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CRIS.py: A Versatile and High-throughput Analysis Program for CRISPR-based Genome Editing.
CRISPR-Cas9 technology allows the creation of user-defined genomic modifications in cells and whole organisms. However, quantifying editing rates in pools of cells or identifying correctly edited clones is tedious. Targeted next-generation sequencing provides a high-throughput platform for optimizing editing reagents and identifying correctly modified clones, but the large amount of data produced can be difficult to analyze. Here, we present CRIS.py, a simple and highly versatile python-based program which concurrently analyzes next-generation sequencing data for both knock-out and multiple user-specified knock-in modifications from one or many edited samples. Compared to available NGS analysis programs for CRISPR based-editing, CRIS.py has many advantages: (1) the ability to analyze from one to thousands of samples at once, (2) the capacity to check each sample for multiple sequence modifications, including those induced by base-editors, (3) an output in an easily searchable file format enabling users to quickly sort through and identify correctly targeted clones.
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Multiplexed engineering and precision gene editing in cellular immunotherapy.
The advent of cellular immunotherapy in the clinic has entirely redrawn the treatment landscape for a growing number of human cancers. Genetically reprogrammed immune cells, including chimeric antigen receptor (CAR)-modified immune effector cells as well as T cell receptor (TCR) therapy, have demonstrated remarkable responses across different hard-to-treat patient populations. While these novel treatment options have had tremendous success in providing long-term remissions for a considerable fraction of treated patients, a number of challenges remain. Limited in vivo persistence and functional exhaustion of infused immune cells as well as tumor immune escape and on-target off-tumor toxicities are just some examples of the challenges which restrain the potency of today's genetically engineered cell products. Multiple engineering strategies are being explored to tackle these challenges.The advent of multiplexed precision genome editing has in recent years provided a flexible and highly modular toolkit to specifically address some of these challenges by targeted genetic interventions. This class of next-generation cellular therapeutics aims to endow engineered immune cells with enhanced functionality and shield them from immunosuppressive cues arising from intrinsic immune checkpoints as well as the hostile tumor microenvironment (TME). Previous efforts to introduce additional genetic modifications into immune cells have in large parts focused on nuclease-based tools like the CRISPR/Cas9 system or TALEN. However, nuclease-inactive platforms including base and prime editors have recently emerged and promise a potentially safer route to rewriting genetic sequences and introducing large segments of transgenic DNA without inducing double-strand breaks (DSBs). In this review, we discuss how these two exciting and emerging fields-cellular immunotherapy and precision genome editing-have co-evolved to enable a dramatic expansion in the possibilities to engineer personalized anti-cancer treatments. We will lay out how various engineering strategies in addition to nuclease-dependent and nuclease-inactive precision genome editing toolkits are increasingly being applied to overcome today's limitations to build more potent cellular therapeutics. We will reflect on how novel information-rich unbiased discovery approaches are continuously deepening our understanding of fundamental mechanisms governing tumor biology. We will conclude with a perspective of how multiplexed-engineered and gene edited cell products may upend today's treatment paradigms as they evolve into the next generation of more potent cellular immunotherapies.
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CRISPR/Cas9 targeted editing of UDP-rhamnose: Rhamnosyltransferase gene decreases its functions in acteoside biosynthesis and pest resistance in Rehmarmia glutinosa.
UDP-rhamnose: rhamnosyltransferases (URTs)in Rehmarmia glutinosa (RgURT1-RgURT4)may catalyze two key downstream steps of acteoside biosynthesis. Moreover, they were identified from Rehmarmia glutinosa and preliminarily characterized, but their bioinformatics analysis and functions remain to be further explored. The present study mainly focused on investigating their bioinformatics function prediction, genotype-dependent expression, and roles for acteoside biosynthesis and pest resistance with CRISPR/Cas9 technology.Some key findings were as follows:they had a low identity but a typical PSPG box of rhamnosyltransferases, belonging to Glycosyltansferase-GTB type superfamily; They could be expressed depending on genotype,but RgURT4 expression is the highest; Based on RgURT4, two sgRNAs were designed and cloned into pBWA(V)HS-zmpl vector to construct a pBWA(V)HS-Cas9-RgURT vector. It was transferred to Rehmarmia glutinosa using Agrobacterium-mediated transformation so that hygromycin-resistant R. glutinosa plants were obtained. Sequencing indicated that CRISPR/Cas9 targeted editing resulted in base replacements in RgURT4,while its expression was decreased among these edited plants; A few of them had yellower leaves with white dots, lower acteoside and a little higher decaffeoylacteoside than WTs; Tetranychus cinnbarinus among them was observed by stereomicroscope. The results demonstrated that CRISPR/Cas9-mediated RgURT4 editing reduced the acteoside content and pest resistance but decaffeoylacteoside content of Rehmarmia glutinosa. This study will contribute to the function analyses of rhamnosyltransferases gene and downstream steps of acteoside biosynthesis as well as its CRISPR-Cas9-based molecular breeding.
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Generating a Heat-Tolerance Mouse Model.
