The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger ...nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.
Background: CRISPR/Cas9-directed cleavages may result in genomic deletion.
Results: CRISPR/Cas9-produced genomic deletion frequency is inversely related to deletion size, with large deletions and inversions practicable and biallelic deletions exceeding probabilistic expectation.
Conclusion: Biallelic, large genomic deletions are efficiently engineered in mammalian cells by CRISPR/Cas9.
Significance: CRISPR/Cas9-mediated genomic deletion represents a robust method for loss-of-function studies in mammalian cells.
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The Hyperparathyroidism with Jaw-Tumours syndrome is caused by mutations of the
gene: it has been suggested that early onset of the disease and high Ca
levels may predict the presence of a
mutation. ...We searched for large deletions at the
locus in patients with: HPT-JT (nr 2), atypical adenoma (nr 7) or sporadic parathyroid carcinoma (nr 11) with a specific MLPA and qRT-PCR assays applied on DNA extracted from whole blood. A Medline search in database for all the papers reporting a
gene mutation, clinical/histological diagnosis, age at onset, Ca
, PTH levels for familial/sporadic cases was conducted with the aim to possibly identify biochemical/clinical markers predictive, in first diagnosis, of the presence of a
gene mutation. A novel genomic deletion of the first 10 exons of the
gene was found in a 3-generation HPT-JT family, confirmed by SNP array analysis. A classification tree built on the published data, showed the highest probability of having a
mutation in subjects with age at the onset < 41.5 years (44/47 subjects, 93.6%, had the mutation). Whereas the lowest probability was found in subjects with age at the onset ≥ 41.5 years and Ca
levels <13.96 mg/dL (7/20 subjects, 35.0%, had the mutation, odds ratio = 27.1,
< 0.001). We report a novel large genomic CDC73 gene deletion identified in an Italian HPT-JT family. Age at onset < 41.5 ys and Ca
> 13.96 mg/dL are predictive for the presence of a CDC73 genetic lesion.
In
, large genomic deletions have been carried out for genome reduction, antibiotic overproduction, and heterologous protein overexpression. In view of the eco-friendliness of
, it is critical that ...engineering preserves its food-grade status and avoids leaving foreign DNA in the genome. Existing methods of generating large genomic deletions leave antibiotic resistance markers or display low mutation efficiency. In this study, we introduced a clustered regularly interspaced short palindromic repeat-derived genome engineering technique to develop a highly efficient method of generating large genomic deletions in
without any trace of foreign DNA. Using our system, we produced 38 kb plipastatin-synthesizing
operon deletion with 80% efficiency. The significant increase in mutation efficiency was due to plasmids-delivered
-originated SpCas9, target-specific sgRNA and a donor DNA template, which produces SpCas9/sgRNA endonuclease complex continuously for attacking target chromosome until the mutagenic repair occurs. Our system produced single-gene deletion in
(∼100%), point mutation (∼68%) and GFP gene insertion (∼97%) in
and demonstrated its broad applicability for various types of site-directed mutagenesis in
.
Abstract African swine fever virus (ASFV) causes a devastating disease affecting domestic and wild pigs. ASF was first introduced in Sardinia in 1978 and until 2019 only genotype I isolates were ...identified. A remarkable genetic stability of Sardinian ASFV isolates was described, nevertheless in 2019 two wild boar isolates with a sustained genomic deletion (4342 base pairs) were identified (7303WB/19, 7212WB/19). In this study, we therefore performed in vitro experiments with monocyte-derived macrophages (moMФ) to unravel the phenotypic characteristics of these deleted viruses. Both 7303WB/19 and 7212WB/19 presented a lower growth kinetic in moMФ compared to virulent Sardinian 26544/OG10, using either a high (1) or a low (0.01) multiplicity of infection (MOI). In addition, flow cytometric analysis showed that both 7303WB/19 and 7212WB/19 presented lower intracellular levels of both early and late ASFV proteins. We subsequently investigated whether deleted virus variants were previously circulating in wild boars in Sardinia. In the four years preceding the last genotype I isolation (February 2015–January 2019), other eight wild boar isolates were collected, all belonging to p72 genotype I, B602L subgroup X, but none of them presented a sustained genomic deletion. Overall, we observed the deleted virus isolates in Sardinia only in 2019, at the end of a strong eradication campaign, and our data suggest that it might possess an attenuated phenotype in vivo. A better understanding of ASFV evolution in endemic territories might contribute to development of effective control measures against ASF.
