Table 1.

Study characteristics of CRISPR/Cas approaches in DM1.

Study	CRISPR/Cas Strategy, Cas Type, (CTG•CAG)n Length	sgRNA 1	Delivery	DM1 Biomarkers Examined	Cell Types Used
(CTG•CAG)n Excision
Van Agtmaal et al., 2017 [18]	(CTG•CAG)n excision hSpCas9 Unaffected: n = 5/13 DM1: n = ~550/2600	Repeat-flanking sgRNAs Upstream: 11 bp Downstream: 51 bp	Nucleofection: Cas9 and sgRNA expression plasmids	DMPK, SIX5 and DM1-AS RNA expression DMPK protein expression DMPK RNA subcellular distribution (CUG)n RNA foci and MBNL1 foci BIN-1 ex11 and DMD ex78 splicing Myogenic differentiation	Human: Immortalized unaffected and DM1 myoblasts Murine: Immortalized DM500 myoblasts
Provenzano et al., 2017 [22]	(CTG•CAG)n excision eSpCas9 Unaffected: n = 5/13 DM1: n = 300–1000	Repeat-flanking sgRNAs Upstream: 189 bp Downstream: 305 bp	Lipofection and nucleofection: Cas9 and sgRNA expression plasmids	DMPK RNA expression (CUG)n RNA foci and MBNL1 foci SERCA1 ex22 and INSR ex11 splicing Myogenic differentiation DMPK protein expression	Human: HEK293FT cells Immortalized unaffected and DM1 inducible MYOD1-expressing fibroblasts
Dastidar et al., 2018 [21]	(CTG•CAG)n excision hSpCas9 Unaffected: n = 5/13 DM1: n = 1000–1700	Repeat-flanking sgRNAs Upstream: 24 bp Downstream: 51 bp	Lentiviral transduction: CMV-hspCas9-EF1-GFP virus Nucleofection: Cas9/sgRNA RNP complexes	DMPK RNA expression (CUG)n RNA foci andMBNL1 foci SERCA1 ex22 splicing	Human: DM1 iPSC-derived myogenic cells DM1 primary myoblasts DM1 iPSCs
Wang et al., 2018 [23]	(CTG•CAG)n excision SpCas9 and SaCas9 Unaffected: n = 5 DM1: n ≥ 2000	Repeat-flanking sgRNAs Upstream: <220 bp Downstream: <220 bp	Lipofection: Cas9 and sgRNA expression plasmids	(CUG)n and (CAG)n RNA foci	Human: HEK293FT cells DM1 iPSC-derived neural stem cells
Yanovsky-Dagan et al., 2019 [24]	(CTG•CAG)n excision SpCas9 Unaffected: n = 5 DM1: n = 2000	Repeat-flanking sgRNAs Upstream: 11 bp Downstream: 47 bp	Transfection: Cas9 and sgRNA expression plasmids	SIX5 expression DM1 locus CpG hypermethylation H3K9me3 enrichment	Human: HEK293T cells DM1 hESCs
Insertion Poly(A) Signal Cassette
Wang et al., 2018 [23]	Insertion poly(A) signal cassette SpCas9n Unaffected: n = 5 DM1: n ≥ 2000	Paired sgRNAs between stop codon and repeat Donor template: 5′ HR arm: 97 bp 3′ HR arm: 184 bp	Lipofection: Cas9n and sgRNA expression plasmids and donor template	(CUG)n RNA foci DMPK RNA subcellular distribution MAPT ex3, MBNL1/2 ex7, SERCA1 ex22 and INSR ex11 splicing Myogenic differentiation	Human: DM1 iPSCs DM1 iPSC-derived neural stem cells
dCas9-Mediated Repeat Transcription Inhibition, Repeat RNA Visualization and Degradation
Pinto et al., 2017 [25]	(CTG)n transcription block dSpCas9 and dSaCas9 (CTG•CAG)n plasmids n = 0/12/40/240/480/960 (interrupted) DM1: n ≥ 2000	(CAG)6 sgRNA	Transfection: plasmids expressing dCas9 and sgRNAs Transduction: AAV2/6-dSaCas9-sgRNA and AAV2/9-dSaCas9-sgRNA	(CUG)n RNA foci (CUG)n RNA expression Multiple splice modes (RNA-seq) RAN translation Expression of (CUG)n and (CAG)n repeat-containing transcripts Myotonia	Human: Transiently transfected HEK293T and HeLa cells DM1 primary myoblasts Murine: HSALR mice (EDL muscle ex vivo, tibialis anterior and gastrocnemius in vivo)
Batra et al., 2017 [6]	(CUG)n RNA visualization and degradation 666dSpCas9, dSpCas9-GFP and PIN-dSpCas9 (CTG•CAG)n plasmids n = 105 n = 960 (interrupted) DM1: n ≥ 2700	(CAG)n sgRNA	Lipofection: Cas9 and sgRNA expression plasmids Lentiviral transduction: U6-sgRNA and EFS-PIN-dCas9	(CUG)n RNA expression (CUG)n RNA foci MBNL1 foci Multiple splice modes (RNA-seq) Expression of (CUG)n and (CAG)n repeat-containing transcripts	Human: DM1 primary myoblasts Primate: COS-M6 cells

¹ bp upstream/downstream indicate CRISPR/Cas9 cleavage distance in base pairs from first/last triplet in the repeat.