Table I.

Strategies using the CRISPR/Cas9 system for the treatment of diseases.

Disease	In vitro analysis	In vivo analysis	Result	Conclusion	Refs.
AIDS	CRISPR/Cas9 transfected WT iPSCs to generate homozygous CCR5−/− CC	NOD/SCID/IL2Rγnull; NSG mice were engrafted with R5−/−42 generated from CD34+ HSPCs by ZFN HIV+ patients were engrafted with CCR5−/− CD4 T cells by ZFN	i) Resistance to HIV-1 infection In vivo animal model: Suppression of HIV-1 Replication iii) In vivo human model: HIV-1 replication could not be controlled	CRISPR appears more promising than ZFN due to long-term effect and ability to mutate both CCR5 alleles	108-112, 114-116
Neurodegenerative diseases	i) Exon 5 of PSEN2 gene in iPSCs derived from AD patients were corrected using CRISPR/Cas9	ii) Healthy PSEN2N141 iPSC-derived BFCNs underwent CRISPR/Cas9 gene correction and were transplanted into AD mice	i) Reversal in elevated amyloid plaques (Aβ42/40) ii) Neurological development	Reduction in AD neuropathological symptoms. Further analysis required for in vivo human therapy	70-74
DMD	i) Exon 44 knock-in in DMD gene in iPSCs of DMD patients using TALENs and CRISPR ii) 45-55 exon removal of DMD gene in iPSC-derived cardiomyocytes	iii) Targeting exon 23 in DMD gene in mdx mice	ii) Normal function of miR32 iii) Gene correction, however causing indels due to more NHEJ repair compared to HDR	Larger size deletion by CRISPR corrected errors on factors affecting dystrophin function. However, due to off-target effects, further analysis to modify CRISPR/Cas9 is necessary to reduce off-target effects	101-103
Haemophilia	i) iPSCs of HB patient were transfected with CRISPR/Cas9 and differentiated into Hepatocytes ii) Modified HDR cassette containing coding sequence of WT at cFIX mutation locus and modified codon at TALEN/Cas9 binding site	iii) AAV8 using CRISPR-SaCas9 in hepatocytes of HB mice to restore F9 gene creating DSB and inserting cDNA to intron iv) Injection of in vitro generated hepatocytes into NOD/SCID mice	ii) Enhance HDR activation leading to decrease in off0target Repair iii) Genotypic correction and phenotypic improvement iv) Secretion of human FIX in mice	Improvements in reduction of off-target effects caused by CRISPR, which is promising for further analysis of CRISPR for treatment in humans	122-125
ASD	i) RNP-induced Cas9 on NPCs of Ai9 tdTomato mouse	ii) AAV9 with MCO was injected into Mecp2 KO mice iii) RNP-induced Cas9 in NPCs of Ai9 tdTomato mouse from hippocampus, striatum and cortex iv) CRISPR-Gold with Cas9 or Cpf1 on Thy1- YFP and Ai9 mice	ii) Behavioural development i and iii) Significant genome editing iv) Reduction in XFS	High dose of Mecp2 in liver cells causing liver metabolism dysfunction However, CRISPR-Gold showed minimal off-target effects and no effects on immune system98	129-134
SCD	Using pX330 plasmid with CRISPR/Cas9 that contains truncated sgRNA G10 to target first exon in HBB gene in human HSPCs from SCD patients	-	Highly efficient gene correction and reduction in mortality	CRISPR showed fewer off-target effects and better HDR function compared with genome editing by ZFN	120-123

CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR-associated protein; AIDS, acquired immune deficiency syndrome; HIV, human immunodeficiency virus; DMD, Duchenne muscular dystrophy; ASD, autism spectrum disorder; SCD, sickle cell disease; WT, wild-type; iPSCs, induced pluripotent stem cells; CCR5, CXC chemokine receptor 5; NOD/SCID/IL2Rγ^null, non-obese diabetic/severe combined immunodeficient/interleukin 2Rγ^null (NSG); PSEN2, presenilin 2; AD, Alzheimer's disease; ZFN, zinc-finger nucleases; BFCNs, basal forebrain cholinergic neurons; TALENs, transcription activator-like effector nucleases; NHEJ, non-homologous end joining; HDR, homology-directed repair; FIX, coagulation factor IX; cFIX, canine FIX; AAV, adeno-associated virus; DSB, double-strand break; RNP, ribonucleoprotein; Mecp2, methyl CpG binding protein 2 gene; MCO, brain isoform of Mecp2; HBB, haemoglobin subunit β; HSPCs, hematopoietic stem/progenitor cells.