Genome editing is an effective tool for the functional examination of bacterial genes and for live attenuated vaccine construction. Here, we report a method to edit the genomic DNA of
Bacillus ...anthracis
and
Bacillus cereus
using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 system. Using two prophages in
B. anthracis
as targets, large-fragment deletion mutants were achieved with rates of 100 or 20%. In
B. cereus
, we successfully introduced precise point mutations into
plcR
, with phenotypic assays showing that the resulting mutants lost hemolytic and phospholipase enzyme activities similar to
B. anthracis
, which is a natural
plcR
mutant. Our study indicates that CRISPR/Cas9 is a powerful genetic tool for genome editing in the
Bacillus cereus
group, and can efficiently modify target genes without the need for residual foreign DNA such as antibiotic selection markers. This system could be developed for use in the generation of marker-free live anthrax vaccines or for safer construction of microbiological candidate-based recombinant
B. cereus
.
Escherichia coli K‐12, being one of the best understood and thoroughly analyzed organisms, is the preferred platform for genetic and biochemical research. Among all genetic engineering approaches ...applied on E. coli, the homologous recombination approach is versatile and precise, which allows engineering genes or large segments of the chromosome directly by using polymerase chain reaction (PCR) products or synthetic oligonucleotides. The previously explained approaches for random insertion and deletions were reported as technically not easy and laborious. This study, first, finds the minimum length of homology extension that is efficient and accurate for homologous recombination, as 30 nt. Second, proposes an approach utilizing PCR products flanking ambiguous NNN‐sequence (30‐nt) extensions, which facilitate the homologous recombination to recombine them at multiple regions on the genome and generate insertion–deletion mutations. Further analysis found that these mutations were varying in number, that is, multiple genomic regions were deleted. Moreover, evaluation of the phenotype of all the multiple random insertion–deletion mutants demonstrated no significant changes in the normal metabolism of bacteria. This study not only presents the efficiency of ambiguous sequences in making random deletion mutations, but also demonstrates their further applicability in genomics.
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Prior studies have demonstrated that patients with chromosome 22q11.2 deletion syndrome (22q11.2DS) have lower platelet counts (PC) compared to non‐deleted populations. They also have an increased ...mean platelet volume. The mechanism for this has been postulated to be haploinsufficiency of the GPIBB gene. We examined platelet parameters, deletion size and factors known to influence counts, including status of thyroid hormone and congenital heart disease (CHD), in a population of 825 patients with 22q11.2DS. We also measured surface expression of GPIB‐IX complex by flow cytometry. The major determinant of PC was deletion status of GP1BB, regardless of surface expression or other factors. Patients with nested distal chromosome 22q11.2 deletions (those with GP1BB present) had higher PCs than those with proximal deletions where GP1BB is deleted. Patients with 22q11.2DS also demonstrated an accelerated PC decrease with age, occurring in childhood. These data demonstrate that genes within the proximal deletion segment drive PC differences in 22q11.2DS and suggest that PC reference ranges may need to be adjusted for age and deletion size in 22q11.2DS populations. Bleeding did not correlate with either platelet count or GPIb expression. Further studies into drivers of expression of GPIb and associations with severe thrombocytopenia and immune thrombocytopenia are needed to inform clinical care.
We analyzed platelet parameters of 825 participants with 22q11.2 deletion syndrome and found accelerated platelet count decreases occurring in childhood. Genetic analysis revealed that haploinsufficiency of GP1BB was associated with the decrease, but we found no further correlation with GPIb expression levels.
