ABSTRACT Class 2 CRISPR–Cas proteins have been widely developed as genome editing and transcriptional regulating tools. Class 1 type I CRISPR–Cas constitutes ~60% of all the CRISPR–Cas

systems. However, only type I–B and I–E systems have been used to control mammalian gene expression and for genome editing. Here we demonstrate the feasibility of using type I–F system to

regulate human gene expression. By fusing transcription activation domain to _Pseudomonas aeruginosa_ type I–F Cas proteins, we activate gene transcription in human cells. In most cases,

type I–F system is more efficient than other CRISPR-based systems. Transcription activation is enhanced by elongating the crRNA. In addition, we achieve multiplexed gene activation with a

ENGINEERED MINIMAL TYPE I CRISPR-CAS SYSTEM FOR TRANSCRIPTIONAL ACTIVATION AND BASE EDITING IN HUMAN CELLS Article Open access 23 August 2024 HARNESSING NONCANONICAL CRRNA FOR HIGHLY

EFFICIENT GENOME EDITING Article Open access 07 May 2024 INTRODUCTION Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (_cas_) genes-based defence

systems protect bacteria and archaea against phage and other foreign genetic elements1,2,3. Since the identification of increasing number of _cas_ genes, the CRISPR–Cas systems have been

classified into two Classes (Class 1 and Class 2) and six types (Type I–VI)4 based on the different arrangements of _cas_ genes and the subunits of effector complexes5,6,7. Class 2

CRISPR–Cas systems, the best-studied system with single effector protein (e.g., Cas9, Cas12, or Cas13) for foreign DNA or RNA interference, are subdivided into Type II (Cas9), Type V

(Cas12), and Type VI (Cas13). In the past few years, Class 2 CRISPR–Cas systems have revolutionized both basic and clinical researches, enabling more rapid, precise, and robust genome

editing and modifications in cultured cells and animals8,9,10,11,12,13,14,15,16,17. However, there were only a few applications of Class 1 CRISPR–Cas (Type I, Type III and Type IV) system.

Class 1 type I CRISPR–Cas systems are the most prevalent (~60%) in both bacteria and archaea, whereas class 2 only makes up ~10% of all CRISPR–Cas systems18,19. Differing from the Class 2

CRISPR–Cas systems, the Class 1 type I system relies on Cascade (CRISPR-associated complex for antiviral defense complex) for DNA binding, which further recruits Cas3 to degrade the foreign

DNA20. Cascade, which recognizes and binds specific DNA, is a complex consist of multiple Cas proteins and CRISPR RNA (crRNA). CRISPR–Cas expression involves _cas_ genes expression and

CRISPR transcription, yielding a precursor crRNA (pre-crRNA). The pre-crRNA is processed at the repeat regions by Cse33, Cas621 or Csy422 to generate mature crRNA with different

characteristics. Other Cas proteins then bind onto the crRNA and assemble into a functional Cascade23,24,25,26. Cascade discriminates the self and non-self DNAs by recognizing the PAM

(proto-spacer adjacent motif) sequence27, which triggers a conformational change upon binding28,29. The conformational change finally recruits Cas3 for invasive DNA degradation20,30,31,32.

Compared to the widely used class 2 CRISPR–Cas systems, the multiple-subunit class 1 type I CRISPR–Cas system has distinct properties, for example, generating large fragment deletion in

genome editing with Cas333,34, and multiple subunits for different Cas protein–effector fusion strategies35. These differences between the class 1 and class 2 CRISPR–Cas system may

contribute to the advantages of Class 1 CRISPR–Cas system in some applications. Accroding to recent classification studies, there are seven subtypes (I–A to I–G) in type I CRISPR–Cas

system7,36. In recent years, the type I–A37, I–B38,39, I–E40, and I–F41,42 CRISPR–Cas have been used for prokaryotic gene engineering in _Sulfolobus islandicus_ (I–A)_, Clostridium

pasteurianum_ (I–B)_, Lactobacillus crispatus_ (I–E)_, Zymomonas mobilis_ (I–F), and _Pseudomonas aeruginosa_ (I–F). Besides, type I–B43 and type I–E44,45,46 Cascades can work as

transcription repressor in _Sulfolobus islandicus_ (I–B) _and Escherichia coli_ (I–E). Furthermore, type I–E and I–B CRISPR–Cas systems have been used in human cells33,34,35,47 and plants48

for gene editing and transcription regulation. Therefore, developing tools based on type I CRISPR–Cas system might provide alternative tools for genome editing and gene regulation. Type I–F

CRISPR–Cas system is among the well-studied CRISPR–Cas systems. It has fewer Cascade components than type I–E CRISPR–Cas system (4 vs 5), which will be easier to be controlled and delivered.

