CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level

Abstract CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform.

[1]  Max A. Horlbeck,et al.  Nucleosomes impede Cas9 access to DNA in vivo and in vitro , 2016, eLife.

[2]  Paul Shinn,et al.  Retroviral DNA Integration: Viral and Cellular Determinants of Target-Site Selection , 2006, PLoS pathogens.

[3]  Eric S. Lander,et al.  Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras , 2017, Cell.

[4]  E. Lander,et al.  Identification and characterization of essential genes in the human genome , 2015, Science.

[5]  Michael Poidinger,et al.  Enhancers Are Major Targets for Murine Leukemia Virus Vector Integration , 2014, Journal of Virology.

[6]  E. Lander,et al.  Genetic Screens in Human Cells Using the CRISPR-Cas9 System , 2013, Science.

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

[8]  A. Amon,et al.  Short- and long-term effects of chromosome mis-segregation and aneuploidy , 2015, Nature Reviews Molecular Cell Biology.

[9]  Zuoshang Xu,et al.  An enhanced U6 promoter for synthesis of short hairpin RNA. , 2003, Nucleic acids research.

[10]  G. Church,et al.  Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach , 2015, Nature Methods.

[11]  Joshua M. Korn,et al.  CRISPR Screens Provide a Comprehensive Assessment of Cancer Vulnerabilities but Generate False-Positive Hits for Highly Amplified Genomic Regions. , 2016, Cancer discovery.

[12]  Jennifer A. Doudna,et al.  DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 , 2014, Nature.

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

[14]  Neville E. Sanjana,et al.  Improved vectors and genome-wide libraries for CRISPR screening , 2014, Nature Methods.

[15]  Tessa G Montague,et al.  Internal guide RNA interactions interfere with Cas9-mediated cleavage , 2016, Nature Communications.

[16]  S. Elledge,et al.  Functional identification of optimized RNAi triggers using a massively parallel sensor assay. , 2011, Molecular cell.

[17]  Mazhar Adli,et al.  Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease , 2014, Nature Biotechnology.

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

[19]  Kristopher T. Jensen,et al.  Chromatin accessibility and guide sequence secondary structure affect CRISPR‐Cas9 gene editing efficiency , 2017, FEBS letters.

[20]  T. Golub,et al.  Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting. , 2016, Cancer discovery.

[21]  Meagan E. Sullender,et al.  Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation , 2014, Nature Biotechnology.

[22]  Neville E. Sanjana,et al.  High-throughput functional genomics using CRISPR–Cas9 , 2015, Nature Reviews Genetics.

[23]  D. W. Emery.  The use of chromatin insulators to improve the expression and safety of integrating gene transfer vectors. , 2011, Human gene therapy.

[24]  Ian Wheeldon,et al.  Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica. , 2016, ACS synthetic biology.

[25]  Ji Luo.  CRISPR/Cas9: From Genome Engineering to Cancer Drug Discovery. , 2016, Trends in cancer.

[26]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

[27]  J. Doudna,et al.  The new frontier of genome engineering with CRISPR-Cas9 , 2014, Science.

[28]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[29]  Derek Y. Chiang,et al.  The landscape of somatic copy-number alteration across human cancers , 2010, Nature.

[30]  Neville E. Sanjana,et al.  Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells , 2014, Science.

[31]  W. Lim,et al.  Nucleosome breathing and remodeling constrain CRISPR-Cas9 function , 2016, eLife.

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

[33]  B. van Steensel,et al.  Easy quantitative assessment of genome editing by sequence trace decomposition , 2014, Nucleic acids research.

[34]  Jennifer A. Doudna,et al.  Conformational control of DNA target cleavage by CRISPR–Cas9 , 2015, Nature.