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Using CRISPR-Cas9 Genome Editing to Enhance Cell Based Therapies for the Treatment of Diabetes Mellitus

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Abstract

Human pluripotent stem cells (hPSCs) have the ability to form all cell types of the body, making them an excellent potential source of insulin-producing pancreatic β-cells for diabetes treatment. To generate these cells in vitro requires a complete understanding of the normal process of pancreas development: an objective greatly aided by CRISPR-Cas9 genome-editing technology. First identified as the adaptive immune system of bacteria, CRISPR-Cas9 uses RNA to specifically target a DNA endonuclease to the genome, generating a double-strand break that can either be repaired by the error-prone NHEJ or via HDR. From the first demonstration that CRISPR-Cas can be programmed to cleave DNA in 2012, the field has advanced fast and now includes examples of targeting in many model organisms as well as gene knockout or reporter hPSC lines that will aid in the production of specific cell types, such as pancreatic β-cells.

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Abbreviations

AAV:

Adeno-associated virus

Cas:

CRISPR-associated

CRISPR:

Clustered regularly-interspaced short palindromic repeats

dCas9:

Dead Cas9

DHFR:

Dihydrofolate reductase

DSB:

Double-strand break

gRNA:

Guide RNA

GUIDE-seq:

Genome-wide unbiased identification of double-strand breaks enabled by sequencing

HDR:

Homology-directed repair

hESC:

Human embryonic stem cells

iCRISPR:

Induced CRISPR

iPSC:

Induced pluripotent stem cell

MODY4:

Mature-onset diabetes of the young 4

Ngn3:

Neurogenin3

NHEJ:

Nonhomologous end-joining

PAM:

Protospacer-adjacent motif

Pdx1:

Pancreatic and duodenal homeobox 1

TALEN:

Transcription activator-like effector nuclease

tracrRNA:

Trans-activating crRNA

ZFN:

Zinc-finger nuclease

References

  1. Rezania A, Bruin JE, Arora P, Rubin A, Batushansky I, Asadi A, et al. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol. 2014;32(11):1121–33.

    Article  CAS  PubMed  Google Scholar 

  2. Pagliuca FW, Millman JR, Gürtler M, Segel M, Van Dervort A, Ryu JH, et al. Generation of functional human pancreatic β cells in vitro. Cell. 2014;159(2):428–39.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ding Q, Lee Y-K, Schaefer EAK, Peters DT, Veres A, Kim K, et al. A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell. 2012;12(2):238–51.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Gaj T, Gersbach CA, Barbas III CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013;31(7):397–405.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hockemeyer D, Jaenisch R. Gene targeting in human pluripotent cells. Cold Spring Harb Symp Quant Biol. 2010;75:201–9.

    Article  CAS  PubMed  Google Scholar 

  6. Ishino Y, Shinagawa H, Makino K, Amemura M, 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(12):5429–33.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Jansen R, Embden JDAV, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43(6):1565–75.

    Article  CAS  PubMed  Google Scholar 

  8. Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327(5962):167–70.

    Article  CAS  PubMed  Google Scholar 

  9. Makarova KS, Aravind L, Grishin NV, Rogozin IB, Koonin EV. A DNA repair system specific for thermophilic Archaea and bacteria predicted by genomic context analysis. Nucleic Acids Res. 2002;30(2):482–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005;151(Pt 8):2551–61.

    Article  CAS  PubMed  Google Scholar 

  11. Pourcel C, Salvignol G, 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(Pt 3):653–63.

    Article  CAS  PubMed  Google Scholar 

  12. Mojica FJM, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005;60(2):174–82.

    Article  CAS  PubMed  Google Scholar 

  13. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315(5819):1709–12.

    Article  CAS  PubMed  Google Scholar 

  14. Deveau H, Barrangou R, Garneau JE, Labonté J, Fremaux C, Boyaval P, et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J Bacteriol. 2008;190(4):1390–400.

    Article  CAS  PubMed  Google Scholar 

  15. Horvath P, Romero DA, Coute-Monvoisin AC, Richards M, Deveau H, Moineau S, et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J Bacteriol. 2008;190(4):1401–12.

