gRNA Target Research Articles

Oncogenic Cooperation Between IL7R-JAK-STAT Pathway Mutations.

Oncogenic Cooperation Between IL7R-JAK-STAT Pathway Mutations.

Distributed hybrid-indexing of compressed pan-genomes for scalable and fast sequence alignment.

Computational pan-genomics utilizes information from multiple individual genomes in large-scale comparative analysis. Genetic variation between case-controls, ethnic groups, or species can be discovered thoroughly using pan-genomes of such subpopulations. Whole-genome sequencing (WGS) data volumes are growing rapidly, making genomic data compression and indexing methods very important. Despite current space-efficient repetitive sequence compression and indexing methods, the deployed compression methods are often sequential, computationally time-consuming, and do not provide efficient sequence alignment performance on vast collections of genomes such as pan-genomes. For performing rapid analytics with the ever-growing genomics data, data compression and indexing methods have to exploit distributed and parallel computing more efficiently. Instead of strict genome data compression methods, we will focus on the efficient construction of a compressed index for pan-genomes. Compressed hybrid-index enables fast sequence alignments to several genomes at once while shrinking the index size significantly compared to traditional indexes. We propose a scalable distributed compressed hybrid-indexing method for large genomic data sets enabling pan-genome-based sequence search and read alignment capabilities. We show the scalability of our tool, DHPGIndex, by executing experiments in a distributed Apache Spark-based computing cluster comprising 448 cores distributed over 26 nodes. The experiments have been performed both with human and bacterial genomes. DHPGIndex built a BLAST index for n = 250 human pan-genome with an 870:1 compression ratio (CR) in 342 minutes and a Bowtie2 index with 157:1 CR in 397 minutes. For n = 1,000 human pan-genome, the BLAST index was built in 1520 minutes with 532:1 CR and the Bowtie2 index in 1938 minutes with 76:1 CR. Bowtie2 aligned 14.6 GB of paired-end reads to the compressed (n = 1,000) index in 31.7 minutes on a single node. Compressing n = 13,375,031 (488 GB) GenBank database to BLAST index resulted in CR of 62:1 in 575 minutes. BLASTing 189,864 Crispr-Cas9 gRNA target sequences (23 MB in total) to the compressed index of human pan-genome (n = 1,000) finished in 45 minutes on a single node. 30 MB mixed bacterial sequences were (n = 599) were blasted to the compressed index of 488 GB GenBank database (n = 13,375,031) in 26 minutes on 25 nodes. 78 MB mixed sequences (n = 4,167) were blasted to the compressed index of 18 GB E. coli sequence database (n = 745,409) in 5.4 minutes on a single node.

Efficient multiplexed gene regulation in Saccharomyces cerevisiae using dCas12a.

CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for Saccharomyces cerevisiae and systematically evaluate its design features. This includes the role of the NLS position, use of repression domains, and the position of the gRNA target. Our optimal system is comprised of dCas12a E925A with a single C-terminal NLS and a Mxi1 or a MIG1 repression domain, which enables up to 97% downregulation of a reporter gene. We also extend this system to allow for inducible regulation via an RNAP II-controlled promoter, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous β-carotene pathway. Together these findings offer valuable insights into the design constraints of dCas12a-based CRISPRi and enable new avenues for flexible and efficient gene regulation in S. cerevisiae.

Highly Efficient Temperature Inducible CRISPR-Cas9 Gene Targeting in Drosophila suzukii.