Creating mouse models of human genetic disease (Gurumurthy and Lloyd, Dis Models Mech 12(1):dmm029462, 2019) and livestock trait (Schering et al. Arch Physiol Biochem 121(5):194-205, 2015; Habiela et al. J Gen Virol 95 (Pt 11):2329-2345, 2014) have been proven to be a useful tool for understanding the mechanism behind the phenotypes and fundamental and applied research in livestock. A single base pair deletion of prolactin receptor (PRLR) has an impact on hair morphology phenotypes beyond its classical roles in lactation in cattle, the so-called slick cattle (Littlejohn et al. Nat Commun 5:5861, 2014). Here, we generate a knock-in mouse model by targeting the specific locus of PRLR gene using Cas9-mediated genome editing via homology-directed repair (HDR) in mouse zygotes. The mouse model carrying the identical PRLR mutation in slick cattle may provide a useful animal model to study the pathway of thermoregulation and the mechanism of heat-tolerance in the livestock.
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Broadening prime editing toolkits using RNA-Pol-II-driven engineered pegRNA.
The prime editor is a versatile tool for targeted precise editing to generate point mutations, small insertions, or small deletions in eukaryotes. However, canonical PE3 system is less efficient, notably in primary cells or pluripotent stem cells. Here, we employed RNA polymerase II promoter instead of RNA polymerase III promoter, whose application is limited by specific DNA contexts, to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs) and, together with other optimizations, achieved efficient targeting with poly(T)-containing pegRNAs, as well as combinatorial and conditional genetic editing. We also found simultaneous suppression of both DNA mismatch repair and DNA damage response could achieve efficient and accurate editing in human embryonic stem cells. These findings relieve the restrictions of RNA polymerase III (RNA-Pol-III)-based base editors and broadened the applications of prime editing.
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Glycosylase base editors enable C-to-A and C-to-G base changes.
Current base editors (BEs) catalyze only base transitions (C to T and A to G) and cannot produce base transversions. Here we present BEs that cause C-to-A transversions in Escherichia coli and C-to-G transversions in mammalian cells. These glycosylase base editors (GBEs) consist of a Cas9 nickase, a cytidine deaminase and a uracil-DNA glycosylase (Ung). Ung excises the U base created by the deaminase, forming an apurinic/apyrimidinic (AP) site that initiates the DNA repair process. In E. coli, we used activation-induced cytidine deaminase (AID) to construct AID-nCas9-Ung and found that it converts C to A with an average editing specificity of 93.8% ± 4.8% and editing efficiency of 87.2% ± 6.9%. For use in mammalian cells, we replaced AID with rat APOBEC1 (APOBEC-nCas9-Ung). We tested APOBEC-nCas9-Ung at 30 endogenous sites, and we observed C-to-G conversions with a high editing specificity at the sixth position of the protospacer between 29.7% and 92.2% and an editing efficiency between 5.3% and 53.0%. APOBEC-nCas9-Ung supplements the current adenine and cytidine BEs (ABE and CBE, respectively) and could be used to target G/C disease-causing mutations.
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Split complementation of base editors to minimize off-target edits.
Base editors (BEs) empower the efficient installation of beneficial or corrective point mutations in crop and human genomes. However, conventional BEs can induce unpredictable guide RNA (gRNA)-independent off-target edits in the genome and transcriptome due to spurious activities of BE-enclosing deaminases, and current improvements mostly rely on deaminase-specific mutagenesis or exogenous regulators. Here we developed a split deaminase for safe editing (SAFE) system applicable to BEs containing distinct cytidine or adenosine deaminases, with no need of external regulators. In SAFE, a BE was properly split at a deaminase domain embedded inside a Cas9 nickase, simultaneously fragmenting and deactivating both the deaminase and the Cas9 nickase. The gRNA-conditioned BE reassembly conferred robust on-target editing in plant, human and yeast cells, while minimizing both gRNA-independent and gRNA-dependent off-target DNA/RNA edits. SAFE also substantially increased product purity by eliminating indels. Altogether, SAFE provides a generalizable solution for BEs to suppress off-target editing and improve on-target performance.
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Emerging ethical perspectives in the clustered regularly interspaced short palindromic repeats genome-editing debate.
This paper provides an overview of the ethical issues in the international clustered regularly interspaced short palindromic repeats (CRISPR) genome editing debate from March 2015 to September 2016. We present the regulatory framework for embryo research in the UK, and explain why CRISPR is not a significant break with the past. We discuss the ethical issues arising from CRISPR applications beyond human embryos, namely the use of gene drive-engineered mosquitoes to eradicate diseases, engineering nonhuman animals to harvest organs for human transplant and engineering crops. We discuss the experiments that have demonstrated the technical feasibility of cultivating embryos in vitro for up to 14 days, and possibly beyond this limit, and the ethical issues arising from the proposal to extend the limit beyond 14 days.
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Highly parallel profiling of the activities and specificities of Cas12a variants in human cells.
Several Cas12a variants have been developed to broaden its targeting range, improve the gene editing specificity or the efficiency. However, selecting the appropriate Cas12a among the many orthologs for a given target sequence remains difficult. Here, we perform high-throughput analyses to evaluate the activity and compatibility with specific PAMs of 24 Cas12a variants and develop deep learning models for these Cas12a variants to predict gene editing activities at target sequences of interest. Furthermore, we reveal and enhance the truncation in the integrated tag sequence that may hinder off-targeting detection for Cas12a by GUIDE-seq. This enhanced system, which we term enGUIDE-seq, is used to evaluate and compare the off-targeting and translocations of these Cas12a variants.
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Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation.
Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.
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