gRNA Library Research Articles

CAR T Cell Cytotoxicity Is Dependent on Death Receptor-Driven Apoptosis

▪Background: T cells engineered to express chimeric antigen receptors targeting the B-cell antigen CD19 (CART19) have demonstrated impressive results in the treatment of lymphoid cancers. Despite these promising outcomes, a significant subset of patients relapse after initial response. To investigate the molecular pathways that drive relapse, we performed an unbiased, CRISPR/Cas9-mediated genome-wide knockout screen in the acute lymphoblastic leukemia (ALL) cell line Nalm6, and found that loss of CD19 was the primary driver of relapse after initial response. This finding is consistent with clinical observations that antigen loss is a primary driver of late disease recurrence, however it fails to address the molecular etiology of intrinsic resistance, which affects ~50% of patients with non-Hodgkin lymphoma and ~20% of patients with ALL, or of late antigen-independent relapse, which accounts for ~40% of relapses in ALL. Identification of the mechanisms regulating CART19 susceptibility is an essential first step in overcoming resistance to this powerful therapy. We hypothesized that genetic alteration in ALL cells were responsible for mediating intrinsic, CD19-independent resistance. To investigate this, we conducted a genome-wide loss of function screen in a model designed to evaluate intrinsic resistance to CART19.Methods: Using a lentiviral guide RNA (gRNA) library containing four distinct gRNAs targeting each human gene (~80,000 gRNAs total), we enabled genome-wide knockout in Nalm6, whereby each target cell lost function of only one gene. This gene-modified cell pool was then exposed to either CART19 or control T cells at a low effector:target ratio (0.25:1) to model the expected in vivo E:T ratio. At 24h, surviving Nalm6 cells were collected and gRNA from these cells underwent next-generation sequencing. Sequenced samples were processed using three distinct genome-scale knockout screen algorithms (MAGeCK, permutation-based non-parametric analysis and ScreenBeam). This pipeline allowed identification of (i) significantly enriched gRNA, postulated to mediate loss of gene function that confers resistance to CART19, and (ii) significantly depleted guides, postulated to mediate loss of gene function that confers sensitivity to CART19. The role of identified genes was then validated in in vitro and in vivo studies.Results: Analysis of gRNA sequencing data from our screen (Figure 1) revealed that the three genes whose loss of function most significantly promoted resistance to CART19 were BID, FADD and CASP8, all of which are key regulators of death receptor-driven apoptosis. TNFRSF10B, encoding the death receptor TRAIL-R2, was also significantly enriched. Interestingly, amongst the 10 genes whose loss most significantly sensitized to CART19 were TRAF2, BIRC2 and CFLAR, all negative regulators of death receptor activity. Pathway analysis of the top 50 genes (25 enriched, 25 depleted) demonstrated significant enrichment in the death receptor pathway, with a false discovery rate of 3.79x10-7.We proceeded to functionally validate the role of BID and FADD in mediating resistance to CART19 by deleting these genes in Nalm6 using de novo designed gRNAs. Strikingly, BIDKO and FADDKO cells were highly resistant to CART19 cytotoxicity in vitro as compared to wild-type Nalm6. Resistance was evident as early as 6 hours after co-culture and was maintained for at least 7 days. Observed resistance to CART19 directly correlated to fraction of KO cells present, suggesting that gene loss was mechanistically responsible for failed CART19 cytotoxicity. We further evaluated the impact of BID or FADD loss on anti-leukemic activity of CART19 in our Nalm6 xenograft model. We observed that BIDKO or FADDKO significantly impaired the anti-leukemic activity of CART19 in vivo.Conclusions: CART19 can cure select patients with B-cell cancers, while others experience transient or no clinical benefit. Using a genome-wide loss of function screen, we identified that death receptor-associated proteins are centrally involved in regulating CART19 cytotoxicity, and that loss of these molecules leads to intrinsic resistance to CART19. These findings are, to our knowledge, the first to characterize the role of death receptors as critical regulators of CART19 cytotoxicity, and suggest that tumor cell modulation of death receptor signaling may drive both inherent resistance and antigen-independent relapse. DisclosuresJune:Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Gill:Novartis: Research Funding; Carisma Therapeutics: Equity Ownership; Extellia: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ruella:University of Pennsylvania: Patents & Royalties.

