possible.
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Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells.
CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.
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Guide Picker is a comprehensive design tool for visualizing and selecting guides for CRISPR experiments.
BACKGROUND: Guide Picker ( https://www.deskgen.com/guide-picker/ ) serves as a meta tool for designing CRISPR experiments by presenting ten different guide RNA scoring functions in one simple graphical interface. It allows investigators to simultaneously visualize and sort through every guide targeting the protein-coding regions of any mouse or human gene. RESULTS: Utilizing a multidimensional graphical display featuring two plots and four axes, Guide Picker can analyze all guides while filtering based on four different criteria at a time. Guide Picker further facilitates the CRISPR design process by using pre-computed scores for all guides, thereby offering rapid guide RNA generation and selection. CONCLUSIONS: The ease-of-use of Guide Picker complements CRISPR itself, matching a powerful and modular biological system with a flexible online web tool that can be used in a variety of genome editing experimental contexts.
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Data imbalance in CRISPR off-target prediction.
For genome-wide CRISPR off-target cleavage sites (OTS) prediction, an important issue is data imbalance-the number of true OTS recognized by whole-genome off-target detection techniques is much smaller than that of all possible nucleotide mismatch loci, making the training of machine learning model very challenging. Therefore, computational models proposed for OTS prediction and scoring should be carefully designed and properly evaluated in order to avoid bias. In our study, two tools are taken as examples to further emphasize the data imbalance issue in CRISPR off-target prediction to achieve better sensitivity and specificity for optimized CRISPR gene editing. We would like to indicate that (1) the benchmark of CRISPR off-target prediction should be properly evaluated and not overestimated by considering data imbalance issue; (2) incorporation of efficient computational techniques (including ensemble learning and data synthesis techniques) can help to address the data imbalance issue and improve the performance of CRISPR off-target prediction. Taking together, we call for more efforts to address the data imbalance issue in CRISPR off-target prediction to facilitate clinical utility of CRISPR-based gene editing techniques.
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