The spotted-wing Drosophila (Drosophila suzukii Matsumura) is native to eastern Asia, but has become a global threat to fruit production. In recent years, CRISPR/Cas9 targeting was established in this species allowing for functional genomic and genetic control studies. Here, we report the generation and characterization of Cas9-expressing strains of D. suzukii. Five independent transgenic lines were generated using a piggyBac construct containing the EGFP fluorescent marker gene and the Cas9 gene under the control of the D. melanogaster heat shock protein 70 promoter and 3’UTR. Heat-shock (HS) treated embryos were analyzed by reverse transcriptase PCR, revealing strong heat inducibility of the transgenic Cas9 expression. By injecting gRNA targeting EGFP into one selected line, 50.0% of G0 flies showed mosaic loss-of-fluorescence phenotype, and 45.5% of G0 flies produced G1 mutants without HS. Such somatic and germline mutagenesis rates were increased to 95.4% and 85.7%, respectively, by applying a HS. Parental flies receiving HS resulted in high inheritance of the mutation (92%) in their progeny. Additionally, targeting the endogenous gene yellow led to the lack of pigmentation and male lethality. We discuss the potential use of these efficient and temperature-dependent Cas9-expressing strains for the genetic studies in D. suzukii.

Population modification strategies for malaria vector control are uniquely resilient to observed levels of gene drive resistance alleles.

Cas9/guide RNA (gRNA)-based gene drive systems are expected to play a transformative role in malaria elimination efforts., whether through population modification, in which the drive system contains parasite-refractory genes, or population suppression, in which the drive system induces a severe fitness load resulting in population decline or extinction. DNA sequence polymorphisms representing alternate alleles at gRNA target sites may confer a drive-resistant phenotype in individuals carrying them. Modeling predicts that, for observed levels of SGV at potential target sites and observed rates of de novo DRA formation, population modification strategies are uniquely resilient to DRAs. We conclude that gene drives can succeed when fitness costs incurred by drive-carrying mosquitoes are low enough to prevent strong positive selection for DRAs produced de novo or as part of the SGV and that population modification strategies are less prone to failure due to drive resistance.

CRISPR/Cas13d-mediated efficient KDM5B mRNA knockdown in porcine somatic cells and parthenogenetic embryos.

An efficient mRNA knockdown strategy is needed to explore gene function in cells and embryos, especially to understand the process of maternal mRNA decay during early embryo development. Cas13, a novel RNA-targeting CRISPR effector protein, could bind and cleave complementary single-strand RNA, which has been employed for mRNA knockdown in mouse and human cells and RNA-virus interference in plants. Cas13 has not yet been reported to be used in pigs. In the current study, we explored the feasibility of CRISPR/Cas13d-mediated endogenous RNA knockdown in pigs. KDM5B, a histone demethylase of H3K4me3, was downregulated at the transcriptional level by 50% with CRISPR/Cas13d in porcine fibroblast cells. Knockdown of KDM5B-induced H3K4me3 expression and decreased the abundance of H3K27me3, H3K9me3, H3K4ac, H4K8ac, and H4K12ac. These changes affected cell proliferation and cell cycle. Furthermore, stable integration of the CRISPR/Cas13d system into the porcine genome resulted in the continuous expression of Cas13d and persistent knockdown of KDM5B. Finally, the RNA-targeting potential of Cas13d was further validated in porcine parthenogenetic embryos. By microinjection of Cas13d mRNA and gRNA targeting KDM5B into porcine oocytes, the expression of KDM5B was downregulated, the abundance of H3K4me3 increased as expected, and the expression of embryonic development-related genes was changed accordingly. These results indicate that CRISPR/Cas13d provides an easily programmable platform for spatiotemporal transcriptional manipulation in pigs.

