Gene Editing Group
Head: Dr. Manuel Kaulich
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