Identification of Genetic Pathways Controlling Resistance to Standard Combination Chemotherapy in Acute Myeloid Leukemia

Introduction: Resistance to chemotherapy, manifesting as refractory or relapsed disease, remains the largest cause of mortality in acute myeloid leukemia (AML). Clonal evolution through the acquisition of new mutations, or selection of resistant clones, appears responsible in many cases of relapsed disease. We used a genome-wide CRISPR-Cas9 loss of function screen to identify deleted genes, which mediate resistance to Cytarabine (AraC) and anthracycline, Doxorubicin (Dox) in AML.Methods: Dose response was defined using MTS viability assays, with synergism determined using the Chou-Talalay method. For the screen, Cas9-expressing OCI-AML3 cells were transduced with the Brunello gRNA library, which contains 76,441 gRNAs targeting 19,114 genes (Doench et al. 2016, Nat Biotech). Cells were treated with continuous, intermediate-dose AraC/Dox (A/D) or with intermittent, high-dose A/D. Cell viability was monitored by trypan blue exclusion. gRNA representation was measured prior to, and at the end of treatment, using next-generation sequencing. Individual genomic targets were examined sequentially, using multiple gRNAs and correlated with effects on proliferation and cell cycle. Validation was performed in vivo, in independent cell lines (MV4;11) and in silico using bioinformatics on human clinical datasets. Differences in mean values were compared with t-tests.Results: AraC and Dox were synergistic at ratios 10:1, 20:1 and 40:1; the most synergistic ratio (40:1) was used for the screen. By 12 days, both continuous and intermittent dosing eliminated non-transduced controls and the bulk of library-transduced cells. Rapid resistance was seen in AraC monotherapy (10 days), however using combined AraC and Dox, a resistant population emerged by 20 days, mimicking dynamics of relapsed or refractory disease. Resistant populations also showed increased IC50 in vitro and resistance to in vivo A/D when transplanted into immunodeficient NRGS mice, compared to native cell lines (higher white cell counts (3.9 vs 1.9 x 109/L, p = 0.013), lower platelets (597 vs 978 x 109/L, p = 0.024) and higher bone marrow human CD45 chimerism (12.6 vs 1.7%, p = 0.007)).Deoxycytidine kinase (DCK) and cyclin-dependent kinase inhibitor 2a (CDKN2A) were the top hits, out of ~10 genes identified. DCK catalyses the rate-limiting step in metabolising AraC to its active form, and accordingly, AML cells with CRISPR-mediated DCK deletion were resistant to AraC, but also to combination A/D, demonstrating that resistance to a single agent may result in therapeutic failure of combination regimens.CDKN2A transcriptionally encodes p14ARF and p16INK4A, using common exons 2 and 3, but a distinct exon 1 and alternate reading frame. Enriched CDKN2A gRNAs identified in the screen were directed at common exon 2 and p16INK4A unique exon 1, with a p14ARF exon 1 gRNA not being represented in the Brunello library. p16INK4A regulates cell cycle and proliferation by inhibiting G1-S transition and p14ARF prevents p53 degradation, promoting apoptosis. CDKN2A is frequently deleted in multiple types of cancer. Functionally, CDKN2A exon 2 knockout, expected to disrupt both p14ARF and p16INK4A functions, led to chemotherapy resistance through enhanced proliferation and prevention of cell cycle arrest after low-dose and high-dose chemotherapy treatment. To delineate differential contributions of p14ARF and p16INK4A deletion to this resistance phenotype, we deleted p16INK4A unique exon 1. p16INK4A exon 1 deletion also conferred a proliferative advantage in low-dose chemotherapy, but not high-dose, suggesting that concurrent impaired function of p14ARF is required to confer resistance to high-dose chemotherapy. Finally, gene expression data from 3 independent AML patient cohorts (Verhaak, et al. 2009, Haematologica; Metzeler, et al. 2008, Blood; Bullinger, unpublished) demonstrated that low CDKN2A expression conferred inferior survival, confirming the clinical relevance of CDKN2A loss-of-function in AML.Conclusion: This study demonstrates the utility of genome-wide CRISPR screens to functionally capture genetic heterogeneity and evolution through chemotherapy treatment in AML. This approach can identify clinically relevant gene mutations and therapeutic vulnerabilities. DisclosuresBullinger:Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lane:Novartis: Consultancy; Celgene: Consultancy; Janssen: Consultancy, Research Funding.