DEVELOPMENT OF A CRISPR-BASED ANTIVIRAL SYSTEM AGAINST BOHV-1

<h3>Background</h3> Bovine Herpesvirus type 1 (BoHV-1) infection has no cure and infected animals become lifelong carriers, greatly impacting health and productivity of herds worldwide. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas (CRISPR associated) tool has been widely explored for the development of antiviral strategies due to its ability to precisely edit genetic sequences. In this manner, we have designed a CRISPR-based antiviral system against BoHV-1 through the tetracycline-inducible expression of the Cas9 nuclease and a guide RNA (gRNA) homologous to a viral sequence in genetically modified bovine cells. <h3>Methods</h3> The in silico design of the antiviral system was based on the literature. BoHV-1 and bovine (Bos taurus taurus) genome were obtained from GenBank and basic molecular vectors from Addgene. Sequence analysis of appropriate targets were performed with Snapgene and Serial Cloner. Primers for cloning were obtained using the NCBI tool Primer BLAST. The gRNA for bovine DNA cleavage was selected from literature considering cleavage efficiency and potential off-target effects. The gRNA for the viral genome cleavage was designed using the CRISPR gRNA design tool from ATUM and CHOPCHOP. <h3>Results</h3> The developed molecular vector allows the delivery of the necessary machinery to perform a cleavage in the chromosome 25 of the bovine DNA between the FSCN1 and the ACTB genes, where the sequence of the antiviral mechanism will be inserted by homologous recombination (HR). The cleavage site has already been reported as safe for the insertion of sequences into the bovine genome. The HR fragment has the Cas9 nuclease sequence and a tetracycline-inducible expression system capable of conditioning the expression of the nuclease gene to the presence of the antibiotic. In addition, the fragment has a sequence for the expression of a gRNA targeting an essential sequence for virus replication, the UL52 gene. The expressed Cas9 enzyme and gRNA form a ribonucleoprotein complex capable of cleaving only the virus genome leading to its inactivation and preventing its replication in the genetically modified cells stimulated by tetracycline. <h3>Conclusion</h3> The mechanism and elements developed explore new applications of the CRISPR-Cas tool to combat viral diseases that currently have no cure. The ease of altering the gRNA target makes the proposed system highly flexible and can possibly be applied to different diseases caused by viruses.

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

BackgroundThe homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells.ResultsBy introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template.ConclusionThe allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template.This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

Calmodulinopathy Correction using CRISPR Interference in a Cardiac Microtissue Model

Calmodulinopathies, caused by mutations in calmodulin (CaM), a ubiquitous Ca2+ sensor, can lead to life-threatening cardiac arrhythmias, such as long QT syndrome (LQTS). In this study, we created a calmodulinopathy model in 3D cardiac microtissues (CMTs), using cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs) with the D130G-CALM2 mutation, and used CRISPR interference (CRISPRi) to rescue the disease phenotype. Lentiviruses that encode a guide RNA (gRNA) targeting CALM2 (both wildtype and mutant) and a Krüppel associated box (KRAB) transcriptional repressor were transduced to CMTs and their electrophysiological and mechanical properties were analyzed. Results show that D130G-CALM2 CMTs reproduced key features of calmodulinopathy associated LQTS, including prolongation of action potential duration (APD), electrical alternans and longer pacing cycle lengths at which loss of 1:1 capture occurred compared to uncorrected mutant CMTs. After transduction with CALM2 gRNA and KRAB lentivirus, we observed significant knockdown of CALM2 mRNA, shortened APD, increased loading rate during contraction and relaxation, and decreased time to peak and relaxation time over a range of pacing frequencies in CMTs. In conclusion, by demonstrating correction of disease electrophysiological and mechanical phenotype by CRISPRi in 3D tissue models, this study provides a proof of concept for the utility of using viral transduction for CRISPRi in cardiac muscle to correct the tissue phenotype.

