The next generation of CRISPR–Cas technologies and

applications

Adrian Pickar-Oliver

1,3

, Charles A. Gersbach

1,2,3,4

Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA

Department of Surgery, Duke University Medical Center, Durham, NC 27708, USA

Center for Genomic and Computational Biology, Duke University, Durham, NC 27708, USA

Abstract

The prokaryote-derived CRISPR–Cas genome editing systems have transformed our ability to

manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse

species. The ease of use and robustness of this technology have revolutionized genome editing for

research spanning from fundamental science to translational medicine. Initial successes have

inspired efforts to discover new systems for targeting and manipulating nucleic acids, including

those from Cas9, Cas12, Cascade and Cas13 orthologs. Genome editing by CRISPR–Cas can

utilize non-homologous end joining (NHEJ) and homologous-directed repair (HDR) for DNA

repair, as well as single-base editing enzymes. In addition to targeting DNA, CRISPR–Cas-based

RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-

inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to

regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, these

advances are considerably advancing our understanding of biology and propelling CRISPR–Cas-

based tools towards clinical use in gene and cell therapies.

INTRODUCTION

The ability to modulate and edit genetic information is crucial for studying gene function

and uncovering biological mechanisms. Since the first demonstration of producing specific

DNA fragments with restriction enzymes in 1971, scientists have been harnessing

prokaryotic molecules for gene editing

. In addition to restriction enzymes

, classes of

DNA-modifying tools include recombinases

and programmable nucleases such as

Corresponding author: charles.gersbach@duke.edu.

Author contributions

Both authors equally contributed to researching data for the article, the discussion of content, writing of the manuscript its editing

before submission.

Competing interests

CAG and AKP are inventors on patent applications related to CRISPR technologies. CAG is a scientific advisor to Element Genomics,

Locus Biosciences, and Sarepta Therapeutics.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Reviewer information

Nature Reviews Molecular Cell Biology

thanks J. Chen, R. Platt and other anonymous reviewer(s) for their contribution to the peer

review of this work.

HHS Public Access

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Nat Rev Mol Cell Biol

. 2019 August ; 20(8): 490–507. doi:10.1038/s41580-019-0131-5.

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meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and

clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated

endonuclease (Cas) systems

. DNA-binding proteins that modify specific loci have

tremendously advanced science, biotechnology and medicine. However, the complexity of

developing modular DNA-binding proteins to bind at custom targets often requires protein

engineering expertise. In the past decade, the CRISPR–Cas9 technology has transformed

genome engineering by removing the need for any expertise in engineering custom targeted

DNA-binding proteins, because the target specificity of CRISPR–Cas9 relies on base pairing

of nucleic acids rather than protein–DNA recognition.

In nature, the CRISPR-Cas system is a prokaryotic adaptive immunity mechanism used to

cleave invading nucleic acids

. An assortment of CRISPR-Cas systems exists across diverse

species of bacteria and archaea, which differ in their components and mechanisms of action.

For example, class 1 CRISPR–Cas systems comprise multi-protein effector complexes

whereas class 2 systems have a single effector protein; overall there are 6 CRISPR–Cas

types and at least 29 subtypes

6-8

, and this list of types and subtypes is undergoing rapid

expansion. All CRISPR-Cas systems rely on CRISPR RNA (crRNA) or, in experimental

CRISPR–Cas9 systems, on the guide RNA (gRNA) for guidance and targeting specificity

(Figure 1). Following hybridization of the spacer [G] part of the crRNA to a target sequence

that is positioned next to a protospacer adjacent motif (PAM) (or a protospacer flanking

sequence (PFS) in type VI systems), the Cas nuclease cleaves the target nucleic acid. Thus,

site-specific cleavage at any locus containing a PAM or PFS can be achieved by retargeting

CRISPR-Cas systems with designed crRNAs containing appropriate spacer sequences. The

discovery and development of type II CRISPR-Cas9 systems and the ease of their use have

led to rapid adoption and development of a great range of applications, spanning from

fundamental to translational science and medicine

. In turn, the early successes have

inspired efforts to discover new CRISPR–Cas systems and develop novel genome

engineering applications.