The type I–F CRISPR–Cas system was first discovered as CRISPR subtype Ypest from _Yersinia pestis_49,50. The Cascade components of type I–F CRISPR–Cas system were also named as Csy (CRISPR

subtype Ypest) subunits, which includes Csy1 (Cas8f1), Csy2 (Cas5f1), Csy3 (Cas7f1), and Csy4 (Cas6f)7,26 (Fig. 1a). In addition, the Cascade of type I–F variant (type I–Fv, or type I–F2)

CRISPR–Cas system, derived from type I–F system, consists of only three subunits: Cas5fv (Cas5f2), Cas6f, and Cas7fv (Cas7f2)4,7 (Fig. 1a). The type I–F and type I–Fv Cascade recognizes

5′-CC PAM on the non-target strand for target binding51,52. Their crRNAs consist of 8-nt 5′ handle for Csy1 and Csy2 binding, 32-nt spacers bound by six copies of Csy3 for target

recognition, and 20-nt 3′ hairpin for Csy4 binding and pre-crRNA processing22. Recently, type I–F CRISPR–Cas system has been used for genome engineering in _Zymomonas mobilis_41 and

_Pseudomonas aeruginosa_42. However, there has not been any report on the exploitation of the type I–F or type I–Fv CRISPR–Cas system for genome manipulation application in human cells yet.

In this study, we explore the possibility of developing programmable type I–F and type I–Fv CRISPR tools for transcription activation in mammalian cells. In contrast to type I–E and I–B,

_Pseudomonas aeruginosa_ type I–F and _Shewanella putrefaciens_ type I–Fv systems require fewer subunits for dsDNA targeting in bacteria53,54. Also, the multiple subunits in type I–F and

type I–Fv might provide different combinations for tagging and increase signal strength when genetic modulators are fused to different subunits. By fusing the VPR (VP64-p65-Rta)

transcription activation domain to the type I–F Cascade subunit Csy3, we achieve both exogenous (e.g., GFP expression) and endogenous (e.g., _HBB, HBG1/2, SOX2, OCT4_, _IL1B_, and _IL1R2_)

gene activation in HEK293T cells. Interestingly, by changing the spacer length of crRNA, we can enhance the activation level of target genes. As is the case for class 2 systems, we can

achieve multiplex gene activation through a customized CRISPR array from a single vector. Finally, the type I–F CRISPR–Cas system can activate target genes specifically without altering the

expression of any predicted off-target genes. These data demonstrate the feasibility of using type I–F CRISPR–Cas system for programmable transcription activation and may have important

implications in their adaptation for genome editing. RESULTS TYPE I–F CRISPR–CAS MAINTAINS ACTIVITY IN HUMAN CELLS Csy1, Csy2, Csy3, and Csy4 constitute the Cascade complex in the

_Pseudomonas aeruginosa_ type I–F CRISPR–Cas system (PaeCascade) (Fig. 1a)26,53,55. Csy1 mediates PAM recognition (5′-CC-3′) at the 5′ end of the protospacer. Csy1 and Csy2 bind to the 5′

handle of the crRNA. Multiple Csy3 binds to the crRNA, serving as the backbone of the complex (Fig. 1b). Each Csy3 binds to 6-nt of the crRNA spacer with the precise number of Csy3 subunits

determined by the length of the crRNA spacer56 (from 14 to 50-nt), resulting in 3–9 copies of Csy357. Csy4 binds to the crRNA 3′ hairpin structure and is responsible for pre-crRNA maturation

(Fig. 1b). In comparison, _Shewanella putrefaciens_ type I–F variant Cascade (SpuCascade) contains only three subunits (Cas5fv, Cas6f, and Cas7fv) (Fig. 1a), leading to its more open

configuration (Supplementary Fig. 1)54. Here, Cas5fv plays an important role in PAM recognition and dsDNA unwinding. Casf7v is involved in crRNA-target ssDNA duplex and non-target ssDNA

binding to stabilize the complex, while Cas6f participates in pre-crRNA processing and crRNA hairpin binding (Fig. 1c). We first expressed and purified PaeCascade and SpuCascade complexes in

_E. coli_ to test their dsDNA binding ability by electrophoretic mobility shift assays (EMSA). As shown in Fig. 1b, c, both PaeCascade and SpuCascade complexes could shift the dsDNA target

probe (crRNA) in vitro. Next, we examined the expression of individual PaeCascade and SpuCascade subunits in 293T cells (Supplementary Fig. 2). While the level of expression differed between

subunits, they could all be readily expressed in mammalian cells. Both Csy4 and Cas6f are involved in crRNA maturation by processing the direct repeat (DR) of pre-crRNA55,57. We, therefore,

tested the activities of ectopically expressed Csy4 and Cas6f using HEK293T cells transiently expressing a DR-GFP fusion sequence (DR-GFP) (Supplementary Fig. 3a). When DR-GFP was

co-expressed with Csy4 or Cas6f, the percentages of GFP positive cells were drastically reduced (Supplementary Fig. 3b), indicating successful cleavage of the DR-GFP fusion mRNA. TARGETED

TRANSCRIPTION ACTIVATION BY TYPE I–F CRISPR–CAS To better examine PaeCascade and SpuCascade, we introduced rtTA (reverse tetracycline-controlled transactivator) expression cassette and eGFP

expression cassette controlled by a minimal CMV promoter plus six copies of the tetracycline-responsive element (TRE) into HEK293T cells by lentiviral vector (TRE-eGFP reporter) (Fig. 1d).