96 Validation of the candidate mutation responsible for embryonic lethality in Holstein haplotype 2 carriers

Holstein haplotype 2 (HH2) is embryonic lethal and carried by 1.21% of the US Holstein population. Using next-generation sequencing, we identified a high-impact frameshift mutation in intraflagellar protein 80 (IFT80) as the putative causal mutation. In bovine embryos, IFT80 expression begins at the 8-cell stage and decreases by the blastocyst stage. We hypothesised that the loss of function of IFT80 early in development causes the lethal phenotype. The aim of this study was to mimic the mutation observed invivo using a CRISPR-Cas9 approach to determine its effect on embryo development. Two guide RNAs (gRNAs) were designed to disrupt exon 11 (Ex11), one before and one after the known IFT80 mutation site, creating a 317-nucleotide (nt) cut to facilitate genotyping. Then, gRNAs annealed to a tracr-Cas9mRNA complex were delivered to 1-cell embryos by microinjection. Each replicate contained control embryos injected with only Cas9mRNA and treated embryos injected with gRNAs targeting IFT80. Embryos from each group were collected at the 8-cell stage for genotyping and gene expression analysis (n=47), or on Day 8 to validate genotypes of embryos left to develop (n=50). DNA sequences containing gRNA target sequences were amplified and visualised on an agarose gel. IFT80 expression was determined in biallelic embryos (n=13) using quantitative PCR and normalized to GAPDH. Primers were designed for the transcript regions before and after gRNAs target sequences, exons 9 and 12, respectively. Expression data were analysed using SAS software (v. 9.4; SAS Institute Inc.) using PROC GLM and LSMEANS to determine expression differences. Biallelic samples (n=9) were Sanger-sequenced (SS) and aligned with the reference sequence to determine exact cut sites. Protein amino acid (AA) sequences were predicted using SS data. Protein models were constructed using the I-Tasser platform, and then aligned and visualised using PyMol 2.4. Biallelic edits showed a significant decrease in exon 12 expression (P&amp;lt;0.05), and no difference in exon 9 compared with controls (P&amp;gt;0.05), indicating that the transcript was severely affected downstream of the edited sites. The reference protein model contained 777 AA, whereas the biallelic sample with the most accurate cut sites yielded a 385-AA protein, indicating that the mutation severely altered protein conformation and possible function. Embryos injected with CRISPR-Cas9 targeting Ex11 arrested at the 8-cell stage and failed to form blastocysts. Day 8 embryos were genotyped (n=24) and 58% were biallelic, 21% were monoallelic, and 21% appeared wild-type. Given the high rate of edits, the observed embryonic arrest is likely due to disruption of IFT80, and wild-type embryos may contain small edits not visible by gel. In conclusion, generation of CRISPR-Cas9 IFT80 knockouts demonstrated that the frameshift mutation in Ex11 results in a seemingly nonfunctional protein that is responsible for the embryonic lethality seen in HH2 carriers. Future research is needed to determine how IFT80 regulates embryonic development. This research was supported by USDA-NIFA National Needs Fellowship, USDA-NIFA AFRI Grant No. 2019-67015-28998.

A catalogue of biochemically diverse CRISPR-Cas9 orthologs

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Abstract 1826: CRISPR mediated KRAS mutant gene editing in pancreatic and colorectal cancer therapy