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• Development of a small-scale CRISPR/AsCpf1 screen in CHO.• Usage of paired gRNAs enables full deletion of coding or noncoding genomic regions.• Growth perturbing paired gRNAs identified.• Key ...points for considerations in future screens identified.
Chinese hamster ovary (CHO) cells are the most widely used host for the expression of therapeutic proteins. Recently, significant progress has been made due to advances in genome sequence and annotation quality to unravel the black box CHO. Nevertheless, in many cases the link between genotype and phenotype in the context of suspension cultivated production cell lines is still not fully understood. While frameshift approaches targeting coding genes are frequently used, the non-coding regions of the genome have received less attention with respect to such functional annotation. Importantly, for non-coding regions frameshift knock-out strategies are not feasible. In this study, we developed a CRISPR-mediated screening approach that performs full deletions of genomic regions to enable the functional study of both the translated and untranslated genome.
An in silico pipeline for the computational high-throughput design of paired guide RNAs (pgRNAs) directing CRISPR/AsCpf1 was established and used to generate a library tackling process-related genes and long non-coding RNAs. Next generation sequencing analysis of the plasmid library revealed a sufficient, but highly variable pgRNA composition. Recombinase-mediated cassette exchange was applied for pgRNA library integration rather than viral transduction to ensure single copy representation of pgRNAs per cell. After transient AsCpf1 expression, cells were cultivated over two sequential batches to identify pgRNAs which massively affected growth and survival. By comparing pgRNA abundance, depleted candidates were identified and individually validated to verify their effect.
Graphical workflow for a CRISPR/AsCpf1 paired gRNA (pgRNA) genomic deletion screen in Chinese hamster ovary (CHO) cells. pgRNA library ordered as a single-stranded oligonucleotide pool, followed by PCR amplification and cloning into the delivery plasmid backbone. Next, stably pgRNA expressing pre-screening cell pools were generated. By the application of the AsCpf1 endonuclease, genomic deletions were induced, and cell pools were screened for the desired phenotype. In parallel, pre-screening cell pools were treated with Cas9 resulting in no alteration of the genome. Comparison of pgRNA abundance between AsCpf1 and Cas9 treated samples after screening procedure identified phenotype modifying hits. Critical pooled CRISPR screening steps are marked by exclamation marks. Display omitted
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Huntington’s disease (HD) is caused by an expanded CAG repeat in huntingtin (HTT). Since HD is dominant and loss of HTT leads to neurological abnormalities, safe therapeutic strategies require ...selective inactivation of mutant HTT. Previously, we proposed a concept of CRISPR-Cas9 using mutant-specific PAM sites generated by SNPs to selectively inactivate mutant HTT. Aiming at revealing suitable targets for clinical development, we analyzed the largest HD genotype dataset to identify target PAM-altering SNPs (PAS) and subsequently evaluated their allele specificities. The gRNAs based on the PAM sites generated by rs2857935, rs16843804, and rs16843836 showed high levels of allele specificity in patient-derived cells. Simultaneous use of two gRNAs based on rs2857935-rs16843804 or rs2857935-rs16843836 produced selective genomic deletions in mutant HTT and prevented the transcription of mutant HTT mRNA without impacting the expression of normal counterpart or re-integration of the excised fragment elsewhere in the genome. RNA-seq and off-target analysis confirmed high levels of allele specificity and the lack of recurrent off-targeting. Approximately 60% of HD subjects are eligible for mutant-specific CRISPR-Cas9 strategies of targeting one of these three PAS in conjunction with one non-allele-specific site, supporting high applicability of PAS-based allele-specific CRISPR approaches in the HD patient population.
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CRISPR-Cas9 to selectively inactivate mutant huntingtin may produce robust therapeutic benefits in Huntington’s disease (HD). We revealed PAM-altering SNPs that permit allele-specific CRISPR-Cas9 in HD (rs2857935, rs16843804, and rs16843836). Targeting those sites in combinations using dual gRNAs prevented the transcription of mutant HTT mRNA without impacting the normal counterpart.
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