When dCas9-VPR (dCas9 fused to transcription activator VP64-p65-Rta58) was co-transfected with gRNAs targeting the TRE sequence into TRE-eGFP reporter cells, percentages of GFP positive

cells were significantly increased, indicating successful targeting of dCas9-VPR to the promoter and transcriptional activation of eGFP (Supplementary Fig. 4). With the TRE-eGFP reporter

cells, we wanted to test whether PaeCascade and SpuCascade can bind dsDNA and induce transcription activation in mammalian cells. We next fused VPR to each of the codon-optimized PaeCascade

and SpuCascade subunits and generated polycistronic all-in-one expression vectors of the Cascade complexes. To test possible effects due to configuration differences, we generated vectors

with the same subunits in different sequences (Fig. 1e, f and Supplementary Fig. 5). Then we tested their activity in the TRE-eGFP reporter cells together with a TRE-targeting crRNA. Three

configurations of the ectopically expressed PaeCascade complex (1243-VPR, 1234-VPR, and 3241-VPR) were able to activate GFP expression in ~10% of the cells (Fig. 1e). In contrast, despite

having fewer subunits, none of the SpuCascade vectors could activate GFP expression (Fig. 1f). Such differences reaffirm the notion that Cascade complexes have distinct properties from one

another and warrant further mechanistic studies. Given the complicate chromatin structure of eukaryote into consideration (e.g., histone binding, different histone modification, and etc.),

such distinct properties may due to their difference of PAM recognition mechanism (e.g., DNA minor groove vs major groove) and DNA helicase activity54. In the following sections, we will

focus on type I–F PaeCascade and investigate how to use it to effectively and efficiently activate transcription. Given the presence of multiple copies of Csy3 in a functional Cascade

complex, Csy3 may become limiting during complex assembly if all subunits are encoded by a single transcript (Fig. 1e). To address this possibility, we devised a helper-activator strategy

(Fig. 1g). Here, two helper vectors encode the subunits in pairs (Csy1/2 or Csy3/4). Activator vectors encode the subunits in pairs and have one of the subunits fused to VPR, resulting in

four different activators in all (Supplementary Fig. 6). The activator vectors were then co-transfected into the TRE-eGFP reporter cells in combination with a helper vector and TRE-targeting

crRNA (Fig. 1f). Among all the fusion types in the helper-activator 2-vector system, Csy1-VPR, Csy2-VPR, Csy3-VPR, and Csy4-VPR, only the Csy3-VPR fusion 2-vector system have a higher

activating efficiency than the all-in-one 1234-VPR vector system. Further experiments with another two plasmid system, in which Csy1, Csy2, and Csy4 were expressed by P2A fusion in one

plasmid and Csy3-VPR in another, showed highest _HBB_ and _HBG_ activation level in molar ratio = 1:3 (Supplementary Fig. 7a). However, its activation efficiency in _HBG_ was not as good as

Csy3-VPR fusion helper-activator 2-vector system (Supplementary Fig. 7b). Therefore, we decided to use the Csy3-VPR fusion helper-activator 2-vector system for further studies. And in the

hitherto described experiments, Csy3-VPR refers to the Csy3-VPR fusion helper-activator 2-vector system. These data clearly showed that type I–F PaeCascade could be utilized to activate

reporter gene expression. ENDOGENOUS GENE ACTIVATION BY TYPE I–F CRISPR–CAS Unlike most endogenous genes, multiple copies of TREs targeted by Cascade/crRNA existed in the TRE-eGFP reporter

cells. To investigate PaeCascade-mediated transcriptional activation of endogenous genes, we designed a crRNA against ~200 bp upstream of the transcriptional start site (TSS) of the

hemoglobin β protein coding gene (_HBB_). We co-transfected the crRNA expressing vector into HEK293T cells with the Csy3-VPR helper-activator vectors described above. Again, cells expressing

the combination with Csy3-VPR fusion showed the highest transcription activation activity at the HBB locus (~15 fold higher than control cells) (Fig. 2a). For convenience, PaeCascade-VPR

referred to Csy3-VPR in the test below. To determine how PaeCascade VPR fusion complex may differentially activate gene expression at different loci, we picked six genes (_HBB_, _HBG_,