An In Vivo Genome-Wide CRISPR Screen Identifies Novel Dependencies for Blast Crisis Chronic Myelogenous Leukemia

Poorly differentiated aggressive myeloid diseases such as Acute Myelogenous Leukemia (AML) and blast crisis Chronic Myelogenous Leukemia (bcCML) are often resistant to standard therapy and associated with significantly poor survival in both children and adults. There is thus a significant need for a better understanding of the mechanisms that drive disease progression and for finding novel therapeutic targets. Thus, to determine the molecular effectors of myeloid leukemia growth in vivo, we carried out a genome-wide CRISPR/Cas9 dropout screen using the lentiviral Brie gRNA library. This library targets 19,674 genes, and has on average 3 gRNAs for each gene, and 1000 control non-targeting gRNAs. We carried out this whole-genome screen in a mouse model of Cas9+ blast crisis CML (bcCML) driven by BCR-ABL/ NUP98-HOXA9 since this represents a very aggressive phase of myeloid cancer where 90% of the leukemic blasts are undifferentiated and cancer stem cell-like. This in vivo screen led to the identification of 3636 genes essential for leukemic growth and propagation in the bone marrow of recipient mice, constituting pathways such as metabolism, protein translation and DNA replication. The genes that were significantly depleted included known drivers of myeloid cancer progression and regulators of myeloid cancer stem cells (for example, Brd4, Kdm1a, Pafah1b1/Lis1, Rptor), indicating that our screening strategy can successfully identify functional drivers of cancer growth.While intrinsic signals that drive myeloid cancer progression are well described, little is known about how interactions with the surrounding microenvironment can control leukemic growth and propagation. Our whole-genome screen identified ~130 cell surface genes that are significantly depleted in the bcCML stem cells transplanted in vivo. Since environmental factors commonly signal through receptors on the surface of leukemic cells, this subset is likely to include most, if not all, genetic effectors of niche driven signals required for in vivo growth and propagation of aggressive myeloid leukemia cells. Of these 130 genes, several have earlier been shown by us and others to be essential for myeloid cancer progression including Itgb1, Cxcr4 and Cd44. We are currently testing the functional contribution of novel candidate cell surface molecules, which can integrate signals from the environment, on the in vivo growth and progression of myeloid malignancies. We anticipate that these studies will provide a basis for testing antibody-mediated therapeutic inhibition of specific microenvironmental signals on myeloid leukemia growth and propagation. DisclosuresNo relevant conflicts of interest to declare.

Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells.

CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful tool for functional genomics with manifold applications. Genome-wide guide-RNA (gRNA) libraries allow facile generation of a pool of cells, each harboring a gene knockout mutation that can be used for the study of gene function, pathway analysis or the identification of genes required for cellular fitness. Furthermore, CRISPR genetic screens can be applied for the discovery of genes whose knockout sensitizes cells to drug treatments or mediates drug resistance. Here, we provide a detailed protocol discussing the necessary steps for the successful performance of pooled CRISPR-Cas9 screens.

A validated gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome.

