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mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.
As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation.
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A genetic system for biasing the sex ratio in mice.
Biasing the sex ratio of populations of different organisms, including plants, insects, crustacean, and fish, has been demonstrated by genetic and non-genetic approaches. However, biasing the sex ratio of mammalian populations has not been demonstrated genetically. Here, we provide a first proof of concept for such a genetic system in mammals by crossing two genetically engineered mouse lines. The maternal line encodes a functional Cas9 protein on an autosomal chromosome, whereas the paternal line encodes guide RNAs on the Y chromosome targeting vital mouse genes. After fertilization, the presence of both the Y-encoded guide RNAs from the paternal sperm and the Cas9 protein from the maternal egg targets the vital genes in males. We show that these genes are specifically targeted in males and that this breeding consequently self-destructs solely males. Our results pave the way for a genetic system that allows biased sex production of livestock.
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Calcium signaling consequences of RyR2 mutations associated with CPVT1 introduced via CRISPR/Cas9 gene editing in human-induced pluripotent stem cell-derived cardiomyocytes: Comparison of RyR2-R420Q, F2483I, and Q4201R.
BACKGROUND: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) created from patients with catecholaminergic polymorphic ventricular tachycardia 1 (CPVT1) have been used to study CPVT1 arrhythmia. OBJECTIVE: The purpose of this study was to evaluate the Ca2+ signaling aberrancies and pharmacological sensitivities of 3 CRISPR/Cas9-introduced CPVT1 mutations located in different molecular domains of ryanodine receptor 2 (RyR2). METHODS: CRISPR/Cas9-engineered hiPSC-CMs carrying RyR2 mutations-R420Q, Q4201R, and F2483I-were voltage clamped, and their electrophysiology, pharmacology, and Ca2+ signaling phenotypes measured using total internal reflection fluorescence microscopy. RESULTS: R420Q and Q4201R mutant hiPSC-CMs exhibit irregular, long-lasting, spatially wandering Ca2+ sparks and aberrant Ca2+ releases similar to F2483I unlike the wild-type myocytes. Large sarcoplasmic reticulum (SR) Ca2+ leaks and smaller SR Ca2+ contents were detected in cells expressing Q4201R and F2483I, but not R420Q. Fractional Ca2+ release and calcium-induced calcium release gain were higher in Q4201R than in R420Q and F2483I hiPSC-CMs. JTV519 was equally effective in suppressing Ca2+ sparks, waves, and SR Ca2+ leaks in hiPSC-CMs derived from all 3 mutant lines. Flecainide and dantrolene similarly suppressed SR Ca2+ leaks, but were less effective in decreasing spark frequency and durations. CONCLUSION: CRISPR/Cas9 gene editing of hiPSCs provides a novel approach in studying CPVT1-associated RyR2 mutations and suggests that Ca2+-signaling aberrancies and drug sensitivities may vary depending on the mutation site.
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CRISPR-offinder: a CRISPR guide RNA design and off-target searching tool for user-defined protospacer adjacent motif.
Designing efficient and specific CRISPR single-guide RNAs (sgRNAs) is vital for the successful application of CRISPR technology. Currently, a growing number of new RNA-guided endonucleases with a different protospacer adjacent motif (PAM) have been discovered, suggesting the necessity to develop a versatile tool for designing sgRNA to meet the requirement of different RNA-guided DNA endonucleases. Here, we report the development of a flexible sgRNA design program named "CRISPR-offinder". Support for user-defined PAM and sgRNA length was provided to increase the targeting range and specificity. Additionally, evaluation of on- and off-target scoring algorithms was integrated into the CRISPR-offinder. The CRISPR-offinder has provided the bench biologist a rapid and efficient tool for identification of high quality target sites, and it is freely available at https://sourceforge.net/projects/crispr-offinder-v1-2/ or http://www.biootools.com.
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Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant.
Precise editing of genomic DNA can be achieved upon repair of CRISPR-induced DNA double-stranded breaks (DSBs) by homology-directed repair (HDR). However, the efficiency of this process is limited by DSB repair pathways competing with HDR, such as non-homologous end joining (NHEJ). Here we individually express in human cells 204 open reading frames involved in the DNA damage response (DDR) and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of CRISPR-mediated HDR. By defining the RAD18 domains required to promote HDR, we derive an enhanced RAD18 variant (e18) that stimulates CRISPR-mediated HDR in multiple human cell types, including embryonic stem cells. Mechanistically, e18 induces HDR by suppressing the localization of the NHEJ-promoting factor 53BP1 to DSBs. Altogether, this study identifies e18 as an enhancer of CRISPR-mediated HDR and highlights the promise of engineering DDR factors to augment the efficiency of precision genome editing.
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Super-Enhancers at the Nanog Locus Differentially Regulate Neighboring Pluripotency-Associated Genes.
