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Methods and advances in RNA characterization and design.
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Optimization of CRISPR/Cas System for Improving Genome Editing Efficiency in Plasmodium falciparum.
Studies of molecular mechanisms and related gene functions have long been restricted by limited genome editing technologies in malaria parasites. Recently, a simple and effective genome editing technology, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system, has greatly facilitated these studies in many organisms, including malaria parasites. However, due to the special genome feature of malaria parasites, the manipulation and gene editing efficacy of the CRISPR/Cas system in this pathogen need to be improved, particularly in the human malaria parasite, Plasmodium falciparum. Herein, based on the CRISPR/Cas9 system, we developed an integrating strategy to generate a Cas9i system, which significantly shortened the time for generation of transgenic strains in P. falciparum. Moreover, with this Cas9i system, we have successfully achieved multiplexed genome editing (mutating or tagging) by a single-round transfection in P. falciparum. In addition, we for the first time adapted AsCpf1 (Acidaminococcus sp. Cpf1), an alternative to Cas9, into P. falciparum parasites and examined it for gene editing. These optimizations of the CRISPR/Cas system will further facilitate the mechanistic research of malaria parasites and contribute to eliminating malaria in the future.
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Gene editing strategies to treat lysosomal disorders: The example of mucopolysaccharidoses.
Lysosomal storage disorders are a group of progressive multisystemic hereditary diseases with a combined incidence of 1:4,800. Here we review the clinical and molecular characteristics of these diseases, with a special focus on Mucopolysaccharidoses, caused primarily by the lysosomal storage of glycosaminoglycans. Different gene editing techniques can be used to ameliorate their symptoms, using both viral and nonviral delivery methods. Whereas these are still being tested in animal models, early results of phase I/II clinical trials of gene therapy show how this technology may impact the future treatment of these diseases. Hurdles related to specific hard-to-reach organs, such as the central nervous system, heart, joints, and the eye must be tackled. Finally, the regulatory framework necessary to advance into clinical practice is also discussed.
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Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells.
BACKGROUND: Delivery of CRISPR/Cas RNPs to target cells still remains the biggest bottleneck to genome editing. Many efforts are made to develop efficient CRISPR/Cas RNP delivery methods that will not affect viability of target cell dramatically. Popular current methods and protocols of CRISPR/Cas RNP delivery include lipofection and electroporation, transduction by osmocytosis and reversible permeabilization and erythrocyte-based methods. METHODS: In this study we will assess the efficiency and optimize current CRISPR/Cas RNP delivery protocols to target cells. We will conduct our work using molecular cloning, protein expression and purification, cell culture, flow cytometry (immunocytochemistry) and cellular imaging techniques. DISCUSSION: This will be the first extensive comparative study of popular current methods and protocols of CRISPR/Cas RNP delivery to human cell lines and primary cells. All protocols will be optimized and characterized using the following criteria i) protein delivery and genome editing efficacy; ii) viability of target cells after delivery (post-transduction recovery); iii) scalability of delivery process; iv) cost-effectiveness of the delivery process and v) intellectual property rights. Some methods will be considered 'research-use only', others will be recommended for scaling and application in the development of cell-based therapies.
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Negative DNA supercoiling induces genome-wide Cas9 off-target activity.
CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.
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Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans.
CRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (≤200 bp) are used to insert the 5' and 3' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a double-stranded DNA (dsDNA) donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal CRISPR RNAs (crRNAs) and PCR products. We have also used Nested CRISPR in a nonessential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic single guide RNAs (sgRNAs), but did not observe a significant increase in efficiency. To streamline the approach, we combined all step 1 and step 2 reagents in a single injection and were successful in three of five loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.
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CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes.
Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.
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CRISPR-Cas experiments for schools and the public.
The "gene scissors" CRISPR-Cas currently revolutionize the field of molecular biology with an enormous impact on society due to the broad application potentials in biomedicine, biotechnology and agriculture. We have developed simple CRISPR-Cas experiments that can serve to introduce pupils, students and non-scientists alike to the fascinating power of targeted gene editing. The experimental course is divided into two parts. In part 1, we target plasmid borne lacZ to convert blue E. coli to white E. coli. In part 2, we analyse the CRISPR-Cas9 mediated double strand breaks in the lacZ gene by a) colony PCR, b) colony cracking gel or c) restriction digest of the plasmids. Experimental work is embedded in short theoretical lecture parts that provide background of CRISPR-Cas and a step-by-step tutorial for the practical work. Though the experiment is robust, inexpensive and simple it should be noted that guidance by an expert instructor is required. Based on our experience, a full day lab course has a positive influence on the participants' attitude towards research in general. This is true for high school students as well as non-scientists (age groups 16-70 years).
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Targeted genomic translocations and inversions generated using a paired prime editing strategy.
A variety of cancers have been found to have chromosomal rearrangements, and the genomic abnormalities often induced expression of fusion oncogenes. To date, a pair of engineered nucleases including ZFNs, TALENs, and CRISPR-Cas9 nucleases have been used to generate chromosomal rearrangement in living cells and organisms for disease modeling. However, these methods induce unwanted indel mutations at the DNA break junctions, resulting in incomplete disease modeling. Here, we developed prime editor nuclease-mediated translocation and inversion (PETI), a method for programmable chromosomal translocation and inversion using prime editor 2 nuclease (PE2 nuclease) and paired pegRNA. Using PETI method, we successfully introduced DNA recombination in episomal fluorescence reporters as well as precise chromosomal translocations in human cells. We applied PETI to create cancer-associated translocations and inversions such as NPM1-ALK and EML4-ALK in human cells. Our findings show that PETI generated chromosomal translocation and inversion in a programmable manner with efficiencies comparable of Cas9. PETI methods, we believe, could be used to create disease models or for gene therapy.
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TP53-dependent toxicity of CRISPR/Cas9 cuts is differential across genomic loci and can confound genetic screening.
CRISPR/Cas9 gene editing can inactivate genes in a precise manner. This process involves DNA double-strand breaks (DSB), which may incur a loss of cell fitness. We hypothesize that DSB toxicity may be variable depending on the chromatin environment in the targeted locus. Here, by analyzing isogenic cell line pair CRISPR experiments jointly with previous screening data from across ~900 cell lines, we show that TP53-associated break toxicity is higher in genomic regions that harbor active chromatin, such as gene regulatory elements or transcription elongation histone marks. DSB repair pathway choice and DNA sequence context also associate with toxicity. We also show that, due to noise introduced by differential toxicity of sgRNA-targeted sites, the power of genetic screens to detect conditional essentiality is reduced in TP53 wild-type cells. Understanding the determinants of Cas9 cut toxicity will help improve design of CRISPR reagents to avoid incidental selection of TP53-deficient and/or DNA repair deficient cells.
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