_SOX2_, _OCT4_, _IL1B_, and _IL1R2_) and designed crRNAs targeting different promoter regions (−500 bp to −100 bp upstream TSS) in each locus. In all cases, PaeCascade-VPR was able to

activate endogenous gene expression to varying degrees (Fig. 2b), with the region 100–200 bp upstream of TSS being the best targets (Fig. 2c). And the fold activation of each gene was highly

correlated to their basal expression level, with the weaker expressed genes showed greater fold change (Fig. 2d). These findings indicated that the type I–F PaeCascade complex could

robustly activate endogenous gene transcription. Is PaeCascade-VPR more efficient than gene activation tools based on other CRISPR systems? To answer this question, we compared

PaeCascade-VPR system to the other gene activation tools (dCas9-VPR, dAsCas12a-VPR, and type I–E EcoCascade-VPR). We designed crRNAs or gRNAs of these systems targeting to the same loci of

_HBB_, _HBG_, _SOX2_, and _IL1B_ (Fig. 3a). The results showed that dCas9-VPR had the highest transcription activity for _HBB_ when targeting 170 bp upstream TSS (Fig. 3b). Except for the

_HBB_ -170bp TSS locus, PaeCascade-VPR appeared to outperform dCas9-VPR at activating transcription for gene loci examined (Fig. 3b–e). In all the loci tested, PaeCascade-VPR showed higher

activating efficiency than dAsCas12a-VPR and EcoCascade-VPR (Fig. 3b–e). These data suggested that PaeCascade-VPR may be more efficient than canonical dCas9-VPR and other CRISPR-based

systems, at least at certain gene loci, and represented a worthy addition to molecular tools that could modulate gene expression. ENHANCING TRANSCRIPTION ACTIVATION THROUGH CRRNA ENGINEERING

Since the spacer length of PaeCascade crRNA may be extended (beyond the canonical 32-nt) to accommodate more Csy3 subunits (more Csy3-VPR)53,54, we investigated the effect of spacer length

on PaeCascade-VPR activity at the _HBB_, _HBG_, and _SOX2_ loci (Fig. 4a). Given that the minimal length for Cys3 binding is 6-nt, we varied the length of spacers by multiples of six. In

each case, crRNAs with longer spacers (e.g., 50 and 56-nt) led to more efficient transcriptional activation (Fig. 4a), pointing to a simple yet effective way to regulate and tune endogenous

gene expression through enriching VPR in a certain locus. To test whether Cascade-mediated transcriptional activation could be further manipulated, we co-transfected two crRNAs that target

the same locus with the PaeCascade-VPR complex into cells. Among the six genes tested (_HBB_, _HBG_, _SOX2_, _OCT4_, _IL1B_, and _IL1R2_) (Distances between crRNAs: _HBB_ crRNA1-crRNA2: 27 

bp; _HBG_ crRNA1-crRNA2: 55 bp; _SOX2_ crRNA1-crRNA2: 96 bp; _OCT4_ crRNA1-crRNA2: 79 bp; _IL1B_ crRNA1-crRNA2: 71 bp; _IL1R2_ crRNA1-crRNA2: 65 bp), synergistic activation could be observed

at four loci (Fig. 4b), indicating that simultaneous targeting of the PaeCascade-VPR complex to multiple regions of a promoter may enhance its activity. Not surprisingly, the distance

between the two crRNA target regions also had an impact on the extent of transcriptional activation. We designed pairs of crRNAs with different distances and tested their ability to activate

_HBG_ expression (Fig. 4c). A distance about 50–75 bp appeared optimal for the _HBG_ gene in this case. These observations underlined the multiple ways by which PaeCascade-VPR may be

further improved as a robust and efficient tool for gene expression modulation. MULTIPLEXED GENE ACTIVATION BY CUSTOMIZED CRISPR ARRAYS The _Pseudomonas aeruginosa_ CRISPR arrays, which

contain tandem spacers linked by direct repeats (DRs), are transcribed and then processed by Csy4 to generate mature crRNAs that can target different sites22. We, therefore, reasoned that

using customized CRISPR arrays driven by a single Pol. III promoter (e.g., hU6) might allow PaeCascade-VPR to bind multiple regulatory sites and achieve more efficient single gene

activation. To this end, we constructed a vector that should yield a single transcript with spacer 1 and 2 that was subsequently processed by Csy4 into two mature crRNAs (Fig. 5a). Then, we

constructed the CRISPR array expressing vectors to produce two crRNAs that target the gene loci of _HBB_, _HBG_, and _SOX2_ in HEK293T (Fig. 5b). In each case, introducing a single construct

containing the CRISPR array could provide a transcriptional activation level comparable to that using two individual crRNA vectors (Fig. 5b). Furthermore, the same strategy could be used to

produce spacers that target different genes (at least three genes) simultaneously and effectively activate gene transcription (Fig. 5c). The ability of PaeCascade-VPR to activate multiplex

genes simultaneously with a customized CRISPR array in a single construct instead of individual crRNAs in independent constructs enormously simplified the activation system, which increased

the transfection efficiency and makes it not necessary to express and deliver multiplex gRNAs independently in comparison with type II CRISPR system. These data pointed to PaeCascade-VPR as

a powerful and flexible system with much untapped potential for research applications compared to the much better-studied type 2 systems. MISMATCH AND OFF-TARGET ANALYSIS OF PAECASCADE-VPR