    Article  CAS  PubMed  Google Scholar 

  16. Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006;1:7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Garneau JE, Dupuis M-È, Villion M, Romero DA, Barrangou R, Boyaval P, et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010;468(7320):67–71.

    Article  CAS  PubMed  Google Scholar 

  18. Heler R, Marraffini LA, Bikard D. Adapting to new threats: the generation of memory by CRISPR‐Cas immune systems. Mol Microbiol. 2014;93(1):1–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Haft DH, Selengut J, Mongodin EF, Nelson KE. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput Biol. 2005;1(6):e60.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Kunin V, Sorek R, Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol. 2007;8(4):R61.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Grissa I, Vergnaud G, Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics. 2007;8(1):172.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Hale CR, Majumdar S, Elmore J, Pfister N, Compton M, Olson S, et al. Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Mol Cell. 2012;45(3):292–302.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Pougach K, Semenova E, Bogdanova E, Datsenko KA, Djordjevic M, Wanner BL, et al. Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol Microbiol. 2010;77(6):1367–79.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pul Ü, Wurm R, Arslan Z, Geißen R, Hofmann N, Wagner R. Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol Microbiol. 2010;75(6):1495–512.

    Article  CAS  PubMed  Google Scholar 

  25. Makarova KS, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, et al. Evolution and classification of the CRISPR–Cas systems. Nat Rev Microbiol. 2011;9(6):467–77.

    Article  CAS  PubMed  Google Scholar 

  26. Carte J, Christopher RT, Smith JT, Olson S, Barrangou R, Moineau S, et al. The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus. Mol Microbiol. 2014;93(1):98–112.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hochstrasser ML, Doudna JA. Cutting it close: CRISPR-associated endoribonuclease structure and function. Trends Biochem Sci. 2015;40(1):58–66.

    Article  CAS  PubMed  Google Scholar 

  28. Barrangou R, Marraffini LA. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol Cell. 2014;54(2):234–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Gasiunas G, Sinkunas T, Siksnys V. Molecular mechanisms of CRISPR-mediated microbial immunity. Cell Mol Life Sci. 2013;71(3):449–65.

    Article  PubMed Central  CAS  Google Scholar 

  30. Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012;482(7385):331–8.

    Article  CAS  PubMed  Google Scholar 

  31. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011;471(7340):602–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. van der Oost J, Westra ER, Jackson RN, Wiedenheft B. Unravelling the structural and mechanistic basis of CRISPR–Cas systems. Nat Rev Microbiol. 2014;12(7):479–92.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science. 2008;322:1843–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mojica FJM, Diez-Villasenor C, Garcia-Martinez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology. 2009;155(3):733–40.

    Article  CAS  PubMed  Google Scholar 

  35. Marraffini LA, Sontheimer EJ. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature. 2010;463(7280):568–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Jore MM, Lundgren M, van Duijn E, Bultema JB, Westra ER, Waghmare SP, et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol. 2011;18(5):529–36.

    Article  CAS  PubMed  Google Scholar 

  37. van der Oost J, Jore MM, Westra ER, Lundgren M, Brouns SJJ. CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem Sci. 2009;34(8):401–7.

    Article  PubMed  CAS  Google Scholar 

  38. Semenova E, Jore MM, Datsenko KA. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc Natl Acad Sci U S A. 2011;108(25):10098–103.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yosef I, Goren MG, Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 2012;40(12):5569–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–21.

    Article  CAS  PubMed  Google Scholar 

  41. Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res. 2013;41(20):e188.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Westra ER, Semenova E, Datsenko KA, Jackson RN, Wiedenheft B, Severinov K, et al. Type I-E CRISPR-Cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition. PLoS Genet. 2013;9(9):e1003742.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Fineran PC, Gerritzen MJH, Suarez-Diez M, Kunne T, Boekhorst J, van Hijum SAFT, et al. Degenerate target sites mediate rapid primed CRISPR adaptation. Proc Natl Acad Sci U S A. 2014;111(16):E1629–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Gudbergsdottir S, Deng L, Chen Z, Jensen JVK, Jensen LR, She Q, et al. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers. Mol Microbiol. 2010;79(1):35–49.

    Article  PubMed  CAS  Google Scholar 

  45. Brouns SJJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH, Snijders APL, et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008;321(5891):960–4.

