Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)—an early step in NHEJ—yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.

[1]  B. Conklin,et al.  Rapid, precise quantification of large DNA excisions and inversions by ddPCR , 2020, Scientific Reports.

[2]  L. Vassilev,et al.  Pharmacologic Inhibitor of DNA-PK, M3814, Potentiates Radiotherapy and Regresses Human Tumors in Mouse Models , 2020, Molecular Cancer Therapeutics.

[3]  Charles D. Yeh,et al.  Advances in genome editing through control of DNA repair pathways , 2019, Nature Cell Biology.

[4]  A. Berger,et al.  RNA isoform screens uncover the essentiality and tumor suppressor activity of ultraconserved poison exons , 2019, Nature Genetics.

[5]  Rory Johnson,et al.  CASPR, an analysis pipeline for single and paired guide RNA CRISPR screens, reveals optimal target selection for long non-coding RNAs , 2019, Bioinform..

[6]  S. Pääbo,et al.  Simultaneous precise editing of multiple genes in human cells , 2019, Nucleic acids research.

[7]  Min H. Kang,et al.  DNA-PK as an Emerging Therapeutic Target in Cancer , 2019, Front. Oncol..

[8]  Rory Johnson,et al.  Hacking the Cancer Genome: Profiling Therapeutically Actionable Long Non-coding RNAs Using CRISPR-Cas9 Screening. , 2019, Cancer cell.

[9]  P. Mangeot,et al.  Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins , 2019, Nature Communications.

[10]  Jacob M. Schreiber,et al.  A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens , 2019, Cell.

[11]  Kendall R. Sanson,et al.  Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities , 2018, Nature Communications.

[12]  Ying Liu,et al.  Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites , 2018, Nature Biotechnology.

[13]  S. Yamashita,et al.  Combinatorial CRISPR/Cas9 Approach to Elucidate a Far-Upstream Enhancer Complex for Tissue-Specific Sox9 Expression. , 2018, Developmental cell.

[14]  A. Bradley,et al.  Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements , 2018, Nature Biotechnology.

[15]  Waseem Akhtar,et al.  Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks , 2018, Molecular cell.

[16]  M. Porteus,et al.  Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human β-globin locus. , 2018, Blood.

[17]  Aaron T. L. Lun,et al.  Specificity of RNAi, LNA and CRISPRi as loss-of-function methods in transcriptional analysis , 2017, bioRxiv.

[18]  John G Doench,et al.  Am I ready for CRISPR? A user's guide to genetic screens , 2017, Nature Reviews Genetics.

[19]  A. McKenna,et al.  CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions. , 2017, American journal of human genetics.

[20]  Phillip G. Montgomery,et al.  Defining a Cancer Dependency Map , 2017, Cell.

[21]  Ann E. Sizemore,et al.  Computational correction of copy-number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells , 2017, Nature Genetics.

[22]  S. Harnor,et al.  Targeting DNA‐Dependent Protein Kinase for Cancer Therapy , 2017, ChemMedChem.

[23]  Guo-Cheng Yuan,et al.  Dissecting super-enhancer hierarchy based on chromatin interactions , 2017, Nature Communications.

[24]  M. Lieber,et al.  Non-homologous DNA end joining and alternative pathways to double-strand break repair , 2017, Nature Reviews Molecular Cell Biology.

[25]  T. Maricic,et al.  Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells , 2017, bioRxiv.

[26]  Ling-Ling Chen,et al.  SLERT Regulates DDX21 Rings Associated with Pol I Transcription , 2017, Cell.

[27]  B. Li,et al.  A tiling1deletion based genetic screen for cis-regulatory element identification in mammalian cells , 2017, Nature Methods.

[28]  Fangting Wu,et al.  LncRNA AK023948 is a positive regulator of AKT , 2017, Nature Communications.

[29]  Zhongzheng Cao,et al.  Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library , 2016, Nature Biotechnology.

[30]  M. Mann,et al.  Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans , 2016, Nature Communications.

[31]  T. Fuchss,et al.  Abstract 1658: M3814, a novel investigational DNA-PK inhibitor: enhancing the effect of fractionated radiotherapy leading to complete regression of tumors in mice , 2016 .

[32]  S. Richard,et al.  Sam68 functions as a transcriptional coactivator of the p53 tumor suppressor , 2016, Nucleic acids research.

[33]  Julia V. Ponomarenko,et al.  Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion , 2016, bioRxiv.

[34]  B. Conklin,et al.  Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing , 2016, Scientific Reports.

[35]  Y. E. Chen,et al.  RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency , 2016, Nature Communications.

[36]  Dongsheng Duan,et al.  In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy , 2016, Science.

[37]  D. Durocher,et al.  High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities , 2015, Cell.

[38]  Meagan E. Sullender,et al.  Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 , 2015, Nature Biotechnology.

[39]  Roderic Guigó,et al.  DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs , 2015, BMC Genomics.

[40]  Matthew C. Canver,et al.  BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis , 2015, Nature.

[41]  J. Pelletier,et al.  Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing , 2015, Genome Medicine.

[42]  Joana A. Vidigal,et al.  Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries , 2015, Nature Communications.

[43]  A. Visel,et al.  Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions , 2015, Cell.

[44]  Ruedi Aebersold,et al.  A Mass Spectrometric-Derived Cell Surface Protein Atlas , 2015, PloS one.

[45]  Natalia N. Ivanova,et al.  Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells , 2015, Nature Biotechnology.

[46]  Steven Lin,et al.  Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery , 2014, eLife.

[47]  Jun S. Liu,et al.  MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens , 2014, Genome Biology.

[48]  Fangting Wu,et al.  Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines , 2014, Nucleic acids research.

[49]  Todd M. Allen,et al.  Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. , 2014, Cell stem cell.

[50]  Joana A. Vidigal,et al.  In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system , 2014, Nature.

[51]  Yao-Cheng Lin,et al.  Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations , 2014, Nature Communications.

[52]  Bian Hu,et al.  Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9 , 2014, RNA biology.

[53]  Matthew C. Canver,et al.  Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells*♦ , 2014, The Journal of Biological Chemistry.

[54]  R. Jaenisch,et al.  One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering , 2013, Cell.

[55]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[56]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[57]  Adam A. Margolin,et al.  The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity , 2012, Nature.

[58]  N. Hacohen,et al.  Highly parallel identification of essential genes in cancer cells , 2008, Proceedings of the National Academy of Sciences.

[59]  A. Seluanov,et al.  Comparison of nonhomologous end joining and homologous recombination in human cells. , 2008, DNA repair.

[60]  E. Hendrickson,et al.  Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells , 2008, Proceedings of the National Academy of Sciences.

[61]  Thomas D. Schmittgen,et al.  Analyzing real-time PCR data by the comparative CT method , 2008, Nature Protocols.

[62]  J. Kappes,et al.  Evidence that Stable Retroviral Transduction and Cell Survival following DNA Integration Depend on Components of the Nonhomologous End Joining Repair Pathway , 2004, Journal of Virology.

[63]  F. Bushman,et al.  Role of the non‐homologous DNA end joining pathway in the early steps of retroviral infection , 2001, The EMBO journal.

[64]  Howard Y. Chang,et al.  NONCODING RNA: CRISPRi‐based genome‐scale identification of functional long noncoding RNA loci in human cells , 2017 .