SYSTEM Although the DNA-binding property of PaeCascade is crucial for its specificity in mammalian cells, it remains poorly understood. It has been shown that the seed region (first 8-nt of

PAM proximal sequence) within the crRNA is critical for initiating target binding and DNA unwinding26. To further probe the target DNA binding specificity of PaeCascade in mammalian cells,

we generated a series of _HBB_ and _HBG_ targeting crRNA variants with 6-nt mismatches in the 32-nt spacer region (Fig. 6a). Being consistent with previously published data from in vitro

experiments26, mismatches in PAM-proximal regions had the biggest impact on the activity of PaeCascade-VPR, with cells exhibiting the lowest activation levels of _HBB_ and _HBG_ with these

crRNA variants (Fig. 6b). Next, we constructed 32 crRNA variants with single-nucleotide mismatches in the 32-nt spacer to determine the contribution of each position (Fig. 6c). As shown in

Fig. 5d, mismatches at nearly every position reduced the level of gene activation. Again, changes in PAM-distal positions had less impact on _HBB/HBG_ activation than those at PAM-proximal

positions. Intriguingly, mismatches at every 6th position showed far less impact on PaeCascade-VPR activity, regardless of their distance to the PAM (Fig. 6d), consisting with its structure

characteristic53. For type I–F Cascade, the binding of the target strand to crRNA follows a periodic 5 + 1 pattern53. The five consecutive base pairs followed by one base pair gap in which

the unpaired nucleotides of crRNA and target strand kink out in opposite directions53. Therefore, the mismatches in per sixth nucleotide have less impact on target DNA binding and activation

efficiency. These data suggest that target binding by PaeCascade-VPR may be exceptionally sequence specific, with even residues far distal to the PAM playing a role in target DNA binding.

To further investigate the specificity of PaeCascade-VPR system, we searched for the target sites with overlapping target regions of PaeCascade-VPR and dCas9-VPR, which also had potential

off-targets on the TSS of other genes (Fig. 7). To find out the off-target genes, we search for two groups of the potential off-target sites. We searched potential off-target sites with ≤4

mismatches to SpCas9 gRNA as the first group of putative off-targets. Taken the features of PaeCascade crRNA into consideration, mismatches on per 6th bases in PaeCascade crRNA had less

impact on _HBB_ and _HBG_ activation (Fig. 6d), which may be tolerable for target binding. Also, mismatches on 25–32th bases were more tolerable than other bases (Fig. 6b, d). Previously

studies also indicated that PAM-proximal region of type I CRISPR was more important for its binding capacity, and ≥5 mismatches would abolish type I CRISPR interference59,60. So we allowed

mismatches in 6th, 12th, 18th, and 24–32th positions, and found all the possible off-targets with ≤4 mismatches to PaeCascade crRNA as the second group of putative off-targets. Then taking

the two groups together, all the possible off-targets were predicted through sequence similarity, which must also lay on the promoter (≤2 kb upstream or downstream TSS) of a certain gene.

According to the criteria above, we searched for target sites on _HBB_ and _HBG_ promoters. We found three regions with overlapping target sites of PaeCascade and dCas9 for off-target

analysis (Fig. 7). The RNA level of _HBB_ or _HBG_ and their predicted off-target genes were then detected. With the crRNAs or gRNAs targeting to _HBB_ 173 bp upstream TSS, _HBB_ 126 bp

upstream TSS or _HBG_, PaeCascade-VPR and dCas9-VPR can increase the transcription level of _HBB_ and _HBG_ as expected (Fig. 7). For both PaeCascade-VPR and dCas9-VPR, no off-target

activations can be detected in all the putative off-target genes (Fig. 7). These results indicated that the type I–F PaeCascade-VPR system is comparable to dCas9-VPR and may have a high

specificity as a transcription activator in human cells. DISCUSSION In this study, we demonstrated that the type I–F CRISPR–Cas system could be repurposed to activate endogenous gene

expression in human cells. Fusing the Csy3 subunit of type I–F PaeCascade to transcription domain (VPR) led to a crRNA-dependent reporter and endogenous gene activation (Figs. 1, 2). And at

most target genes, PaeCascade-VPR was much efficient than dCas9-VPR, dAsCas12a-VPR, and EcoCascade-VPR (Fig. 3b–e). Besides, having each Csy subunit expressed independently further improved

activation efficiency (Fig. 1g). Moreover, compared to dCas9-VPR, the activation efficiency could be further improved by extending the spacer length of crRNA to recruit more Csy3-VPR protein

to target genes (Fig. 4a). Customized CRISPR arrays enabled efficient multiplex gene activation in human cells (Fig. 5). Saturated mutation of crRNA spacer sequence revealed that target DNA

