CRISPR-Cas3 induces broad and unidirectional genome editing in human cells - PubMed (original) (raw)
doi: 10.1038/s41467-019-13226-x.
Kazuto Yoshimi 3 4 5, Yuya Okuzaki 6, Peter Gee 6, Yayoi Kunihiro 3, Ekasit Sonpho 4 7, Huaigeng Xu 6, Noriko Sasakawa 6, Yuki Naito 8 9, Shinichiro Nakada 10, Takashi Yamamoto 11, Shigetoshi Sano 2, Akitsu Hotta 12, Junji Takeda 13 14, Tomoji Mashimo 15 16 17
Affiliations
- PMID: 31811138
- PMCID: PMC6897959
- DOI: 10.1038/s41467-019-13226-x
CRISPR-Cas3 induces broad and unidirectional genome editing in human cells
Hiroyuki Morisaka et al. Nat Commun. 2019.
Abstract
Although single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5'-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.
Conflict of interest statement
The authors declare no competing interests.
Figures
Fig. 1
CRISPR-Cas3 system mediates DNA cleavage in human cells. a Type I-E CRISPR effector is composed of crRNA, Cas3, and a large Cascade complex, which contains Cas5, Cas6, multiple Cas7, Cas8 (Cse1) recognizing the PAM, and two Cas11 (Cse2). b Schematic of the single strand annealing (SSA) assay used to evaluate DNA cleavage and annealing activity. After the transfection of 293T cells with individual Cas, crRNA, and reporter plasmids, dual luciferase activities (Firefly (Fluc) as a reporter and Renilla (Rluc) as the internal control) were sequentially measured (see Supplementary Fig. 2a). c Efficiencies of two plasmid sequences of pre-crRNA, pLRSR, which includes a leader, repeats and a single spacer, and pRSR, which includes repeats and a spacer, both transcribe pre-crRNA, and plasmids of mat-crRNA, pSR (see Supplementary Fig. 3b). Data are presented as mean ± SD. RLU relative light units. *P < 0.05, **P < 0.01, ANOVA with post-hoc Tukey test. d A series of SSA assays lacking specified components of the Cascade-Cas3 effector complex. Lipofection of the pre-crRNA and six Cas (3, 5–8, and 11)-coding plasmids was performed in 293T cells. crRNAnt nontarget crRNA. e Effect of PAM sequences on Cas3-mediated DNA cleavage activity in 293T cells (see Supplementary Table 1). f Effect of a single mismatch (gray) for 32-nt spacer sequences on Cas3-mediated DNA cleavage activity (see Supplementary Table 1). g Effect of Cas3 mutants in the HD nuclease domain (H74A) or in SF2-helicase domain motif 1 (K320N) or motif III (S483/T485A). h Comparison of DNA cleavage activity between Class 1 E. coli type I-E_, S. putrefaciens_ type I-F_, P. furiosus_ type I-G (Cas3), and Class 2 S.pyogenes type II-A (Cas9) (see Supplementary Table 1 and Supplementary Fig. 4). Source data are in the Source Data file.
Fig. 2
CRISPR-Cas3 facilitates a large deletion at endogenous targeted loci in 293T cells. a Schematic of the CRISPR-Cas3 system targeting human EMX1 and CCR5 loci. Primer sets (red arrows) used for a 3.7 kb PCR product of EMX1 and a 9.7 kb PCR product of CCR5 are shown. b Electrophoresis of the PCR products. CRISPR-Cas3 targeting AAG PAM or ATG PAM, but not TTT PAM, mediates deletions (see Supplementary Fig. 5a). c Comparison of the editing efficiency between Cas3 (red) and Cas9 (blue) via NGS of the PCR amplicons at various target sites (see Supplementary Table 3). d Cas3-mediated DNA deletion patterns via microarray-based capture sequencing at EMX1 and CCR5 loci in 293T cells. The deletions (blue bars) are aligned at the starting point of the distal end. e Microarray-based capture sequencing with Cas3/crRNAs (#1–6) targeting a 1-Mb region at the CCR5 loci (see Supplementary Fig. 11 for EMX1 loci). Cas3-mediated DNA deletion patterns are aligned with human genome assembly hg38. Dual crRNA (#1 and #5 or #6) are used for sticking in the interval region. f A surrogate reporter assay using mCherry-2A-EGFP plasmids to characterize Cas3-mediated knockouts in f or knock-ins in g. crRNA #1 (red arrows)/sgRNA (blue arrows) were designed at EGFP-coding sequences, and crRNAs/sgRNAs #2 and #3 were outside the coding sequences (see Supplementary Table 9). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, t tests with Bonferroni corrections. Percentage of CRISPR-mediated knockout cells for long-range deletions (mCherry(−)GFP(−)) and for small indel mutations (mCherry(+)GFP(−)). g CRISPR-mediated gene knock-in assay by flow cytometry. Cas3- or Cas9-mediated nucleotide substitution by dsDNA-dependent HDR restores GFP expression (see Supplementary Table 11) in 293T cells. Percentage of CRISPR-mediated knock-in cells (mCherry(+)GFP(+)). Source data are in the Source Data file.
Fig. 3
Off-target analysis of the CRISPR-Cas3 system. a Schematic of discordant read pairs and splitting reads by paired-end NGS. Cas3-mediated large mutations were detected by discordant and split read analyses, but Cas9-mediated small mutations were only detected by splitting read analysis. b WGS of 293T cells mediated by the CRISPR-Cas3 system targeting EMX1 on chromosome 2. *P < 0.05, Repetitive Grubbs test. c POT sites detected by GGGenome searches. Number of consecutive perfect matches or 32-nt partial matches flanking PAM sequences (AAG, TAG, AAC, GAG, AGG, and ATG) are shown. Every sixth position from the 5′ end of the PAM sequences was regarded as a match. d Microarray-based capture sequencing for POT sites selected by GGGenome in c. An off-target site of Cas9 targeting the EMX1 loci showed a high score and a variety of indel mutations. Source data are in the Source Data file.
Fig. 4
Cas3-mediated DMD exon skipping in induced pluripotent stem cells. a Schematic of Cas3 (red)- or Cas9 (blue)-mediated DMD exon skipping by the luciferase reporter assay. b Efficiency of the DMD exon skipping in 293 T cells by polycistronic plasmid vectors expressing CRISPR-Cas3 (see Supplementary Fig. 17a). Data are shown as mean ± s.e.m *P < 0.05, ANOVA with post-hoc Tukey test. c Scheme of skeletal muscle cell differentiation from iPSCs by doxycycline (Dox)-induced MYOD1 expression. bar indicates 100 µm. d RT-PCR analysis of the DMD exon 45 skipping in skeletal muscle cells differentiated from subclones of DMD patient-derived iPSCs treated with Cas3 (#3-22 and #9-3). The iPSC line Ex44 KI is a control line previously generated by the insertion of Exon 44 in front of Exon 45 using SpCas9. e The restored DMD protein detected by using Wes ProteinSimple in skeletal muscle cells differentiated from the exon skipping subclones of DMD-iPSCs. The iPSC line Ex44 KI was used as a positive control. Source data are in the Source Data file.
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