Super-enhancers are tissue-specific cis-regulatory elements that drive expression of genes associated with cell identity and malignancy. A cardinal feature of super-enhancers is that they are transcribed to produce enhancer-derived RNAs (eRNAs). It remains unclear whether super-enhancers robustly activate genes in situ and whether their functions are attributable to eRNAs or the DNA element. CRISPR/Cas9 was used to systematically delete three discrete super-enhancers at the Nanog locus in embryonic stem cells, revealing functional differences in Nanog transcriptional regulation. One distal super-enhancer 45 kb upstream of Nanog (-45 enhancer) regulates both nearest neighbor genes, Nanog and Dppa3. Interestingly, eRNAs produced at the -45 enhancer specifically regulate Dppa3 expression by stabilizing looping of the -45 enhancer and Dppa3. Our work illustrates that genomic editing is required to determine enhancer function and points to a method to selectively target a subset of super-enhancer-regulated genes by depleting eRNAs.
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Triple deletion of clpC, porB, and mepA enhances production of small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide in Corynebacterium glutamicum.
In our previous work, a two-plasmid CRISPR/Cas9 system was constructed for genome editing in Corynebacterium glutamicum. To increase the transformation efficiency and simplify the plasmid curing steps, an all-in-one CRISPR/Cas9 system was constructed for efficient genome editing. In addition, to research proteolysis during the production of recombinant proteins and generate a host for enhanced expression of recombinant proteins, the system was used to delete three genes, clpC, porB, and mepA in C. glutamicum CGMCC1.15647, which encoded the Clp protease subunit ClpC, anion selective channel protein B, and metallopeptidase A, respectively. After the evaluation of different plasmids and hosts, small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide (SUMO-NT-proBNP), an important protein used for the diagnosis of mild heart failure was successfully expressed in the triple mutant ΔclpCΔporBΔmepA, which exhibit threefold higher levels of protein expression compared with the wild-type. In conclusion, we created a simplified CRISPR tool for genome editing in C. glutamicum, provided a method to generate a host for enhanced expression of recombinant proteins and successfully expressed SUMO-NT-proBNP in C. glutamicum. This tool and method will greatly facilitate genetic engineering and metabolic optimization of this important platform organism.
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Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid.
Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.
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Ligand-binding domains of nuclear receptors facilitate tight control of split CRISPR activity.
Cas9-based RNA-guided nuclease (RGN) has emerged to be a versatile method for genome editing due to the ease of construction of RGN reagents to target specific genomic sequences. The ability to control the activity of Cas9 with a high temporal resolution will facilitate tight regulation of genome editing processes for studying the dynamics of transcriptional regulation or epigenetic modifications in complex biological systems. Here we show that fusing ligand-binding domains of nuclear receptors to split Cas9 protein fragments can provide chemical control over split Cas9 activity. The method has allowed us to control Cas9 activity in a tunable manner with no significant background, which has been challenging for other inducible Cas9 constructs. We anticipate that our design will provide opportunities through the use of different ligand-binding domains to enable multiplexed genome regulation of endogenous genes in distinct loci through simultaneous chemical regulation of orthogonal Cas9 variants.
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CRISPR targeting of SNPs associated with age-related macular degeneration in ARPE-19 cells: a potential model for manipulating the complement system.
Age-related Macular degeneration (AMD) is a major cause of vision loss and is linked to several predisposing single nucleotide polymorphisms (SNPs). CRISPR-mediated genome editing offers the potential to target negatively associated SNPs in an allele-specific manner, necessitating the need for a relevant cell model. The ARPE-19 cell line, with its stable monolayer growth and retinal pigment epithelium (RPE) characteristics, serves as an ideal model for AMD studies. Chronic inflammation and complement system dysregulation are implicated in AMD pathogenesis. Most genetic variations associated with AMD are in complement genes, suggesting their regulatory role. In this study, we conducted targeted PCRs to identify AMD-related SNPs in ARPE-19 cells and used CRISPR constructs to assess allele-specific activity. Guide RNA sequences were cloned into an EF-1-driven SpCas9 vector and packaged into lentivirus. Targeting efficiencies were evaluated with TIDE analysis, and allele-specificity was measured with NGS analysis 30 days post-transduction. Our results showed varying targeting efficiencies depending on guide RNA efficacy. For example, TIDE analysis of CFH SNPs rs1061170 and rs1410996 revealed efficiencies of 35.5% and 33.8%, respectively. CFB SNP rs4541862 showed efficiencies from 3% to 36.7%, and rs641153 ranged from 3.4% to 23.8%. Additionally, allele-specific targeting of AMD-related SNPs rs1061170, rs1410996, rs4541862, and rs641153 ranged from 48% to 52% in heterozygous differentiated ARPE-19 cells. These findings demonstrate the potential to manipulate the complement system in an AMD model by targeting disease-associated SNPs in an allele-specific manner, offering a promising therapeutic approach.
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