CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.This work was funded by the Ministerio de Economía y Competitividad (BIO2011-24417)

Replication Protein A in the Maintenance of Genome Stability

High fidelity double strand break repair is paramount for the maintenance of genome integrity and faithful passage of genetic information to the following generation. Homologous recombination (HR) and non-homologous end joining (C-NHEJ) have evolved as the two major pathways for the efficient and accurate repair of double strand breaks (DSBs). In addition, a minor Ku- and Ligase IV-independent end-joining pathway has been identified and implicated in the formation of chromosomal translocations. This alternative end-joining pathway occurs by bridging the break ends through annealing between short microhomologies, hence the name microhomology-mediated end joining (MMEJ). In addition to these defined DSB repair pathways, a broken DNA end possesses immense mutagenic potential to generate chromosomal rearrangements. Diverse and complex rearrangements are a commonly observed feature amongst cancer cells. The focus of this thesis is to examine the role of Replication Protein A (RPA) in binding single-stranded DNA (ssDNA) repair intermediates to promote error free repair and to prevent mutagenic chromosomal deletions and rearrangements. RPA is a highly conserved, heterotrimeric ssDNA binding protein with a ubiquitous role in all DNA transactions involving ssDNA intermediates. RPA promotes resection at DSBs to facilitate HR and abrogation of this function has severe consequences. Defective RPA can lead to the formation of secondary structures and impair loading of homology search proteins such as Rad52 and Rad51. Using a chromosomal end-joining assay, we demonstrate that hypomorphic rfa1 mutants exhibit elevated frequencies of MMEJ by up to 350-fold. Biochemical characterization of RPAt33 and RPAt48 complexes show these mutants are compromised for their ability to prevent spontaneous annealing and the removal of secondary structures to fully extend ssDNA. These results demonstrate that annealing between MHs defines a critical control to regulate MMEJ repair. Therefore, RPA bound to ssDNA intermediates shields complementary sequences from annealing to promote error-free HR and prevents repair by mutagenic MMEJ, thereby preserving genomic integrity. RPA also impedes intrastrand annealing between short inverted repeat sequences to prevent the formation of foldback structures. Foldbacks have been proposed to drive palindromic gene amplification, a genome destabilizing rearrangement that can disrupt the protein expression equilibrium and is a prevalent phenomenon within tumor cells. Palindromic duplications are elevated ~1000-fold in rfa1-t33 sae2Δ and rfa1-t33 mre11-H125N mutants compared to sae2Δ or mre11-H125N, yet we did not detect these events in the hypomorphic rfa1-t33 mutant. This suggests that Mre11 and Sae2 play critical roles in preventing palindromic amplification through regulation of the Mre11 structure-specific endonuclease to process DNA foldbacks (also called DNA hairpins). Therefore, Mre11-Sae2 together with RPA prevent palindromic gene amplification. Together, these data focus the spotlight on RPA playing active central and supporting roles to sustain genome stability. This additionally raises that notion that secondary structures are potent instigators and mediators of many genome rearrangements and their prevention by RPA is absolutely crucial

Perks and considerations when targeting functional non-coding regions with CRISPR/Cas9

