Gene Editing Research Review An Overview of Recent Gene Editing Research Publications Featuring Illumina® Technology

3 For Research Use Only. Not for use in diagnostic procedures.

An overview of recent publications featuring Illumina technology

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.

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

4 Gene Editing Research For Research Use Only. Not for use in diagnostic procedures.

INTRODUCTION

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology

is a revolutionary gene editing method in which a programmable RNA guides a

nuclease to find a specific target location in the genome. This simple approach

replaces the laborious and expensive protein-based DNA editing techniques, such

as the use of zinc finger proteins (ZNFs) or transcription activator–like effector

nucleases (TALENs). From the time of the initial publications describing its application

for genome editing in both prokaryotic1, 2 and eukaryotic cells,3, 4 the use of CRISPR-

Cas technology has spread exponentially to laboratories worldwide.5 Potential

applications are in the fields of basic research, therapeutics, agriculture, and

environmental research. In 2016, 2 years after gene editing was first introduced in

human trials,6 the National Institutes of Health (NIH) approved the first application of

CRISPR-Cas technology in a human trial.7, 8 This decision holds promise for future

treatment of rare genetic diseases, human immunodeficiency virus (HIV) infection,

and hematologic malignancies through cell replacement.

The CRISPR-Cas system originates from the prokaryotic adaptive immune system that

targets and cuts invading genetic elements from phages or plasmids.9-15

The efficacy and safety of any DNA editing tool are highly dependent on specificity.

Some studies have found that the use of CRISPR-Cas may lead to undesirable

off-target effects.16-28 For this reason, several groups are developing methods to

detect29-32 and reduce33-45 off-target effects.

The CRISPR Locus and the Mechanism for CRISPR-Cas Technology

Bacteria and archaea use CRISPR-Cas systems for adaptive immunity.

The CRISPR locus consists of short palindromic repeats separated by short

nonrepetitive sequences called “spacers”. Spacers originate from invading

sources of DNA, such as phages, that are copied and incorporated into the

CRISPR locus when an infection occurs. Near this locus, a second locus includes

a set of genes that encode CRISPR-associated endonucleases (Cas), which

introduce cuts in the genome. When a repeat infection occurs from the same

invading DNA, an RNA molecule (CRISPR RNA, or crRNA) will form a complex

with Cas and a transactivating crRNA (tracrRNA) to guide the nuclease to the

exogenous sequence. The Cas-RNA complex will recognize the DNA target that

is complementary to the crRNA and adjacent to a specific 3-nucleotide locus (the

protospacer-adjacent motif, or PAM). The complex then cuts and deactivates the

invading DNA (Figure 1).

1. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA and Charpentier E. A program- mable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816-821.

2. Gasiunas G, Barrangou R, Horvath P and Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012;109:E2579-2586.

3. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819-823.

4. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Sci- ence. 2013;339:823-826.

5. Dow LE. Modeling Disease In Vivo With CRIS- PR/Cas9. Trends Mol Med. 2015;21:609-621.

6. Tebas P, Stein D, Tang WW, et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N Engl J Med. 2014;370:901-910.

7. Reardon S. First CRISPR clinical trial gets green light from US panel. Nature News. 2016;

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 M and Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol. 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 Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43:1565-1575.

12. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J and Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005;60:174-182.

13. Pourcel C, Salvignol G and Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. 2005;151:653-663.

14. Barrangou R, Fremaux C, Deveau H, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315:1709-1712.

15. Brouns SJ, Jore MM, Lundgren M, et al. Small CRISPR RNAs guide antiviral defense in pro- karyotes. Science. 2008;321:960-964.

16. Marraffini LA and Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science. 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.

5 For Research Use Only. Not for use in diagnostic procedures.

An overview of recent publications featuring Illumina technology

Figure 1. The CRISPR locus consists of short palindromic repeats separated by short nonrepetitive sequences, called “spacers”. Spacers originate from invading sources of DNA that are copied and incorporated into the CRISPR locus when an infection occurs. Near this locus, another includes a set of genes that encode Cas endonucleases, enzymes that introduce cuts in the genome.

Although different nucleases are associated with CRISPR activity in different bacteria,

most of the topics in this review refer to Streptococcus pyogenes, which relies on the

endonuclease Cas9. For this reason, the review refers to CRISPR-Cas9 technology.

The CRISPR-Cas9 system requires 2 RNA molecules: crRNA, transcribed from

the DNA spacers, and tracrRNA, whose interaction with crRNA is a structural

requirement for the recruitment of Cas9 (Figure 2). In a landmark study,46 these 2

RNAs were hybridized to create a single-guide RNA (sgRNA). This simplified Cas9-

sgRNA system demonstrated gene editing properties.

Figure 2. Cartoon representation of the molecular structure of Cas9 (transparent) and sgRNA (red) interacting with DNA (blue).

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. CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 2013;41:9584- 9592.

27. Lin Y, Cradick TJ, Brown MT, et al. CRISPR/ Cas9 systems have off-target activity with insertions or deletions betwe