binding by PaeCascade was sensitive to crRNA-DNA mismatch, suggesting that transcription activation by PaeCascade-VPR might be specific (Fig. 6d). And actually, we did not observe any

off-target effects in the putative off-target genes of PaeCascade-VPR (Fig. 7). Taken together, these data prove that PaeCascade-VPR is a good programmable transcription activator in human

cells. We found that all subunits of PaeCascade (Csy1, Csy2, Csy3, and Csy4) could be fused with VPR without disturbing the formation of functional PaeCascade complex (Fig. 1g), which

provides great flexibility on engineering. It is possible that we can activate gene expression with different kinds of effectors: Cascade-TET1 (Ten-Eleven Translocation dioxygenase1) fusion

for DNA demethylation; Cascade-p300 fusion for histone acetylation; Cascade-VP64 or Cascade-VPR (VP64-p65-Rta) fusion for transcriptional factor recruitment, and achieve stronger and more

persisted gene activation through combining these three methods properly61,62,63,64. So, it might be possible to fuse more transcription regulating domains to the PaeCascade complex to

improve activation efficiency or even achieve long term memory activating of endogenous genes. While our manuscript was under preparation, Adrian et al. reported transcription regulation by

type I–B and type I–E CRISPR–Cas system in human cells65. Although type I–B also used four subunits to activate endogenous genes, type I–B tool was not better than dCas9. Furthermore, type

I–B Cas7 (Csy3 equivalent) failed to induce gene activation when fused to transcription activator65. However, transcription activator fused to Csy3 subunit of type I–F CRISPR system showed

the highest activating level (Fig. 1g). It was even better than dCas9 and other transcription activation systems at most (4/5) tested endogenous sites. In addition to gene activation,

PaeCascade subunits might be fused with transcription repressor to repress gene expression, or nuclease domain to cleave target DNA in human genome65. Previous studies of type I CRISPR have

identified an eight nucleotide PAM-proximal seed region (1–5th, 7th, 8th bases)26,59,60, and the imprecise base-pairing at every sixth position within the 32 nucleotide crRNA

sequence53,54,66, owing to structure feature of every sixth base being flipped out of the RNA–DNA duplex upon target binding. Being consistent with these studies, we found that the

PAM-proximal position is crucial for gene activation of PaeCascade-VPR (Fig. 6a). In contrast, every sixth base had a relatively weak influence on its binding (Fig. 6d). Recent studies that

generating long-range deletions in human embryonic stem cells or HEK293T with EcoCascade-Cas3 revealed no prominent off-target effect either by deep sequencing or by whole genome

sequencing33,34. It had been shown that type I–B and type I–E CRISPR–Cas could induce specific targeted transcription activation in human cells without crRNA-dependent off-target effects35.

According to our research data, we could achieve a high transcription activation level without activation of putative off-target genes by type I–F PaeCascade (Fig. 7). These data indicate

that the specificity of type I system is high in mammalian cells. Transcription activation could be used to upregulate therapeutic gene expression. For example, activating _HBB_ or _HBG_

gene expression might be used to treat β-thalassemia. Further studies are needed to investigate the function and the delivery of PaeCascade-VPR in primary cells (e.g., hematopoietic stem

cell) or in vivo. Other aspects, including the cytotoxicity and immunogenicity of type I–F system, should be studied in detail. Further efforts improving the activation efficiency of

PaeCascade-VPR are also important as well. Only then can type I–F PaeCascade-VPR be a tool for therapeutic gene expression activation. In brief, we found that PaeCascade-VPR can induce

targeted gene activation without off-target effects, indicating that PaeCascade-VPR is a good programmable transcription activator in human cells. Regulating of gene expression by Type I–F

CRISPR system broadens the usage of CRISPR system as a gene regulating tools in mammalian cells. METHODS CELL CULTURE HEK293T cells were obtained from ATCC and cultured in Dulbecco’s

modified Eagle medium (Corning, 10-013-CVR) supplemented with 10% fetal bovine serum at 37 °C and 5% CO2 in humidified incubator, with daily medium change. Cells were split every 2–3 days.

All the cells were mycoplasma negative. Transient transfection of HEK293T cells was performed using PEI (Polysciences, 24765-1). Cells were lysed by Trizol 48 h later for qPCR analysis or

collected 72 h later for flow cytometry analysis. PLASMIDS AND VECTORS Type I–F Cascade (from _Pseudomonas aeruginosa_) _E. coli_ expression plasmids were obtained from Addgene