In this review, we discuss recent advances in CRISPR–Cas tools for gene editing and

epigenetic modulation, before describing a diverse range of new CRISPR–Cas functions. We

discuss next-generation applications such as perturbation of the transcriptome and non-

coding genome, single-base editing, genome-wide pooled screens, chromatin reorganization

and therapeutic potential moving towards clinical studies.

Advances in genome editing

CRISPR-Cas systems are modular DNA-binding or RNA-binding proteins that can be

engineered to bind specific sequences by designing crRNAs or gRNAs containing spacers

that complement the target sequence. In addition to binding specific nucleic acid sequences,

these proteins also function as nucleases and thus can be used for programmable genome

editing.

Broader targeting capacity

By harnessing the unique attributes of various CRISPR-Cas systems, such as PAM

specificity, protein size and nuclease activity, a range of CRISPR–Cas-based DNA-targeting

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tools have been developed for genome editing applications. Additionally, the development of

methods for the detection of on-target and off-target interactions has advanced the targeting

specificity of CRISPR-Cas tools (Supplementary information Box 1).

CRISPR-Cas9 tools—Cas9 belongs to the class 2 type II CRISPR systems and is the

most widely used genome editing tool. Specifically,

Streptococcus pyogenes

Cas9 (SpCas9)

was the first to be used outside of prokaryotic cells

and reprogrammed for genome editing

in mammalian cells

11,12

; it remains the most commonly used Cas9. Following DNA target

recognition, SpCas9 typically generates a blunt double-strand break (DSB) (Figure 1a)

DNA targeting by SpCas9 relies on the 20-nucleotides long spacer and on the PAM 5’-

NGG

10,14

. Cas9 systems are dual-RNA-guided: a crRNA is responsible for DNA targeting

and also hybridizes with the trans-activating crRNA (tracrRNA), which is responsible for

forming the complex with Cas9

15,16

. The crRNA and tracrRNA functions can be

recapitulated with an engineered single guide RNA (gRNA)

(Figure 1a).

Recognition of the PAM 5’-NGG (N represents any nucleotide) limits the availability of

SpCas9 target sites in the human genome to an average of one target site for every eight base

pairs

. To increase the availability of target sites, directed evolution [G] approaches have

generated variants with altered PAM specificities (Table 1)

17,18

. For example, an expanded-

PAM SpCas9 variant, xCas9, recognizes 5’-NG, 5’-GAA and 5’-GAT PAM sequences

Another motivation for engineering Cas9 variants is to increase targeting specificity. In fact,

several studies have described mutated Cas9 variants with reduced off-target cleavage

following expression of Cas9 and gRNAs from plasmids

19-22

or their delivery as

ribonucleoprotein (RNP) complexes

. Alternatively, on-target CRISPR-Cas specificity has

been increased by engineering secondary structures in the form of RNA hairpins on the

spacer region of gRNAs, which increase the thermodynamic barrier to crRNA or gRNA

strand invasion at off-target sites while generally maintaining on-target activity

The discovery and development of additional Cas9 orthologs that recognize different PAM

sequences has provided a greater choice of target sites. For example,

Streptococcus

thermophilus

Cas9 recognizes the PAM 5’-NNAGAAW (W represents A or T)

11,25

and

Neisseria meningitidis

Cas9 recognizes 5’-NNNNGATT

26-28

. These Cas9 orthologs have

been repurposed for DNA targeting in bacteria and mammalian cells. Furthermore, the PAM

recognized by

Staphylococcus aureus

Cas9 (SaCas9) is 5’-NNGRRT (R represents A or

. Notably, SaCas9 gene editing efficiencies are comparable to SpCas9 and the smaller

size of SaCas9 (1,053 amino acids compared to 1,368 amino acids of SpCas9) has enabled

its use in size-restricted delivery vectors such as adeno-associated virus (AAV)