    Article  CAS  PubMed  Google Scholar 

  46. Hale C, Kleppe K, Terns RM, Terns MP. Prokaryotic silencing (psi)RNAs in Pyrococcus furiosus. RNA. 2008;14(12):2572–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Carte J, Wang R, Li H, Terns RM, Terns MP. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 2008;22(24):3489–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science. 2010;329(5997):1355–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Sashital DG, Jinek M, Doudna JA. An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3. Nat Struct Mol Biol. 2011;18(6):680–7.

    Article  CAS  PubMed  Google Scholar 

  50. Gesner EM, Schellenberg MJ, Garside EL, George MM, MacMillan AM. Recognition and maturation of effector RNAs in a CRISPR interference pathway. Nat Struct Mol Biol. 2011;18(6):688–92.

    Article  CAS  PubMed  Google Scholar 

  51. Lintner NG, Kerou M, Brumfield SK, Graham S, Liu H, Naismith JH, et al. Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for antiviral defense (CASCADE). J Biol Chem. 2011;286(24):21643–56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Hatoum-Aslan A, Maniv I, Marraffini LA. Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc Natl Acad Sci U S A. 2011;108(52):21218–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Richter H, Zoephel J, Schermuly J, Maticzka D, Backofen R, Randau L. Characterization of CRISPR RNA processing in Clostridium thermocellum and Methanococcus maripaludis. Nucleic Acids Res. 2012;40(19):9887–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Nam KH, Haitjema C, Liu X, Ding F, Wang H, DeLisa MP, et al. Cas5d protein processes Pre-crRNA and assembles into a cascade-like interference complex in subtype I-C/Dvulg CRISPR-Cas system. Structure. 2012;20(9):1574–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;156(5):935–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Wright AV, Sternberg SH, Taylor DW, Staahl BT, Bardales JA, Kornfeld JE, et al. Rational design of a split-Cas9 enzyme complex. Proc Natl Acad Sci U S A. 2015;112(10):2984–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Gasiunas G, Barrangou R. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012;109(39):E2579–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Sternberg SH, Redding S, Jinek M, Greene EC, Doudna JA. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature. 2015;507(7490):62–7.

    Article  CAS  Google Scholar 

  59. Szczelkun MD, Tikhomirova MS, Sinkunas T, Gasiunas G, Karvelis T, Pschera P, et al. Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes. Proc Natl Acad Sci U S A. 2014;111(27):9798–803.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. eLife. 2013;2, e00471.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug II RG, et al. In vivo genome editing using a high-efficiency TALEN system. Nature. 2013;490(7422):114–8.

    Google Scholar 

  62. Campbell JM, Hartjes KA, Nelson TJ, Xu X, Ekker SC. New and TALENted genome engineering toolbox. Circ Res. 2013;113(5):571–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Orlando SJ, Santiago Y, DeKelver RC, Freyvert Y, Boydston EA, Moehle EA, et al. Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res. 2010;38(15):e152.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Wefers B, Meyer M, Ortiz O. Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. Proc Natl Acad Sci U S A. 2013;110(10):3782–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Ding Q, Regan SN, Xia Y, Oostrom LA, Cowan CA, Musunuru K. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell. 2013;12(4):393–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Krentz NAJ, Nian C, Lynn FC. TALEN/CRISPR-mediated eGFP knock-in add-on at the OCT4 locus does not impact differentiation of human embryonic stem cells towards endoderm. PLoS One. 2014;9(12):e114275.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Doyon Y, Vo TD, Mendel MC, Greenberg SG, Wang J, Xia DF, et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods. 2010;8(1):74–9.

    Article  PubMed  CAS  Google Scholar 

  68. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281–308.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. 2014;3, e04766.

    PubMed  PubMed Central  Google Scholar 

  70. Dang Y, Jia G, Choi J, Ma H, Anaya E, Ye C, et al. Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency. Genome Biol. 2015;16:280.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  71. Smith C, Gore A, Yan W, Abalde-Atristain L, Li Z, He C, et al. Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell. 2014;15(1):12–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Veres A, Gosis BS, Ding Q, Collins R, Ragavendran A, Brand H, et al. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell. 2014;15(1):27–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Suzuki K, Yu C, Qu J, Li M, Yao X, Yuan T, et al. Targeted gene correction minimally impacts whole-genome mutational load in human-disease-specific induced pluripotent stem cell clones. Cell Stem Cell. 2014;15(1):31–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2014;33(2):187–97.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  75. Kondo S, Ueda R. Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics. 2013;195(3):715–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Sebo ZL, Lee HB, Peng Y, Guo Y. A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering. Fly. 2014;8(1):52–7.