Over 200 glycosyltransferases are involved in the orchestration of the biosynthesis of the human glycome, which is comprised of all glycan structures found on different glycoconjugates in cells. The glycome is vast, and despite advancements in analytic strategies it continues to be difficult to decipher biological roles of glycans with respect to specific glycan structures, type of glycoconjugate, particular glycoproteins, and distinct glycosites on proteins. In contrast to this, the number of glycosyltransferase genes involved in the biosynthesis of the human glycome is manageable, and the biosynthetic roles of most of these enzymes are defined or can be predicted with reasonable confidence. Thus, with the availability of the facile CRISPR/Cas9 gene editing tool it now seems easier to approach investigation of the functions of the glycome through genetic dissection of biosynthetic pathways, rather than by direct glycan analysis. However, obstacles still remain with design and validation of efficient gene targeting constructs, as well as with the interpretation of results from gene targeting and the translation of gene function to glycan structures. This is especially true for glycosylation steps covered by isoenzyme gene families. Here, we present a library of validated high-efficiency gRNA designs suitable for individual and combinatorial targeting of the human glycosyltransferase genome together with a global view of the predicted functions of human glycosyltransferases to facilitate and guide gene targeting strategies in studies of the human glycome.

Screening Regulatory Element Function with CRISPR/Cas9-based Epigenome Editing.

Genomic regulatory elements that control gene expression play an important role in many traits and diseases. Identifying the regulatory elements associated with each gene or phenotype and understanding the function of that element remain a significant challenge. To address this technological need, we developed CRISPR/Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. This protocol includes detailed instructions for design and cloning of gRNA libraries, construction of endogenous reporter cell lines via CRISPR/Cas9-mediated knock-in of fluorescent proteins, overall screen design, and recovery of the gRNA library for enrichment analysis. This protocol will be generally useful for implementing genome engineering technologies for high-throughput functional annotation of putative regulatory elements in their native chromosomal context.

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Cpf1-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cpf1.

Following the type II CRISPR-Cas9 system, type V CRISPR-Cpf1 endonucleases have been found to be applicable for genome editing in various organisms in vivo. However, there are as yet no web-based tools capable of optimally selecting guide RNAs (gRNAs) among all possible genome-wide target sites. Here, we present Cpf1-Database, a genome-wide gRNA library design tool for LbCpf1 and AsCpf1, which have DNA recognition sequences of 5'-TTTN-3' at the 5' ends of target sites. Cpf1-Database provides a sophisticated but simple way to design gRNAs for AsCpf1 nucleases on the genome scale. One can easily access the data using a straightforward web interface, and using the powerful collections feature one can easily design gRNAs for thousands of genes in short time. Free access at http://www.rgenome.net/cpf1-database/. sangsubae@hanyang.ac.kr.

CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects.

Genome-editing involves the insertion, deletion, or replacement of DNA in the genome of a living organism using “molecular scissors.” Traditional genome editing with engineered nucleases for human stem cells is limited by its low efficiency, high cost, and poor specificity. The CRISPR system has recently emerged as a powerful gene manipulation technique with advantages of high editing efficiency and low cost. Although this technique offers huge potential for gene manipulation in various organisms ranging from prokaryotes to higher mammals, there remain many challenges in human stem cell research. In this review, we highlight the basic biology and application of the CRISPR/Cas9 system in current human stem cell research, discuss its advantages and challenges, and debate the future prospects for human stem cells in regenerative medicine.

Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries

Genome-wide CRISPR-based knockout (CRISPR-KO) screening is an emerging technique which enables systematic genetic analysis of a cellular or molecular phenotype in question. Continuous improvements, such as modifications to the guide RNA (gRNA) scaffold and the development of gRNA on-target prediction algorithms, have since been made to increase their screening performance. We compared the performance of three available second-generation human genome-wide CRISPR-KO libraries that included at least one of the improvements, and examined the effect of gRNA scaffold, number of gRNAs per gene and number of replicates on screen performance. We identified duplicated screens using a library with 6 gRNAs per gene as providing the best trade-off. Despite the improvements, we found that each improved library still has library-specific false negatives and, for the first time, estimated the false negative rates of CRISPR-KO screens, which are between 10% and 20%. Our newly-defined optimal screening parameters would be helpful in designing screens and constructing bespoke gRNA libraries.

Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens.

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

Requirement of glycosylation machinery in TLR responses revealed by CRISPR/Cas9 screening.

