In cancer patient care, the National Cancer Institute (NCI) defines the term drug resistance as the failure of cancer cells to respond to a drug that used to kill or weaken them. Resistance to therapy can be divided into two broad categories: cell intrinsic and acquired mechanisms. Modern technologies such as deep-sequencing have aided in the interrogation of clinical samples and allowed the identification of molecular signatures and genotypes that predict response to a certain drug. From those data, a complex picture emerges, in which drug resistance can result from a wide range of molecular mechanisms: increased rates of drug efflux, alterations in drug metabolism and mutation of drug targets, as well as the activation of survival and inactivation of death pathways. Epigenetic changes as well as the cancer microenvironment have also been identified as important contributors to drug resistance. Hence, the molecular determinants of drug resistance can be complex and require innovative technologies to identify the underlying mechanisms.
The CRISPR/Cas system has been described as a bacterial immune system, targeting viral pathogens such as lytic bacteriophages. The underlying mechanism is based on the fact that bacterial DNA nucleases can recognize and destroy bacteriophage DNA sequences by utilizing small guide RNAs through DNA double-strand cleavage. Interestingly, throughout this process, bacteria incorporate short sequences of the bacteriophage DNA into their own genomic DNA, thereby accumulating an encoded memory of pathogens that previously infected the bacteria. These incorporated short viral DNA sequences are arranged in form of clustered regularly-interspaced short palindromic repeats (CRISPR).
CRISPR/Cas gene editing depends on two components, a protein with DNA-nuclease activity (Cas) and a short RNA oligonucleotide (guide RNA, gRNA) that directs the nuclease activity of Cas enzymes to gRNA-complementary DNA sequences, thereby conferring specificity to the reaction. An additional prerequisite for successful DNA targeting of the Cas-gRNA complex is the presence of a protospacer-adjacent motif (PAM) DNA sequence in the target DNA, for which the exact sequence depends on the bacterial Cas-enzyme. For the most widely used Streptococcus pyogenes Cas9 (SpCas9), this sequence has the format of NGG, where N can be any nucleotide. Most notably, the Cas enzyme can be expressed in human cells and, by providing a human DNA-directed gRNA, induce a highly specific DNA double strand break that cannot be repaired error-free, leading to insertion and deletion (InDel) mutations. Phenotypes of InDel mutations range from in-frame deletions to complete gene knockouts.
While single genetic changes can be used to generate well-controlled model systems, they do not allow for unbiased screenings. To perform genetic screens, a multitude of gRNA sequences can be combined to generate libraries targeting open reading frames (ORFs) or else in the human or other genomes. Major advantages of these genetic screens are their unbiased performance and ease of use applicability to many biological questions.
gRNA library generation - limitations
State-of-the-art knockout gRNA library generation starts with the bioinformatical identification of functional gRNAs for which several online tools have become available (Table 1).
|Table 1: gRNA-design algorithms. Adapted from De Bruyn, et al., PHARMAKON, 2017.|
Identified sequences are extended by adapter sequences, coding for restriction enzyme recognition sites as well as primer annealing sites. Designed sequences are subsequently pooled synthesized, PCR amplified and enzymatically digested. Most commonly, the PCR product is introduced into lentiviral plasmids, requiring an open chromatin species of the target plasmid. Conventionally, gRNAs are pool-cloned by T4 DNA ligase or homology-based recombination procedures, resulting in the final gRNA library.
However, available gRNA libraries suffer from the methods they are generated with. Pooled gRNA cloning requires PCR amplification that introduces a PCR-dependent sequence bias of up to 10.000-fold into the library. This bias translates into high numbers of experimental coverage to ensure the experimental presence of all cloned sequences, making genome-wide screens unfeasible for most laboratories. Furthermore, restriction enzyme or homology-based cloning methods required open target-plasmid DNA. This open form of DNA is a substrate for the intrinsic exonuclease activity of most ligases, resulting in cloning artefacts and dramatically reducing final library quality.
|Table 2: Available genome-wide gRNA-libraries. Adapted from De Bruyn, et al., PHARMAKON, 2017.|
Due to these technical issues, the maximal diversity of gRNA libraries is estimated to be between 100.000 and 300.000 gRNA sequences (Table 2). However, there are 250 million SpCas9 target sites in human genome, hence, conventional cloning methods cannot generate gRNA libraries with sufficient diversity to cover the coding as well as the non-coding human genome.
Therefore, functional genomic interrogations with the CRISPR/Cas system have been mostly focused on the coding genome.
Research in the Kaulich laboratory focusses on understanding malignant cell cycle entry and drug resistance mechanisms. To do so, we develop innovative technologies to functionally interrogate the human coding and non-coding genome.
Dr. Manuel Kaulich
Institute of Biochemistry II
Goethe University Medical School
University Hospital Building 75
60590 Frankfurt am Main / Germany
Office: +49 (0) 69 6301 5450
Lab: +49 (0) 69 6301
Email: kaulich (at) em.uni-frankfurt.de
Hazal studied statistics at the Hacettepe University of Turkey and obtained her bachelor degree in 2018. She continues to work towards her master's degree in bioinformatics also at the Hacettepe University of Turkey. For her master’s thesis, she decided to move to Germany to join our group to work on implementing a graphical interface of combinatorial CRISPR screening data.