Since the CRISPR system was discovered as an adaptive immune response in prokaryotic cells, the past decade has witnessed the engineering and deployment of CRISPR/Cas9 as one of the most efficient and powerful molecular tools. By leveraging the nuclease activity of CRISPR/Cas9, researchers are able to probe the biological functions of genetic elements and dissect molecular interactions by disrupting, activating or inactivating genes. In addition to biological research, the CRISPR/Cas9 toolkit has profoundly revolutionized gene therapy and agricultural products. However, there are many challenges regarding its efficiency, specificity and safety. Continuous efforts are being made to advance techniques and characterize the consequences of genome editing. In this thesis, we describe considerations when targeting genomic regions with CRISPR/Cas9 and provide methods to address some concerns related to efficiency and safety. In Paper I, we introduced a non-hazardous method of transfecting human cells with large-size CRISPR/Cas9 vectors. By co-transfecting small-size vectors (3 kb) to cells, the delivery efficiency of CRISPR/Cas9 vectors (15 kb) and cell viability was significantly increased. The performance of the method has been verified in a number of hard-to-transfect human cell lines with both electroporation- and liposome-based transfection. In Paper II, we revealed the complexity of CRISPR/Cas9-induced on-target genomic alterations by combining an advanced droplet-based target enrichment method followed by long-read sequencing and de novo assembly-based analysis. This approach enabled us to dissect the on-target sequence content in the order of kilobases, which was very challenging with many other available methods. With this tool, we uncovered the co-occurrence of multiple on-target rearrangements including duplication, inversion, as well as integrations of exogenous DNA and clustered interchromosomal rearrangements in CRISPR/Cas9-modified human cells. Furthermore, our study demonstrated that unintended genomic alterations could lead to the expression of DNA derived from both the target region and exogenous sources, as well as affect cell proliferation. In Paper III, we reported a large unexpected genomic deletion in the HAP1 cell line, which is the one of most popular models used in CRISPR/Cas9-mediated experiments. This 287 kb deletion located on Chromosome 10 contains four widely-expressed protein-coding genes including the PTEN gene locus. We detected changes in histone acetylation and transcriptomes in HAP1 cells carrying the deletion. The loss of this genomic locus was not induced by Cas9 off-target nuclease activity. However, the generation of CRISPR/Cas9-modified cells significantly enhanced the frequency of the deletion among cell clones. Furthermore, our analysis indicated that this deletion initially found in HAP1 cells resembled a frequent deletion pattern driven by the PTEN gene in cancer patients. In conclusion, we have presented two methods: one to improve delivery efficiency and another to detect on-target sequence content with higher resolution. Furthermore, we have revealed unintended genomic aberrations at targeted and non-targeted sites. These observations should be taken into consideration when modifying the genome with CRISPR/Cas9, and a comprehensive genomic validation is necessary

Molecular pathology of Diamond Blackfan anaemia

Diamond-Blackfan anaemia (DBA) is one of a rare group of genetic disorders known as ‘inherited bone marrow failure (IBMF) syndromes with bone marrow failure, birth defects and higher propensity to cancer. It is arare autosomal dominant disorder with an incidence of 7 in 106 newborns, DBA is characterized by a defect in erythroid lineage development and a quantitative as well as a qualitative defect in erythroid progenitors. This disorder is inherited in 45% of cases and emerges as de novo in the remaining cases. Diagnosis of DBA is challenging as it is based on several clinical features shared by other IBMF syndromes. Diagnosis of this disorder has remained for many years a challenge. In recent years, the use of Next generation sequencing (NGS) of the 83 ribosomal protein (RP) genes has greatly improved the diagnosis and the future prospect of managing the disease. This new diagnostic approach provides several advantages over other conventional and classic approaches, last but not least that now over 65-80% of DBA cases have been identified to carry a genetic mutation of one of the 83 RP genes, while only few could be analyzed until recently. It also facilitates the analysis of DBA family members to facilitate both the selection of possible donors for transplant and to distinguish inherited from de novo mutations. RP gene mutations leading to reduced amounts of RPs (affecting both the 40S and 60S ribosomal subunits) interfere with the processing of rRNA. Until recently the different rRNA species in DBA patients could only be analyzed using Northern blot technique, with several limitations mainly due to the poor yield of RNA in these patients. To resolve some of these problems, in this study we designed primers specific for 32s rRNA intermediate, 18s, 28s, and 5s rRNA. We studied the relative expression of these genes in DBA compared to healthy controls. We studied rRNA defect in a cohort of forty-eight DBA patients from resting and stimulated T cells. rRNA profile study showed a significant difference in the resting and stimulated T cells from DBA patients compared to controls. We then applied CRISPR CAS 9 technology, electroporation, flowcytometry, and timed cell-sorting to validate two novel heterozygous mutations discovered in our Lab, the RPS17 c.3G>C and RPL11 c.475_476 del AA. We optimized a method to successfully introduce a heterozygous knock in mutation using CRISPR/Cas9 technology in K562 erythroleukemic cell line. Firstly, K562 cells were transfected with LentiCRISPRv2 plasmid that has a specific guide RNA (gRNA) ligated to it using the Amaxa nucleofection system. Seventy-two hours later, single green fluorescent protein (GFP) expressing K562 cells were sorted into 96 wells plates. After three weeks of growing these single clones, they were analyzed and the viability was shown to be 50%. Sanger sequencing was carried out to confirm the presence of heterozygous knock in of the specific mutation. Quantitative real time PCR studies using our designed primers (18S, 28S, 32S and 5.8S rRNA) revealed that both mutations resulted in rRNA processing defect in K562 cell line compared to control. In conclusion, our study lead to the characterization of newly established mutations and we demonstrated that such mutations were responsible and causative of a defect in the production of rRNA in an in vitro model.Open Acces