Abstract Background: KRASG12V and KRASG12D mutations are among the most commonly observed mutations in pancreatic (30% and 51%) and colorectal (27% and 45%) adenocarcinomas and have been associated with poor prognosis. Activating mutations in KRAS play potent roles in cancer initiation, propagation, and maintenance. Although they represent important therapeutic targets, no effective treatment for direct inhibition of KRASG12V and KRASG12D mutants have been reported. We have developed a new potent strategy combing the potency of CRISPR for gene editing with a tumor selective nanocarrier, to impair cancer cell proliferation by directly targeting G12V and G12D mutations of the KRAS oncogene. Methods: ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with CRISPR/Cas9. ADGN nanoparticles containing Cas9mRNA and gRNA targeting either KRASG12D or KRASG12V were evaluated on pancreas (PANC1, PK-45H) and colorectal (LS513, SW480, SW403) cancer cells harboring KRASG12D or KRASG12V mutations. KRAS mutant indel frequency was evaluated by T7E1 method and compared to KRASWT (HT29, HS-69) cells. Cell proliferation was determined by flow cytometry. In-vivo efficacy of IV-administered ADGN/mRNACas9/gRNA complexes was evaluated in PANC1-LUC (KRASG12D) and SW403 (KRASG12V) mouse xenografts. The phosphorylation of ERK and AKT was measured by western blot, KRAS mutation level in the tumor and variation of housekeeping genes were estimated by deep sequencing. Results: We have developed highly specific gRNAs targeting KRASG12V and KRASG12D mutants complexed with ADGN-nanoparticles. These complexes efficiently and selectively silenced KRASG12V and KRASG12D in colorectal and pancreatic cancer cells in vitro and in vivo. Silencing KRAS mutations reduced the cell proliferation by 70% and the phosphorylation of the downstream effector pathways ERK and AKT. ADGN-nanoparticles containing CRISPR/gRNA targeting KRASG12D abolished PANC1 tumor growth and induced KRASG12D gene editing in the tumor with an indel frequency of 76%. ADGN-nanoparticles containing CRISPR/gRNA targeting KRASG12V abolished SW403 tumor growth and induced KRASG12V gene editing in the tumor with an indel frequency of 82%. In contrast, no effect on tumor growth were observed with ADGN-nanoparticles containing non-specific gRNA or gRNA targeting other KRAS mutations. ADGN/mRNACas9/gRNA targeting KRASG12D or KRASG12V abolished pancreatic and colorectal tumor growth respectively in vivo, without inducing any significant toxicity, emergence of off target effects or other KRAS mutations. Conclusions: ADGN-nanoparticle containing mRNACas9/gRNA targeting KRASG12D or KRASG12V were effective in selectively targeting mutated KRAS both in vitro and in vivo. Our study provides a proof-of-concept that ADGN/CRISPR can be applied to target driver mutations of cancers in vivo and permanently disrupt the oncogenic alleles, leading to inhibition of tumor growth. We proposed a new strategy for targeting KRAS oncogenes that can be useful in treatment of pancreatic and colorectal cancers. Citation Format: Gilles Divita, Elodie Czuba, Melanie Guidetti, Audrey Gunenberger, Leslie Tempremant, Veronique Josserand, Neil Desai. CRISPR mediated KRAS mutant gene editing in pancreatic and colorectal cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1826.

Abstract 6137: Development of ARID1A-deficient hepatocellular carcinoma in transgenic pigs

Abstract Hepatocellular carcinoma (HCC) is an aggressive disease with poor response to treatment at advanced stages. Frameshift and nonsense mutations in ARID1A, a subunit of the SWI/SNF chromatin-remodeling complex, are among the most frequent genetic alterations in human HCC. Hence, it is necessary to develop animal models for preclinical assessment of targeted therapies for HCC with ARID1A deficiency. Due to the remarkable physiologic, genetic, and size similarities between pigs and humans, porcine models are excellent candidates for translational HCC studies. Here, we generated a porcine HCC model with ARID1A deficiency using the Oncopig, a transgenic pig that harbors heterozygous Cre recombinase-inducible TP53R167H and KRASG12D transgenes. Porcine HCC cells were developed by transduction of hepatocytes isolated from four Oncopigs with adenoviral vector encoding Cre recombinase. TP53R167H and KRASG12D expression in the developed HCC cell lines was confirmed by RT-PCR, and the hepatocyte origin of the cells was validated by positive arginase-1 staining. To disrupt ARID1A expression, Oncopig HCC cell lines were transfected with recombinant Cas9 nuclease complexed with a gRNA targeting ARID1A. This resulted in &amp;gt;85% ARID1A editing in each of the four cell lines as determined by targeted Illumina sequencing. Single cell clones were isolated and screened for ARID1A mutations. The functional effect of ARID1A deficiency was evaluated in ARID1A-edited cell pools and ARID1A knockout single cell clones. Consistent with its reported tumor suppressor role, deficiency of ARID1A increased Oncopig HCC cell proliferation as measured by MTS assays. Following in vitro characterization, ARID1A-deficient Oncopig HCC cells were injected autologously into subcutaneous sites in Oncopig flanks, resulting in development of masses measuring 1-2 cm within two weeks. These subcutaneous masses were excised and fragmented, and 2 - 3 fragments were engrafted into the liver of the same animal by ultrasound-guided injection. Development of intrahepatic masses was detected by ultrasound imaging as well as CT scans. Following euthanasia of the Oncopigs, the intrahepatic masses were collected for histological and genomic analyses. Subcutaneous and intrahepatic masses were histologically characterized as HCC, and CRISPR-Cas9 mediated ARID1A deficiency was confirmed by targeted Illumina sequencing. To conclude, we have developed a novel large animal model of ARID1A-deficient HCC and demonstrated the feasibility of generating genetically-tailored HCC tumors in Oncopigs. These translational HCC models are promising tools for testing precision medicine approaches and investigating the contribution of clinically-relevant driver mutations on treatment susceptibility. Citation Format: Lobna Elkhadragy, William M. Totura, Kimia Dasteh Goli, Lawrence B. Schook, Ron C. Gaba, Kyle M. Schachtschneider. Development of ARID1A-deficient hepatocellular carcinoma in transgenic pigs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6137.

Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements

In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we lack predictive rules for designing effective gRNA target sites. Here, we identify multiple features of bacterial promoters that impose stringent requirements on CRISPRa target sites. Notably, we observe narrow, 2–4 base windows of effective sites with a periodicity corresponding to one helical turn of DNA, spanning ~40 bases and centered ~80 bases upstream of the TSS. However, we also identify two features suggesting the potential for broad scope: CRISPRa is effective at a broad range of σ70-family promoters, and an expanded PAM dCas9 allows the activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.

Sequential i-GONAD: An Improved In Vivo Technique for CRISPR/Cas9-Based Genetic Manipulations in Mice.

Improved genome-editing via oviductal nucleic acid delivery (i-GONAD) is a technique capable of inducing genomic changes in preimplantation embryos (zygotes) present within the oviduct of a pregnant female. i-GONAD involves intraoviductal injection of a solution containing genome-editing components via a glass micropipette under a dissecting microscope, followed by in vivo electroporation using tweezer-type electrodes. i-GONAD does not involve ex vivo handling of embryos (isolation of zygotes, microinjection or electroporation of zygotes, and egg transfer of the treated embryos to the oviducts of a recipient female), which is required for in vitro genome-editing of zygotes. i-GONAD enables the generation of indels, knock-in (KI) of ~ 1 kb sequence of interest, and large deletion at a target locus. i-GONAD is usually performed on Day 0.7 of pregnancy, which corresponds to the late zygote stage. During the initial development of this technique, we performed i-GONAD on Days 1.4–1.5 (corresponding to the 2-cell stage). Theoretically, this means that at least two GONAD steps (on Day 0.7 and Day 1.4–1.5) must be performed. If this is practically demonstrated, it provides additional options for various clustered regularly interspaced palindrome repeats (CRISPR)/Caspase 9 (Cas9)-based genetic manipulations. For example, it is usually difficult to induce two independent indels at the target sites, which are located very close to each other, by simultaneous transfection of two guide RNAs and Cas9 protein. However, the sequential induction of indels at a target site may be possible when repeated i-GONAD is performed on different days. Furthermore, simultaneous introduction of two mutated lox sites (to which Cre recombinase bind) for making a floxed allele is reported to be difficult, as it often causes deletion of a sequence between the two gRNA target sites. However, differential KI of lox sites may be possible when repeated i-GONAD is performed on different days. In this study, we performed proof-of-principle experiments to demonstrate the feasibility of the proposed approach called “sequential i-GONAD (si-GONAD).”

Are the current gRNA ranking prediction algorithms useful for genome editing in plants?

Introducing a new trait into a crop through conventional breeding commonly takes decades, but recently developed genome sequence modification technology has the potential to accelerate this process. One of these new breeding technologies relies on an RNA-directed DNA nuclease (CRISPR/Cas9) to cut the genomic DNA, in vivo, to facilitate the deletion or insertion of sequences. This sequence specific targeting is determined by guide RNAs (gRNAs). However, choosing an optimum gRNA sequence has its challenges. Almost all current gRNA design tools for use in plants are based on data from experiments in animals, although many allow the use of plant genomes to identify potential off-target sites. Here, we examine the predictive uniformity and performance of eight different online gRNA-site tools. Unfortunately, there was little consensus among the rankings by the different algorithms, nor a statistically significant correlation between rankings and in vivo effectiveness. This suggests that important factors affecting gRNA performance and/or target site accessibility, in plants, are yet to be elucidated and incorporated into gRNA-site prediction tools.