Ronay studied molecular biology, genetics and bioengineering at Sabanci University in Istanbul. In his Master’s Thesis Ronay used CRISPR/Cas9 editing to study transcription factors regulating IL7R gene expression in T lymphocytes. In January 2020, Ronay joined the Gene Editing Group of Dr. Manuel Kaulich as a PhD student to understand the role of cellular communication in oncogene-mediated transformation.
Sandra studies Biomolecular Engineering at the Technical University of Darmstadt. She obtained her bachelor´s degree in 2018 and started her master with focus on cell biology and biochemistry. She joined the Gene Editing Group in October 2019 for her master thesis, where she works on oncogenesis.
Publications from PubMed: 22Keiten-Schmitz J, Wagner K, Piller T, Kaulich M, Alberti S, Müller S. The Nuclear SUMO-Targeted Ubiquitin Quality Control Network Regulates the Dynamics of Cytoplasmic Stress Granules. Mol. Cell 2020. 79 (1) 54-67.e7 Link
Trembinski DJ, Bink DI, Theodorou K, Sommer J, Fischer A, van Bergen A, Kuo CC, Costa IG, Schürmann C, Leisegang MS, Brandes RP, Alekseeva T, Brill B, Wietelmann A, Johnson CN, Spring-Connell A, Kaulich M, Werfel S, Engelhardt S, Hirt MN, Yorgan K, Eschenhagen T, Kirchhof L, Hofmann P, Jaé N, Wittig I, Hamdani N, Bischof C, Krishnan J, Houtkooper RH, Dimmeler S, Boon RA. Aging-regulated anti-apoptotic long non-coding RNA Sarrah augments recovery from acute myocardial infarction. Nat Commun 2020. 11 (1) 2039 Link
Bogucka K, Pompaiah M, Marini F, Binder H, Harms G, Kaulich M, Klein M, Michel C, Radsak MP, Rosigkeit S, Grimminger P, Schild H, Rajalingam K. ERK3/MAPK6 controls IL-8 production and chemotaxis. Elife 2020. 9 Link
Wiechmann S, Maisonneuve P, Grebbin BM, Hoffmeister M, Kaulich M, Clevers H, Rajalingam K, Kurinov I, Farin HF, Sicheri F, Ernst A. Conformation-specific inhibitors of activated Ras GTPases reveal limited Ras dependency of patient-derived cancer organoids. J. Biol. Chem. 2020. 295 (14) 4526-4540 Link
Lim R, Sugino T, Nolte H, Andrade J, Zimmermann B, Shi C, Doddaballapur A, Ong YT, Wilhelm K, Fasse JWD, Ernst A, Kaulich M, Husnjak K, Boettger T, Guenther S, Braun T, Krüger M, Benedito R, Dikic I, Potente M. Deubiquitinase USP10 regulates Notch signaling in the endothelium. Science 2019. 364 (6436) 188-193 Link
Wegner M, Diehl V, Bittl V, de Bruyn R, Wiechmann S, Matthess Y, Hebel M, Hayes MG, Schaubeck S, Benner C, Heinz S, Bremm A, Dikic I, Ernst A, Kaulich M. Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome. Elife 2019. 8 Link
Wegner MS, Schömel N, Gruber L, Örtel SB, Kjellberg MA, Mattjus P, Kurz J, Trautmann S, Peng B, Wegner M, Kaulich M, Ahrends R, Geisslinger G, Grösch S. UDP-glucose ceramide glucosyltransferase activates AKT, promoted proliferation, and doxorubicin resistance in breast cancer cells. Cell. Mol. Life Sci. 2018. 75 (18) 3393-3410 Link
Le Guerroué F, Eck F, Jung J, Starzetz T, Mittelbronn M, Kaulich M, Behrends C. Autophagosomal Content Profiling Reveals an LC3C-Dependent Piecemeal Mitophagy Pathway. Mol. Cell 2017. 68 (4) 786-796.e6 Link
Pitchai GP, Kaulich M, Bizard AH, Mesa P, Yao Q, Sarlos K, Streicher WW, Nigg EA, Montoya G, Hickson ID. A novel TPR-BEN domain interaction mediates PICH-BEND3 association. Nucleic Acids Res. 2017. 45 (19) 11413-11424 Link
Wesely J, Steiner M, Schnütgen F, Kaulich M, Rieger MA, Zörnig M. Delayed Mesoderm and Erythroid Differentiation of Murine Embryonic Stem Cells in the Absence of the Transcriptional Regulator FUBP1. Stem Cells Int 2017. 2017 5762301 Link
van Wijk SJL, Fricke F, Herhaus L, Gupta J, Hötte K, Pampaloni F, Grumati P, Kaulich M, Sou YS, Komatsu M, Greten FR, Fulda S, Heilemann M, Dikic I. Linear ubiquitination of cytosolic Salmonella Typhimurium activates NF-κB and restricts bacterial proliferation. Nat Microbiol 2017. 2 17066 Link
Lönn P, Kacsinta AD, Cui XS, Hamil AS, Kaulich M, Gogoi K, Dowdy SF. Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics. Sci Rep 2016. 6 32301 Link
Meitinger F, Anzola JV, Kaulich M, Richardson A, Stender JD, Benner C, Glass CK, Dowdy SF, Desai A, Shiau AK, Oegema K. 53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration. J. Cell Biol. 2016. 214 (2) 155-66 Link
Tobias IS, Kaulich M, Kim PK, Simon N, Jacinto E, Dowdy SF, King CC, Newton AC. Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation. Biochem. J. 2016. 473 (4) 509-23 Link