    Article  CAS  PubMed  Google Scholar 

  77. Kuscu C, Arslan S, Singh R, Thorpe J, Adli M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol. 2014;32(7):677–83.

    Article  CAS  PubMed  Google Scholar 

  78. Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD, Dadon DB, et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol. 2014;32(7):670–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Knight SC, Xie L, Deng W, Guglielmi B, Witkowsky LB, Bosanac L, et al. Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science. 2015;350(6262):823–6.

    Article  CAS  PubMed  Google Scholar 

  80. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31(9):827–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 2014;24(1):132–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Park J, Bae S, Kim J-S. Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics. 2015;31(24):4014–6.

    PubMed  Google Scholar 

  83. Li W, Xu H, Xiao T, Cong L, Love MI, Zhang F, et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 2014;15(12):554.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  84. Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol. 2014;32(12):1262–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Gratz SJ, Ukken FP, Rubinstein CD, Thiede G, Donohue LK, Cummings AM, et al. Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics. 2014;196(4):961–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013;31(9):839–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Cradick TJ, Fine EJ, Antico CJ, Bao G. CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 2013;41(20):9584–92.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014;32(3):279–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Muller M, Lee CM, Gasiunas G, Davis TH, Cradick TJ, Siksnys V, et al. Streptococcus thermophilus CRISPR-Cas9 systems enable specific editing of the human genome. Mol Ther. 2016;24:636–44.

    Article  PubMed  CAS  Google Scholar 

  90. Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013;10(11):1116–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods. 2014;11(4):399–402.

    Article  CAS  PubMed  Google Scholar 

  93. Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014;32(6):569–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol. 2014;32(6):577–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Fujii W, Kawasaki K, Sugiura K, Naito K. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res. 2013;41(20):e187.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Hara S, Tamano M, Yamashita S, Kato T, Saito T, Sakuma T, et al. Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9. Sci Rep. 2015;5:11221.

    Google Scholar 

  98. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013;10(10):977–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 2013;23(10):1163–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell. 2014;54(4):698–710.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013;41(15):7429–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Thakore PI, D’Ippolito AM, Song L, Safi A, Shivakumar NK, Kabadi AM, et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods. 2015;12(12):1143–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell. 2014;159(3):647–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013;154(2):442–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013;152(5):1173–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Hemphill J, Borchardt EK, Brown K, Asokan A, Deiters A. Optical control of CRISPR/Cas9 gene editing. J Am Chem Soc. 2015;137(17):5642–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. González F, Zhu Z, Shi Z-D, Lelli K, Verma N, Li QV, et al. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell. 2014;15(2):215–26.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  108. Chen Y, Cao J, Xiong M, Petersen AJ, Dong Y, Tao Y, et al. Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell Stem Cell. 2015;17(2):233–44.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  109. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520(7546):186–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163(3):759–71.

    Article  CAS  PubMed  Google Scholar 

  111. Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol. 2015;33(1):102–6.

    Article  CAS  PubMed  Google Scholar 

  112. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014;159(2):440–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature. 2014;514(7522):380–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Jia H, Wang N. Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS One. 2014;9(4):e93806.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  115. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 2013;41(7):4336–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Lo TW, Pickle CS, Lin S, Ralston EJ, Gurling M. Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. Genetics. 2013;195(2):331–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Zhao P, Zhang Z, Ke H, Yue Y, Xue D. Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system. Cell Res. 2014;24(2):247–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Xiao A, Wang Z, Hu Y, Wu Y, Luo Z, Yang Z, et al. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res. 2013;41(14):e141.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Blitz IL, Biesinger J, Xie X, Cho KWY. Biallelic genome modification in F0 Xenopus tropicalis embryos using the CRISPR/Cas system. Genesis. 2013;51(12):827–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Guo X, Zhang T, Hu Z, Zhang Y, Shi Z, Wang Q, et al. Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis. J Embryol Exp Morphol. 2014;141(3):707–14.