Marek’s disease virus as a CRISPR/Cas9 delivery system to defend against avian leukosis virus infection in chickens

The CRISPR/CRISPR-associated protein 9 (Cas9) system is a powerful gene-editing tool originally discovered as an integral mediator of bacterial adaptive immunity. Recently, this technology has been explored for its potential utility in providing new and unique treatments for viral infection. Marek’s disease virus (MDV) and avian leukosis virus subgroup J (ALV-J), major immunosuppressive viruses, cause significant economic losses to the chicken industry. Here, we evaluated the efficacy of using MDV as a CRISPR/Cas9-delivery system to directly target and disrupt the reverse-transcribed products of the ALV-J RNA genome during its infection cycle in vitro and in vivo. We first screened multiple potential guide RNA (gRNA) target sites in the ALV-J genome and identified several optimized targets capable of effectively disrupting the latently integrated viral genome and providing efficient defense against new infection by ALV-J in cells. The optimal single-gRNAs and Cas9-expression cassettes were inserted into the genome of an MDV vaccine strain. The results indicated that engineered MDV stably expressing ALV-J-targeting CRISPR/Cas9 efficiently resisted ALV-J challenge in host cells. These findings demonstrated the CRISPR/Cas9 system as an effective treatment strategy against ALV-J infection. Furthermore, the results highlighted the potential of MDV as an effective delivery system for CRISPR/Cas9 in chickens.

Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts.

We present a new approach to edit both mitochondrial and chloroplast genomes. Organelles have been considered off-limits to CRISPR due to their impermeability to most RNA and DNA. This has prevented applications of Cas9/gRNA-mediated genome editing in organelles while the tool has been widely used for engineering of nuclear DNA in a number of organisms in the last several years. To overcome the hurdle, we designed a new approach to enable organelle genome editing. The plasmids, designated “Edit Plasmids,” were constructed with two expression cassettes, one for the expression of Cas9, codon-optimized for each organelle, under promoters specific to each organelle, and the other cassette for the expression of guide RNAs under another set of promoters specific to each organelle. In addition, Edit Plasmids were designed to carry the donor DNA for integration between two double-strand break sites induced by Cas9/gRNAs. Each donor DNA was flanked by the regions homologous to both ends of the integration site that were short enough to minimize spontaneous recombination events. Furthermore, the donor DNA was so modified that it did not carry functional gRNA target sites, allowing the stability of the integrated DNA without being excised by further Cas9/gRNAs activity. Edit Plasmids were introduced into organelles through microprojectile transformation. We confirmed donor DNA insertion at the target sites facilitated by homologous recombination only in the presence of Cas9/gRNA activity in yeast mitochondria and Chlamydomonas chloroplasts. We also showed that Edit Plasmids persist and replicate in mitochondria autonomously for several dozens of generations in the presence of the wild-type genomes. Finally, we did not find insertions and/or deletions at one of the Cas9 cleavage sites in Chloroplasts, which are otherwise hallmarks of Cas9/gRNA-mediated non-homologous end joining (NHEJ) repair events in nuclear DNA. This is consistent with previous reports of the lack of NHEJ repair system in most bacteria, which are believed to be ancestors of organelles. This is the first demonstration of CRISPR-mediated genome editing in both mitochondria and chloroplasts in two distantly related organisms. The Edit Plasmid approach is expected to open the door to engineer organelle genomes of a wide range of organisms in a precise fashion.

Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA targeting Myostatin (MSTN) genes on the development and gene editing of porcine zygotes.

This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off-target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off-target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non-specific cleavage of off-target sites.