Kaulich M, Dowdy SF. Combining CRISPR/Cas9 and rAAV Templates for Efficient Gene Editing. Nucleic Acid Ther 2015. 25 (6) 287-96 Link
Kaulich M, Lee YJ, Lönn P, Springer AD, Meade BR, Dowdy SF. Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi. Nucleic Acids Res. 2015. 43 (7) e45 Link
Meade BR, Gogoi K, Hamil AS, Palm-Apergi C, van den Berg A, Hagopian JC, Springer AD, Eguchi A, Kacsinta AD, Dowdy CF, Presente A, Lönn P, Kaulich M, Yoshioka N, Gros E, Cui XS, Dowdy SF. Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications. Nat. Biotechnol. 2014. 32 (12) 1256-61 Link
Narasimha AM, Kaulich M, Shapiro GS, Choi YJ, Sicinski P, Dowdy SF. Cyclin D activates the Rb tumor suppressor by mono-phosphorylation. Elife 2014. 3 Link
Kaulich M, Cubizolles F, Nigg EA. On the regulation, function, and localization of the DNA-dependent ATPase PICH. Chromosoma 2012. 121 (4) 395-408 Link
Fava LL, Kaulich M, Nigg EA, Santamaria A. Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint. EMBO J. 2011. 30 (16) 3322-36 Link
Hübner NC, Wang LH, Kaulich M, Descombes P, Poser I, Nigg EA. Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs. Chromosoma 2010. 119 (2) 149-65 Link
Hellenbroich Y, Kaulich M, Opitz S, Schwinger E, Zühlke C. No association of the SCA1 (CAG)31 allele with Huntington's disease, myotonic dystrophy type 1 and spinocerebellar ataxia type 3. Psychiatr. Genet. 2004. 14 (2) 61-3 Link
Several aspects of cell cycle entry have been studied individually; among them are mitogen sensing, metabolic preparation, DNA repair, Cdk’s and the retinoblastoma protein Rb. However, much less is known about the orchestrated organization of these pathways, resulting in cell cycle entry. In an attempt to identify connecting modules of these pathways we performed a genome wide CRISPR/Cas9 knockout screen. This work has given us several new ideas of how these pathways cooperate and serves as a resource for future mechanistic studies
Guide RNA Enrichment after Genome Wide Screen for Drug Resistance. The library is divided into 2 separate libraries (left/orange, right/blue) to simplify the work process. Both graphs display the enrichment of single guide RNA (gRNA) sequences when compared to background.
From this screen we have identified a handful of genes with completely unknown mechanistic links to cell cycle entry. Currently, we are dissecting the molecular mechanisms and are particularly excited about this unknown endeavor.
The cell cycle entry decision process is morphologically unattractive and hence has escaped our efforts to study in single cells. By using targeted gene replacement, we were able to generate an endogenous fluorescent reporter that visualizes the cell cycle entry decision point in single cells. Through the combination of cell biological methods with bioinformatics we were able to demonstrate that single cells can be arrested at various different time points during early G1. This arrest is highly dependent on the activity of cyclin D:Cdk4/6 kinases. In this project we identified early G1 specific substrates of cyclin D:Cdk4/6 and are currently dissecting the molecular mechanisms by which they influence the timing of the restriction point.
Altering endogenous genes/loci in cells is an integral tool of modern cell biology. The ease-of-use of the CRISPR/Cas9 system to introduce genomic DNA breaks at specific sites in vivo has led to its rapid and wide adoption. In the absence of a DNA template, the lesion is repaired by nonhomologous end joining resolving as internal deletions. However, in the presence of a homologous DNA template, homology-directed repair occurs with variable efficiencies. Our previous work has demonstrated that highly efficient gene targeting can be induced by combining CRISPR/Cas9 targeting of genomic loci with recombinant adeno-associated virus (rAAV) to provide a single-stranded homologous DNA template. With this project we aim to further enhance correct recombination rates and design new targeting strategies for inaccessible or difficult to target genomic regions.
|Overview of targeting strategies. We developed different strategies, depending on your application (point mutations, N- or C-terminal tags). Black: untargeted genomic DNA, blue: genomic DNA used as homology, pink: selection cassette for single cell cloning, orange: tag.|