CRISPR/Cas9 mediated knockout of the DES gene alleles with desminopathy-related heterozygous gain-of-function mutations

Наследственные кардиомиопатии характеризуются неблагоприятным прогнозом и низкой пятилетней выживаемостью пациентов с выраженной клиникой. При этом лечение, за исключением хирургического, в основном паллиативное, во многих случаях лишь трансплантация сердца может улучшить состояние пациента и прогноз. Часть наследственных кардиомиопатий ассоциирована с аутосомно-доминантными мутациями в гене DES, кодирующем белок промежуточных филаментов десмин, дефекты в котором ведут к развитию десминопатий с вовлечением наиболее активно работающих мышц - скелетных, миокарда, мышц дыхательной системы. Новые терапевтические подходы, основанные на методах геномного редактирования, могут позволить устранить каузативный генетический дефект. Так как имеются данные об отсутствии клинических симптомов у людей с гетерозиготными нонсенс мутациями в гене DES, по-видимому, имеется возможность снизить тяжесть протекания десминопатий путем нокаута мутантного аллеля в случае гетерозиготной мутации. Целью работы являлась проверка возможности специфического нокаута аллелей гена DES, несущих гетерозиготные мутации, ассоциированные с десминопатиями, методами геномного редактирования. Нами был получен генетический материал трех пациентов с десминопатиями, связанными с мутациями в гене DES (c.330_338del, p.A337P (c.1009G>C) и p.R355P (c.1064G>C)). Направляющие РНК, совместимые с нуклеазами SaCas9 и eSpCas9(1.1), были подобраны, используя онлайн сервис Benchling, и клонированы в плазмиды, несущие соответствующие эндонуклеазы Cas9. Редактирующие плазмиды котрансфицировали в клетки HEK293T вместе с «таргетными» плазмидами, содержащими участки гена DES с мутациями. Анализ характерных для негомологичного соединения концов инделов в выделенной из клеток спустя 48 часов после трансфекции тотальной ДНК проводился посредством TIDE-анализа полученных сиквенсов целевых участков, либо методом Т7Е1 анализа. Наибольшая средняя эффективность 2,22% (до 8,06%) показана при использовании sgRNA на мутацию c.330_338del в комбинации с eSpCas9(1.1). Эффективность других комбинаций направляющих РНК и Cas9 не превышала 3%. Достигнутая эффективность нокаута очевидно недостаточна для коррекции десминопатии на уровне организма. Необходимость специфического нокаутирования мутантных аллелей не позволяет использовать другие направляющие РНК для CRISPR/Cas9, поэтому необходимо совершенствование разработанных систем для повышения их эффективности либо использование новых, более эффективных, направляемых нуклеаз. Hereditary cardiomyopathies are characterized by the generally poor prognosis and low 5-year survival of patients with severe symptoms. Besides surgical approaches, cardiomyopathy therapy mainly palliative and often heart transplantation is the only option to improve patient state and prognosis. Some of these pathologies are associated with the autosomal-dominant DES gene mutations. DES encodes intermediate filaments protein desmin, which defects causes desminopathies involving most active muscles such as skeletal muscles, myocardium and respiratory muscles. New therapeutic based on genome editing approaches could be used to correct causative genetic defect. There are data that heterozygous nonsense mutations in DES gene may be asymptomatic. Thus there is, apparently, a possibility to decrease severity of desminopathy using mutant allele knockout. Purpose. The aim of this work was to test the possibility of specific knockout of the DES gene alleles with heterozygous desminopathy-associated mutations by means of genome editing methods. Materials. We received genetic materials of three patients with desminopathy caused by DES gene mutations (c.330_338del, p.A337P (c.1009G>C) и p.R355P (c.1064G>C)). Guide RNA, compatible with nucleases SaCas9 and eSpCas9(1.1) were designed using online service Benchling and cloned into plasmids with corresponding Cas9 nucleases. Editing plasmids were cotransfected into HEK293T cells with “target” plasmids, containing DES gene sites with mutations. NHEJ-produced indels were assessed using TIDE-analysis with amplified and sequenced sites or using T7E1 analysis. Results. Combination sgRNA for c.330_338del with eSpCas9(1.1) demonstrated most mean efficiency of 2,22% (up to 8,06%). Others combinations of sgRNAs and Cas9 efficiency did not overcome 3%. Conclusions. Achieved knockout efficiency is evidently not enough for organism-level desminopathy correction. The need for specific knockout of mutated alleles does not allow usage of different guide RNAs for CRISPR/Cas9, so it is necessary to improve the developed systems to increase their efficiency or to use new, more efficient, targeted nucleases.