    CAS  Google Scholar 

  121. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013;31(3):227–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Yang H, Wang H, Jaenisch R. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nat Protoc. 2014;9(8):1956–68.

    Article  CAS  PubMed  Google Scholar 

  124. Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014;156(4):836–43.

    Article  CAS  PubMed  Google Scholar 

  125. Shapiro A, Lakey J, Ryan E. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med. 2000;343(4):230–8.

    Article  CAS  PubMed  Google Scholar 

  126. Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol. 2008;26(4):443–52.

    Article  CAS  PubMed  Google Scholar 

  127. Kelly OG, Chan MY, Martinson LA, Kadoya K, Ostertag TM, Ross KG, et al. Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells. Nat Biotechnol. 2011;29(8):750–6.

    Article  CAS  PubMed  Google Scholar 

  128. Rezania A, Bruin JE, Xu J, Narayan K, Fox JK, O’Neil JJ, et al. Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo. Stem Cells. 2013;31(11):2432–42.

    Article  CAS  PubMed  Google Scholar 

  129. Rezania A, Bruin J, Riedel M, Mojibian M. Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. Diabetes. 2012;61(8):2016–29.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Capecchi MR. Altering the genome by homologous recombination. Science. 1989;244(4910):1288–92.

    Article  CAS  PubMed  Google Scholar 

  131. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell. 1998;95(3):379–91.

    Article  CAS  PubMed  Google Scholar 

  133. Radzisheuskaya A, Le Bin CG, dos Santos RL, Theunissen TW, Castro LFC, Nichols J, et al. A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages. Nat Cell Biol. 2013;15(6):579–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Gerrard L, Zhao D, Clark AJ, Cui W. Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency. Stem Cells. 2005;23(1):124–33.

    Article  CAS  PubMed  Google Scholar 

  135. Thyagarajan B, Liu Y, Shin S, Lakshmipathy U, Scheyhing K, Xue H, et al. Creation of engineered human embryonic stem cell lines using phiC31 integrase. Stem Cells. 2008;26(1):119–26.

    Article  CAS  PubMed  Google Scholar 

  136. Zwaka TP, Thomson JA. Homologous recombination in human embryonic stem cells. Nat Biotechnol. 2003;21(3):319–21.

    Article  CAS  PubMed  Google Scholar 

  137. Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol. 2009;27(9):851–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol. 2011;29(8):731–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Rizzino A. Concise review: the Sox2-Oct4 connection: critical players in a much larger interdependent network integrated at multiple levels. Stem Cells. 2013;31(6):1033–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  140. Brafman DA, Moya N, Allen-Soltero S, Fellner T, Robinson M, McMillen ZL, et al. Analysis of SOX2-expressing cell populations derived from human pluripotent stem cells. Stem Cell Rep. 2013;1(5):464–78.

    Article  CAS  Google Scholar 

  141. Fischer Y, Ganic E, Ameri J, Xian X, Johannesson M, Semb H. NANOG reporter cell lines generated by gene targeting in human embryonic stem cells. PLoS One. 2010;5(9), e12533.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  142. Zorn AM, Wells JM. Vertebrate endoderm development and organ formation. Annu Rev Cell Dev Biol. 2009;25(1):221–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Kearns NA, Genga RMJ, Enuameh MS, Garber M, Wolfe SA, Maehr R. Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development. 2014;141(1):219–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Balboa D, Weltner J, Eurola S, Trokovic R, Wartiovaara K, Otonkoski T. Conditionally stabilized dCas9 activator for controlling gene expression in human cell reprogramming and differentiation. Stem Cell Rep. 2015;5(3):448–59.

    Article  CAS  Google Scholar 

  145. Wang P, Rodriguez RT, Wang J, Ghodasara A, Kim SK. Targeting SOX17 in human embryonic stem cells creates unique strategies for isolating and analyzing developing endoderm. Cell Stem Cell. 2011;8(3):335–46.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  146. Jonsson J, Carlsson L, Edlund T, Edlund H. Insulin-promoter-factor 1 is required for pancreas development in mice. Nature. 1994;371(6498):606–9.