(pCsy_complex, 89232). Type I–Fv (from _Shewanella putrefaciens_) Cas7fv, Cas5fv, Cas6fv cassettes were cloned into the pET28a vector (Sigma-Aldrich, 69864-3CN) as a polycistronic operon and

include an N-terminal His-tagged Cas7fv fusion (pET28-type I–Fv). The crRNA sequence was cloned into pACYC184 (NEB, X06403) for bacterial expression. Condon-optimized Cas subunits were

sub-cloned into px601 (Addgene, #61591) (replacing the SaCas9 gene) for transfection into mammalian cells. A site for spacer cloning flanked by two Csy4 direct repeats (DR) or Cas6f direct

repeats was ligated into lentiGuide-Puro (addgene #52963) between BsmBI and EcoRI restriction sites to generate pLenti-crRNA-IF or pLenti-crRNA-IFv vectors. Oligos containing spacer

sequences were annealed and ligated into pLenti-crRNA-IF or pLenti-crRNA-IFv for crRNA expression in mammalian cells. For spacer mutant crRNA cloning, oligos with various of mutant spacer

were annealed and ligated into pLenti-crRNA-IF. Sequences are listed in Supplementary Data 1–4. Sequences of plasmids for expression of PaeCascade-VPR, including pCsy1-Csy2, pCsy3-VPR-Csy4,

and pCsy-crRNA-EV, are listed in Supplementary Data 5. PROTEIN EXPRESSION AND PURIFICATION Type I–F and type I–Fv Cascade were expressed and purified using prokaryotic systems. Briefly, BL21

StarTM (DE3) _E. coli_ cells (Thermo Fisher) were transformed with pCsy_complex (or pET28-type I–Fv) together with pACYC184 vector containing corresponding crRNA. When OD600 reached 0.6,

protein expression was induced by 5 mM IPTG and cultured for another 12 h at 25 °C. Cells were harvested and suspended in buffer A (20 mM HEPES-Na pH 8.0, 250 mM NaCl, 20 mM KCl, 20 mM

MgCl2, 40 mM imidazole), disrupted by sonication and purified using Ni Sepharose 6FF column (GE Healthcare). Size exclusion chromatography was performed on a Superdex 200 Tricon 10/300

column (GE Healthcare) in buffer B (20 mM HEPES-Na pH 7.0, 150 mM NaCl, 1 mM DTT, 1 mM EDTA). Fractions containing the target complex were collected. Protein concentration was measured by

BCA protein assay kit (Thermo Fisher, 23225). ELECTROPHORESIS MOBILITY SHIFT ASSAY (EMSA) Target oligonucleotides used were detailed in Supplementary Data 6. Substrate dsDNA was prepared by

annealing two complementary oligos with a molar ratio of 1:1. 200 nM of substrate DNA were incubated with various amount of purified recombinant protein complex at 37 °C for 30 min in

binding buffer (50 mM HEPES-Na pH 7.0, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 10 IU RNase inhibitor (Thermo Fisher, EO0381)). The products were then separated via non-denaturing TBE-PAGE and

stained by GelredTM (Biotium, 41000). QUANTITATIVE PCR (QPCR) Briefly, total RNA was extracted by TRIZOL (Thermo Fisher) following the manufacture’s instruction and quantified by Nanodrop

1000 (Thermo Fisher). The reverse transcription was carried out using the PrimeScript™RT reagent Kit (TAKARA, RR047Q) following the manufacture’s instruction. Quantitative PCR was carried

out in qTOWER3 system (Analytikjena) using TAKARA TB Green II Real-Time PCR Master Mix following the manufacture’s instruction. Quantitative PCR was performed with indicated primer for

specific genes, and _GAPDH_ served as control. The relative expression level was determined by −ΔΔCt method. qPCR primers are listed in Supplementary Data 7. FLOW CYTOMETRY ANALYSIS Cell was

digested by 0.25% trypsin, and then trypsin digestion was terminated by DMEM containing 10% FBS. Cells were collected and suspended in PBS. The GFP positive cells were detected by CytoFLEX

(Beckman). WESTERN BLOT (WB) Three days post-transfection, cells were lysed in RIPA buffer with protease inhibitor cocktail. Samples were centrifuged at 14,000 × _g_ for 10 min. The

supernatant was harvested and quantified using BCA protein assay kit (Thermo Fisher, 23225) on Victor X5. 25 μg protein was mixed and boiled with 5 × SDS loading buffer. Samples were

separated using SDS-PAGE assay. Protein was transferred to nitrocellulose membranes (Bio-Rad) for 1 hour in transfer buffer at 300 mA. The membranes were blocked at room temperature for 20 

min in 5% milk-TBST and incubated with the primary antibody in 3% BSA-TBST at RT for two hours. Then the membranes were washed in TBST and incubated with secondary antibody in 3% BSA-TBST at