The Toll family of receptors sense microbial products and activate a defense response. The molecular machinery required for the TLR response is not yet fully understood. In the present study, we used a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CAS9 screening system to study TLR responses. We employed a cell line expressing TLR with an NF-κB-driven GFP reporter. The cell line was transduced with a guide RNA (gRNA) library and stimulated with TLR ligands. The cells impaired in GFP induction were sorted, and gRNAs were sequenced. Identified genes were ranked according to the count of sequence reads and the number of gRNA target sites. The screening system worked correctly, as molecules that were already known to be required for the TLR response were identified by the screening. Furthermore, this system revealed that the oligosaccharide transferase complex (OSTC) mediating co-translational glycosylation was required for TLR5, 7 and 9 responses. Protein expression of TLR5, but not an irrelevant molecule (CD44), was abolished by the lack of OSTC, suggesting the essential role of glycosylation in TLR5 protein stability. These results demonstrate that the screening system established here is able to reveal molecular mechanisms underlying the TLR response.

Abstract 3046: Identification of lncRNAs involved in regulation of androgen receptor splice variants in prostate cancer through CRISPR-Cas9-based screen

Abstract Prostate cancer is the second leading cause of death in men in the USA. Despite the high initial response rates to androgen deprivation therapy (ADT), these tumors ultimately develop the resistance, i.e., castration-resistant prostate cancer (CRPC). One of the contributing factors is constitutive expression of androgen receptor (AR) splice variants such as AR3, a major splice variant identified in clinical specimens. However, little is known about long non-coding RNAs (lncRNAs) on regulation of AR splicing. In this study, we generate CRISPR/Cas9-based libraries against a focus group of lncRNAs and an AR3 mini-gene reporter to identify lncRNAs involved in regulation of AR splicing. The principles of this screening system is that only sgRNAs capable of promoting AR3 expression will be enriched in SAM (Synergistic Activation Mediator) library, whereas these clones are lost from the knockout gRNA library after selection. This screen identifies several candidate lncRNA sgRNAs and dual gRNAs (targeting Lnc-DC/ Har1B/ LINC00568/ DHRS4-AS1/ SNHG5). Further characterization confirms the regulation of their corresponding endogenous genes and AR3 expression. Importantly, overexpression of Lnc-DC increases AR3 level and enhances tumor cell growth in response to androgen deprivation. Moreover, while ectopic expression of DHRS4-AS1 increases, DHRS4-AS1 knockout suppresses AR3. Finally, rescue experiments restore the ability of DHRS4-AS1 to induce AR3, further supporting a role for Lnc-DC and DHRS4-AS1 in AR3 regulation. Together, we demonstrate that the CRISPR/Cas9-based screening system provides a powerful tool for lncRNA research. As a result, these lncRNAs may serve as biomarkers to predict the response to ADT or therapeutic targets for treatment of CRPC. Citation Format: Tsui-Ting Ho, Pratirodh Koirala, Nanjiang Zhou, Yin-Yuan Mo. Identification of lncRNAs involved in regulation of androgen receptor splice variants in prostate cancer through CRISPR-Cas9-based screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3046. doi:10.1158/1538-7445.AM2017-3046

PiggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice

CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and unknown tumor suppressor genes (TSGs). Our results demonstrate that PB can be a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries.

A Method to Convert mRNA into a Guide RNA (gRNA) Library without Requiring Previous Bioinformatics Knowledge of the Organism.

While the diversity of species represents a diversity of special biological abilities, many of the genes that encode those special abilities in a variety of species are untouched, leaving an untapped gold mine of genetic information; however, despite current advances in genome bioinformatics, annotation of that genetic information is incomplete in most species, except for well-established model organisms, such as human, mouse, or yeast. A guide RNA (gRNA) library using the clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system can be used for the phenotypic screening of uncharacterized genes by forward genetics. The construction of a gRNA library usually requires an abundance of chemically synthesized oligos designed from annotated genes; if one wants to convert mRNA into gRNA without prior knowledge of the target DNA sequences, the major challenges are finding the sequences flanking the protospacer adjacent motif (PAM) and cutting out the 20-bp fragment. Recently, I developed a molecular biology-based technique to convert mRNA into a gRNA library (Arakawa, 2016) (Figure 1). Here I describe the detailed protocol of how to construct a gRNA library from mRNA. Figure 1.A method to convert mRNA into a gRNA library construction ( Sanjana et al., 2014 ). The scheme of the method is summarized. Each step of D-O is described in detail in the Procedure. Bg, BglII; Xb, XbaI; Bs, BsmBI; Aa, AatII. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2.