Applications of CRISPR-Cas9 as an Advanced Genome Editing System in Life Sciences

Targeted nucleases are powerful genomic tools to precisely change the target genome of living cells, controlling functional genes with high exactness. The clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) genome editing system has been identified as one of the most useful biological tools in genetic engineering that is taken from adaptive immune strategies for bacteria. In recent years, this system has made significant progress and it has been widely used in genome editing to create gene knock-ins, knock-outs, and point mutations. This paper summarizes the application of this system in various biological sciences, including medicine, plant science, and animal breeding.

Recent Advancement and Innovations in CRISPR/Cas and CRISPR Related Technologies: A review

CRISPR genome editing technologies have been improving by every passing day. The initial CRISPR/Cas9 technologies, though emerged an improved version of genome editing in competition with TALENS and ZFNs, was nevertheless not free from technical and off-target effects. Technological improvements overtime start addressing issues with original CRISPR/Cas9 technology. The major areas of improvement targeted nucleases and delivery methods. Overtime the nuclease like Cas9 had some modifications like FokI-dCas9, Truncated guide RNAs (tru-gRNAs), Paired Cas9 nickase, Cpf1, Cas6 with Csm/Csr complex and chemically treated Cas9. In terms of delivery methods the improvements came along after almost all methods including viral methods like Recombinant Adeno Associated Viruses (rAAV), Lentivirus (LV), and bacteriophages. The review summarizes various non-viral gene delivery modes including physical methods like electroporation and chemical methods like nano particles, cell-derived membrane vesicles (CMVs) with upgraded developments. The review also compares various modes of delivering CRISPR gene editing machinery.

Establishment of CRISPR/Cas9-based knock-in in a hemimetabolous insect: targeted gene tagging in the cricket Gryllus bimaculatus

Studies of traditional model organisms like the fruit fly Drosophila melanogaster have contributed immensely to our understanding of the genetic basis of developmental processes. However, the generalizability of these findings cannot be confirmed without functional genetic analyses in additional organisms. Direct genome editing using targeted nucleases has the potential to transform hitherto poorly-understood organisms into viable laboratory organisms for functional genetic study. To this end, here we present a method to induce targeted genome knock-out and knock-in of desired sequences in an insect that serves as an informative contrast to Drosophila, the cricket Gryllus bimaculatus. The efficiency of germ line transmission of induced mutations is comparable to that reported for other well-studied laboratory organisms, and knock-ins targeting introns yields viable, fertile animals in which knock-in events are directly detectable by visualization of a fluorescent marker in the expression pattern of the targeted gene. Combined with the recently assembled and annotated genome of this cricket, this knock-in/knock-out method increases the viability of G. bimaculatus as a tractable system for functional genetics in a basally branching insect.

Deletion of morpholino binding sites (DeMOBS) to assess specificity of morphant phenotypes

Two complimentary approaches are widely used to study gene function in zebrafish: induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce off-target or toxicity-related effects that can be mistaken for true phenotypes. Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness (e.g. use of cryptic splice sites or downstream AUGs). When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5′ untranslated regions would be unlikely to have a significant negative effect on gene expression. Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress morpholino phenotypes does depend on the size and the location of the deletion. Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.

Deletion of MOrpholino Binding Sites (DeMOBS) to Assess Specificity of Morphant Phenotypes

Two complimentary approaches are widely used to study gene function in zebrafish: induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce off-target or toxicity-related effects that can be mistaken for true phenotypes. Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness. When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5’ untranslated regions would be unlikely to have a significant negative effect on gene expression. Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress Morpholino phenotypes does depend on the size and the location of the deletion. Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.

Tracking genome-editing and associated molecular perturbations by SWATH mass spectrometry

Advances in gene editing now allow reverse genetics to be applied to a broad range of biological systems. Ultimately, any modification to coding sequences requires confirmation at the protein level, although immunoblotting is often hampered by antibody quality or availability especially in non-model species. Sequential Window Acquisition of All Theoretical Spectra (SWATH), a mass spectrometry (MS) technology with exceptional quantitative reproducibility and accuracy, offers an ideal alternative for protein-based confirmation. Here, using genome edits in mouse, zebrafish and Bicyclus anynana butterflies produced using either homologous recombination or targeted nucleases, we demonstrate absence of the targeted proteins using SWATH, thus confirming successful editing. We show that SWATH is a robust antibody-independent alternative for monitoring gene editing at the protein level and broadly applicable across diverse organisms and targeted genome manipulation techniques. Moreover, SWATH concomitantly defines the global proteome response in the edited organism, which may provide pertinent biological insights.