RT for one hour and washed in TBST. Blots were visualized using Odyssey finally. The antibodies used for WB were listed below. Rabbit polyclonal anti-GAPDH (Abmart, P30008M) (1:5000

dilution), mouse monoclonal anti-HA antibody (Sigma, H9658) (1:5000 dilution), goat anti-rabbit secondary antibody (Odyssey, 926-32211) (1:5,000 dilution) and the goat anti-mouse secondary

antibody (Odyssey, 926-68070) (1:5,000 dilution). OFF-TARGET PERDITION To predict the putative off-targets for dCas9-VPR, we first searched off-targets with ≤4 mismatches to SpCas9 gRNA. And

for the prediction of PasCascade-VPR, we allowed mismatches in 6th, 12th, 18th, and 24–32nd position, and found all the possible off-targets with ≤4 mismatches to PaeCascade crRNA. Then all

the possible off-target sites were predicted through sequence similarity, which also lay on the promoter (≤2 kb) of a certain gene (UCSC, with Integrated Regulation from ENCODE Tracks and

GeneHancer Regulatory Elements and Gene Interactions). Sequences of all putative off-targets were listed in Supplementary Data 8. SIGNIFICANT ANALYSIS All data were processed and tested

using GraphPad Prism 7.0. For all the data, Gaussian distribution was detected by Shapiro–Wilk normality test. One-way ANOVA (for data having more than two groups) or unpaired _t_ test (for

data having only two groups) was used for data with Gaussian distribution (Normal distribution) and equal SDs. Otherwise, Kruskal–Wallis or Mann–Whitney test was used. Data were displayed as

mean ± S.E.M. Statistical significance level: n.s., not significant; *_P_ < 0.05; **_P_ < 0.01; ***_P_ < 0.001. REPORTING SUMMARY Further information on research design is

available in the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY All relevant data are available upon request. Sequences of plasmids for expression of

PaeCascade-VPR, including pCsy1-Csy2, pCsy3-VPR-Csy4, and pCsy-crRNA-EV, are listed in Supplementary Data 5. The source data for Figs. 1b, c, e, f, g, 2a, b, d, 3b, c, d, e, 4, 5b, c, 6b, d,

7 and Supplementary Figs. 2, 3b, 4b, and 7 are provided as a Source Data file. CHANGE HISTORY * _ 09 JULY 2020 An amendment to this paper has been published and can be accessed via a link

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ACKNOWLEDGEMENTS We would like to thank all the members in Zhou Songyang’s lab for discussions and supports. This work was supported by the National Key R&D Program of China

(2017YFA0102801, 2017YFC1001901, and 2017YFC1001603), the National Natural Science Foundation (91640119, 91019020, 81330055, 31671540, and 31601196), the Guangdong Special Support Program

(2019BT02Y276), the Natural Science Foundation of Guangdong Province (2016A030310206 and 2014A030312011), the Guangzhou Science and Technology Project (201707010085 and 201803010020). AUTHOR

INFORMATION Author notes * These authors contributed equally: Yuxi Chen, Jiaqi Liu. AUTHORS AND AFFILIATIONS * Sun Yat-Sen Memorial Hospital, Sun Yat-sen University; MOE Key Laboratory of

Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China Yuxi Chen, Jiaqi Liu, Shengyao

Zhi, Qi Zheng, Wenbin Ma, Junjiu Huang, Puping Liang & Zhou Songyang * State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060,

China Yuxi Chen, Yizhi Liu & Zhou Songyang * Key Laboratory of Reproductive Medicine of Guangdong Province, the First Affiliated Hospital and School of Life Sciences, Sun Yat-sen

University, Guangzhou, 510275, China Junjiu Huang * Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, 77030,

USA Dan Liu & Zhou Songyang * Guangzhou Regenerative Medicine and Health-Guangdong Laboratory (GRMH-GDL), Guangzhou, 510530, China Zhou Songyang Authors * Yuxi Chen View author

publications You can also search for this author inPubMed Google Scholar * Jiaqi Liu View author publications You can also search for this author inPubMed Google Scholar * Shengyao Zhi View

author publications You can also search for this author inPubMed Google Scholar * Qi Zheng View author publications You can also search for this author inPubMed Google Scholar * Wenbin Ma

View author publications You can also search for this author inPubMed Google Scholar * Junjiu Huang View author publications You can also search for this author inPubMed Google Scholar *

Yizhi Liu View author publications You can also search for this author inPubMed Google Scholar * Dan Liu View author publications You can also search for this author inPubMed Google Scholar

* Puping Liang View author publications You can also search for this author inPubMed Google Scholar * Zhou Songyang View author publications You can also search for this author inPubMed 

Google Scholar CONTRIBUTIONS Z.S., P.L., and Y.C. designed the experiments. D.L. helped with the paper. J.L., Y.C., S.Z., and Q.Z. performed the experiments. Z.S. and P.L. supervised the

research. All authors discussed the results and commented on the paper. CORRESPONDING AUTHORS Correspondence to Puping Liang or Zhou Songyang. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no competing interests. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Communications_ thanks the anonymous reviewer(s) for their contribution to the peer review of

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I–F CRISPR–Cas system as a transcriptional activation tool in human cells. _Nat Commun_ 11, 3136 (2020). https://doi.org/10.1038/s41467-020-16880-8 Download citation * Received: 14 November

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