    Article  CAS  PubMed  Google Scholar 

  147. Offield M, Jetton T, Labosky P, Ray M, Stein R, Magnuson M, et al. PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development. 1996;122(3):983–95.

    CAS  PubMed  Google Scholar 

  148. Schwitzgebel VM, Mamin A, Brun T, Ritz-Laser B, Zaiko M, Maret A, et al. Agenesis of human pancreas due to decreased half-life of insulin promoter factor 1. J Clin Endocrinol Metab. 2003;88(9):4398–406.

    Article  CAS  PubMed  Google Scholar 

  149. Stoffers DA, Zinkin NT, Stanojevic V, Clarke WL, Habener JF. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet. 2004;15(1):106–10.

    Article  Google Scholar 

  150. Stoffers DA, Ferrer J, Clarke WL, Habener JF. Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nat Genet. 1997;17(2):138–9.

    Article  CAS  PubMed  Google Scholar 

  151. Nicolino M, Claiborn KC, Senée V, Boland A, Stoffers DA, Julier C. A novel hypomorphic PDX1 mutation responsible for permanent neonatal diabetes with subclinical exocrine deficiency. Diabetes. 2010;59(3):733–40.

    Article  CAS  PubMed  Google Scholar 

  152. Zhu Z, Verma N, González F, Shi Z-D, Huangfu D. A CRISPR/Cas-mediated selection-free knockin strategy in human embryonic stem cells. Stem Cell Rep. 2015;4(6):1–27.

    Article  CAS  Google Scholar 

  153. Gu G, Dubauskaite J, Melton DA. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development. 2002;129(10):2447–57.

    CAS  PubMed  Google Scholar 

  154. Gradwohl G, Dierich A, LeMeur M. neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci U S A. 2000;97(4):1607–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  155. Schwitzgebel VM, Scheel DW, Conners JR, Kalamaras J, Lee JE, Anderson DJ, et al. Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development. 2000;127(16):3533–42.

    CAS  PubMed  Google Scholar 

  156. Apelqvist Å, Li H, Sommer L, Beatus P, Anderson DJ, Honjo T, et al. Notch signalling controls pancreatic cell differentiation. Nature. 1999;400(6747):877–81.

    Article  CAS  PubMed  Google Scholar 

  157. Pan FC, Wright C. Pancreas organogenesis: from bud to plexus to gland. Dev Dyn. 2011;240(3):530–65.

    Article  CAS  PubMed  Google Scholar 

  158. Piper K, Brickwood S, Turnpenny L. Beta cell differentiation during early human pancreas development. J Endocrinol. 2004;181(1):11–23.

    Article  CAS  PubMed  Google Scholar 

  159. Rubio-Cabezas O, Jensen JN, Hodgson MI, Codner E, Ellard S, Serup P, et al. Permanent neonatal diabetes and enteric anendocrinosis associated with biallelic mutations in NEUROG3. Diabetes. 2011;60(4):1349–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. McGrath PS, Watson CL, Ingram C, Helmrath MA, Wells JM. The basic helix-loop-helix transcription factor NEUROG3 is required for development of the human endocrine pancreas. Diabetes. 2015;64(7):2497–505.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  161. Teo AKK, Gupta MK, Doria A, Kulkarni RN. Dissecting diabetes/metabolic disease mechanisms using pluripotent stem cells and genome editing tools. Mol Metab. 2015;4(9):593–604.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Diabetes Research Group, Child and Family Research Institute, 950 28th Ave. W., Vancouver, BC, Canada, V5Z 4H4

    Nicole A. J. Krentz & Francis C. Lynn

  2. Departments of Surgery/Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada

    Nicole A. J. Krentz & Francis C. Lynn

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  1. Nicole A. J. Krentz
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  2. Francis C. Lynn
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  1. Ottawa, Ontario, Canada

    Kursad Turksen

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Krentz, N.A.J., Lynn, F.C. (2016). Using CRISPR-Cas9 Genome Editing to Enhance Cell Based Therapies for the Treatment of Diabetes Mellitus. In: Turksen, K. (eds) Genome Editing. Springer, Cham. https://doi.org/10.1007/978-3-319-34148-4_8

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