Gene Editing Research Review An Overview of Recent Gene Editing Research Publications Featuring Illumina® Technology TABLE OF CONTENTS 4 Introduction The CRISPR Locus and the Mechanism for CRISPR-Cas Technology 7 Applications of CRISPR-Cas9 Technology Research Applications in the Medical Field Agriculture and Environmental Science 13 Workflow and Specificity Overview of the Procedure On-Target Effects Specificity: What It Means and Why It Matters Method Development Integration of CRISPR-Cas9 Technology in a Research Workflow 24 Bibliography This document highlights recent publications that demonstrate the use of Illumina technologies in single-cell research. To learn more about the platforms and assays cited, visit www.illumina.com. For Research Use Only. Not for use in diagnostic procedures. An overview of recent publications featuring Illumina technology 3 INTRODUCTION Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology 1. Jinek M, Chylinski K, Fonfara I, Hauer M, is a revolutionary gene editing method in which a programmable RNA guides a Doudna JA and Charpentier E. A program- nuclease to find a specific target location in the genome. This simple approach mable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. replaces the laborious and expensive protein-based DNA editing techniques, such 2012;337:816-821. as the use of zinc finger proteins (ZNFs) or transcription activator–like effector 2. Gasiunas G, Barrangou R, Horvath P and Siksnys V. Cas9-crRNA ribonucleoprotein nucleases (TALENs). From the time of the initial publications describing its application complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad 1, 2 3, 4 for genome editing in both prokaryotic and eukaryotic cells, the use of CRISPR- Sci U S A. 2012;109:E2579-2586. Cas technology has spread exponentially to laboratories worldwide.5 Potential 3. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas applications are in the fields of basic research, therapeutics, agriculture, and systems. Science. 2013;339:819-823. environmental research. In 2016, 2 years after gene editing was first introduced in 4. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Sci- human trials,6 the National Institutes of Health (NIH) approved the first application of ence. 2013;339:823-826. CRISPR-Cas technology in a human trial.7, 8 This decision holds promise for future 5. Dow LE. Modeling Disease In Vivo With CRIS- PR/Cas9. Trends Mol Med. 2015;21:609-621. treatment of rare genetic diseases, human immunodeficiency virus (HIV) infection, 6. Tebas P, Stein D, Tang WW, et al. Gene and hematologic malignancies through cell replacement. editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014;370:901-910. The CRISPR-Cas system originates from the prokaryotic adaptive immune system that 7. Reardon S. First CRISPR clinical trial gets green light from US panel. Nature News. 2016; 9-15 targets and cuts invading genetic elements from phages or plasmids. 8. Cyranoski D. CRISPR gene-editing tested in a person for the first time. Nature News. 2016; 9. Ishino Y, Shinagawa H, Makino K, Amemura The efficacy and safety of any DNA editing tool are highly dependent on specificity. M and Nakata A. Nucleotide sequence of the Some studies have found that the use of CRISPR-Cas may lead to undesirable iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and off-target effects.16-28 For this reason, several groups are developing methods to identification of the gene product. J Bacteriol. detect29-32 and reduce33-45 off-target effects. 1987;169:5429-5433. 10. Mojica FJ, Diez-Villasenor C, Soria E and Juez G. Biological significance of a family of regular- ly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol. 2000;36:244-246. 11. Jansen R, Embden JD, Gaastra W and The CRISPR Locus and the Mechanism Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. for CRISPR-Cas Technology Mol Microbiol. 2002;43:1565-1575. 12. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J Bacteria and archaea use CRISPR-Cas systems for adaptive immunity. and Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign The CRISPR locus consists of short palindromic repeats separated by short genetic elements. J Mol Evol. 2005;60:174-182. nonrepetitive sequences called “spacers”. Spacers originate from invading 13. Pourcel C, Salvignol G and Vergnaud G. CRISPR elements in Yersinia pestis acquire sources of DNA, such as phages, that are copied and incorporated into the new repeats by preferential uptake of CRISPR locus when an infection occurs. Near this locus, a second locus includes bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. a set of genes that encode CRISPR-associated endonucleases (Cas), which 2005;151:653-663. introduce cuts in the genome. When a repeat infection occurs from the same 14. Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance invading DNA, an RNA molecule (CRISPR RNA, or crRNA) will form a complex against viruses in prokaryotes. Science. 2007;315:1709-1712. with Cas and a transactivating crRNA (tracrRNA) to guide the nuclease to the 15. Brouns SJ, Jore MM, Lundgren M, et al. Small exogenous sequence. The Cas-RNA complex will recognize the DNA target that CRISPR RNAs guide antiviral defense in pro- karyotes. Science. 2008;321:960-964. is complementary to the crRNA and adjacent to a specific 3-nucleotide locus (the 16. Marraffini LA and Sontheimer EJ. CRISPR protospacer-adjacent motif, or PAM). The complex then cuts and deactivates the interference limits horizontal gene transfer in staphylococci by targeting DNA. Science. invading DNA (Figure 1). 2008;322:1843-1845. 17. Garneau JE, Dupuis ME, Villion M, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010;468:67-71. For Research Use Only. Not for use in diagnostic procedures. 4 Gene Editing Research 18. Deltcheva E, Chylinski K, Sharma CM, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011;471:602-607. 19. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P and Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 2011;39:9275-9282. 20. Yang H, Wang H, Shivalila CS, Cheng AW, Shi L and Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineer- ing. Cell. 2013;154:1370-1379. 21. Cho SW, Kim S, Kim Y, et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 2014;24:132-141. 22. Liang X, Potter J, Kumar S, et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 2015;208:44-53. 23. Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA and Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013;31:839-843. 24. Fu Y, Foden JA, Khayter C, et al. High-fre- quency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013;31:822-826. 25. Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827-832. 26. Cradick TJ, Fine EJ, Antico CJ and Bao G. Figure 1. The CRISPR locus consists of short palindromic repeats separated by short nonrepetitive CRISPR/Cas9 systems targeting beta-globin sequences, called “spacers”. Spacers originate from invading sources of DNA that are copied and and CCR5 genes have substantial off-target incorporated into the CRISPR locus when an infection occurs. Near this locus, another includes a set activity. Nucleic Acids Res. 2013;41:9584- of genes that encode Cas endonucleases, enzymes that introduce cuts in the genome. 9592. 27. Lin Y, Cradick TJ, Brown MT, et al. CRISPR/ Cas9 systems have off-target activity with Although different nucleases are associated with CRISPR activity in different bacteria, insertions or deletions between target DNA most of the topics in this review refer to Streptococcus pyogenes, which relies on the and guide RNA sequences. Nucleic Acids Res. 2014;42:7473-7485. endonuclease Cas9. For this reason, the review refers to CRISPR-Cas9 technology. 28. Zhang H, Zhang J, Wei P, et al. The CRISPR/ Cas9 system produces specific and homozy- gous targeted gene editing in rice in one gen- The CRISPR-Cas9 system requires 2 RNA molecules: crRNA, transcribed from eration. Plant Biotechnol J. 2014;12:797-807. the DNA spacers, and tracrRNA, whose interaction with crRNA is a structural 29. Tsai SQ, Zheng Z, Nguyen NT, et al. GUIDE- seq enables genome-wide profiling of off-tar- requirement for the recruitment of Cas9 (Figure 2). In a landmark study,46 these 2 get cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33:187-197. RNAs were hybridized to create a single-guide RNA (sgRNA). This simplified Cas9- 30. Frock RL, Hu J, Meyers RM, Ho YJ, Kii E and sgRNA system demonstrated gene editing properties. Alt FW. Genome-wide detection of DNA dou- ble-stranded breaks induced by engineered nucleases. Nat Biotechnol. 2015;33:179-186. 31. Crosetto N, Mitra A, Silva MJ, et al. Nucle- otide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods. 2013;10:361-365. 32. Kim D, Bae S, Park J, et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12:237-243, 231 p following 243. 33. Fu Y, Sander JD, Reyon D, Cascio VM and Joung JK.