Mechanisms of iPS cell generation and beyond.

The generation of induced pluripotent stem cells (iPSCs) achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc, transformed our classical views of the cellular epigenetic landscape and delivered a new concept for cell and tissue engineering. In addition to iPSCs, several other cell types have also been generated by master transcription factor (TF)-mediated transdifferentiation. However, the critical molecular mechanisms amongst diverse cellular identity changes are not well understood. Through the investigation of reprogramming mechanisms, we recently revealed that over-expression of constitutive active Smad3 boosted not only iPSC generation, but also 3 other master TF-mediated conversions, from B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons. This demonstrated that there were common mechanisms underlying different master TF-mediated cell conversions. To illuminate such mechanisms further, we have recently performed CRISPR/Cas9-mediated genome-wide knockout screening during reprogramming with a lentiviral gRNA library containing 90,000 gRNAs. This screening provided us with ~15 novel reprogramming roadblock genes as well as ~20 candidate genes essential for the reprogramming process but not for ES cell self-renewal. This data set will be a valuable resource to further understand how overexpression of master TFs alters cellular identity, and to achieve more faithful, efficient cell conversions for regenerative medicine.(Presented at the 1934th Meeting, March 17, 2017).

CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening.

BackgroundThe bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches.ResultsHere we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs.ConclusionsThe simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3268-z) contains supplementary material, which is available to authorized users.

A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism.

The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally making gRNA. I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species.

Abstract 233: Identification of cancer vulnerabilities to metabolic perturbation using genome wide CRISPR screens

Abstract The CRISPR-Cas9 system provides an effective way to introduce targeted loss-of-function mutations in mammalian cells. The advance that the CRISPR-Cas9 technology brings to human genetics sets the stage for identifying cellular fitness genes that operate either globally (core fitness genes) or specifically within a particular genetic background or environmental context (context-specific fitness genes). In tumors, this is the foundation for the concept of synthetic lethality as genes required in tumor cells but not in adjacent normal tissues should make ideal therapeutic targets with high effectiveness and minimal side effects. Towards this goal, we have developed a second-generation CRISPR gRNA library of 176,500 guides targeting 17,661 human protein-coding genes. We used the library to screen five human cell lines, representing a cross-section of wild type and cancer tissues, to identify genes whose knockouts induce significant fitness defects. Our screens accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and identify novel context-specific vulnerabilities. Interestingly, we identified a specific dependency on mitochondrial activity and we validated this using various complex I inhibitors. This strongly supports the idea that oxidative phosphorylation (OXPHOS) dependency - a clear exception to the Warburg effect - is a targetable weakness of some tumors. In order to further understand this metabolic vulnerability, we performed a synthetic lethal screen to identify sensitizers of OXPHOS inhibition. Our screen revealed that inhibition of mitochondrial OXPHOS sensitizes the cells to loss of other metabolic pathways such as glycolysis, pentose phosphate pathway and lipid biosynthesis. Furthermore, loss of the cytosolic aspartate aminotransferase GOT1 was found to be synthetic lethal with perturbation of OXPHOS. This is consistent with the essential role of the electron transport chain in cell proliferation, which is to enable aspartate synthesis. Additionally, we also find novel genes, whose loss of function might alleviate the affect of OXPHOS perturbation. Our findings demonstrate that CRISPR-Cas9 screens enable a high-resolution view of the genetic vulnerabilities of a cell that may represent therapeutic opportunities in cancer. Further, synthetic lethal chemical-genetic screens can reveal novel functional drug combinations, which will enhance the efficacy of targeted therapies. Citation Format: Megha Chandrashekhar, Michael Arreger, Ashwin Seetharaman, Traver Hart, Jason Moffat. Identification of cancer vulnerabilities to metabolic perturbation using genome wide CRISPR screens. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 233.