. More

recently, an even smaller Cas9 ortholog, from

Campylobacter jejuni

(984 amino acids), was

reported to recognize the PAM 5’-NNNVRYM (V represents A, C or G; Y represents C or

and used for targeted genome editing

in vivo

. Additional efforts to identify Cas9

orthologs has resulted in the discovery of CasX (980 amino acids), the smallest Cas9 to

date

CRISPR–Cas12a—Another class II RNA-guided endonuclease that has been

reprogrammed for gene editing in human cells is Cas12a (formerly Cpf1)

. As a type V

system, Cas12a generates a staggered cut with a 5’ overhang at DNA target sites and does

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not use a tracrRNA (Figure 1b). In contrast to the generation of blunt ends by Cas9,

production of staggered ends by Cas12a may be advantageous for applications such as

integrating DNA sequences in a precise orientation. Additionally, Cas12a can cleave crRNA

arrays [G] to generate its own crRNAs. This crRNA processing ability facilitates the use of a

single customized crRNA array for simplified multiplexed genome editing with multiple

crRNAs

Cas12a from

Acidaminococcus

spp. (AsCas12a) and

Lachnospiraceae

spp. (LbCas12a), the

first Cas12a orthologs that were shown to have activity in mammalian cells, recognize the

PAM sequence 5’-TTTV upstream of the target sequence. To improve their genome editing

activity, an enhanced AsCas12a variant (enAsCas12a) has been engineered

. To increase

the targeting range of Cas12a, AsCas12a variants have recently been engineered to

recognize the PAMs 5’-TYCV and 5’-TATV

, or PAMs 5’-VTTV, 5’-TTTT, 5’-TTCN and

5’-TATV

. The unique features and cutting mechanism of Cas12a provides a genome

editing tool that expands the CRISPR toolbox.

Cascade and Cas3—Type I systems of the class 1 category are the most common type of

CRISPR-Cas systems in nature, comprising a multimeric DNA-targeting complex termed

Cascade and the endonuclease Cas3 (Figure 1c). Before recruiting Cas3 to a target DNA

sequence, Cascade must first bind to DNA through PAM and spacer recognition

37-42

Cascade offers greater target site flexibility owing to its promiscuous recognition of PAM

sequences

. Recruitment of Cas3 generates a single-stranded nick followed by target DNA

degradation through 3’ to 5’ exonuclease activity

37,39,44,45

. Both the nickase and helicase

activities of Cas3 are essential for the degradation of foreign DNA in prokaryotes

. The

unique cutting mechanism of Cas3 is being harnessed as an antimicrobial tool by directing

native or exogenous type I systems to bacterial genomes for degradation and subsequent cell

death

. Exploration to repurpose the nickase, helicase and exonuclease activities of Cas3

may lead to new applications in mammalian cells.

In type I systems, crRNA arrays are processed by the Cascade subunit Csy4

. Like Cas12a,

this endonuclease activity has been repurposed for directed RNA processing. For example,

the

Pseudomonas aeruginosa

type I-F Csy4 has been used for generating multiple Cas9

gRNAs in human cells

48,49

Mechanisms and uses of gene editing

Gene editing nucleases, including Cas9, function by generating targeted DNA breaks that

induce the DNA damage response and stimulate repair by various endogenous

mechanisms

. Use of the unique characteristics of the different DNA repair mechanisms

has enabled the development of specific genome editing strategies.

NHEJ versus HDR—Eukaryotes predominantly repair DSBs through the error-prone non-

homologous end joining (NHEJ) pathway, which leads to accumulation of small insertions

or deletions (indels [G]) following repeated cycles of break and repair (Figure 2a).

Alternatively, a repair template with homology to the target site can be delivered with Cas9

to stimulate the error-free homology-directed repair (HDR), but typically at a lower

efficiency than NHEJ-mediated repair (Figure 2b). NHEJ can be used to produce gene

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knockouts (deletions) whereas HDR can be used to introduce a specific change in the

targeted genomic site, such as a point mutation or insertion of a longer segment of DNA.

Increasing the efficiency of HDR following nuclease-mediated DNA breakage is widely

pursued to fully harness the power of genome editing to introduce precise genomic

alterations

51-55

Gene deletions—Following Cas9 cleavage, NHEJ-mediated DNA repair can be harnessed

to create gene knockouts. When targeting a coding exon, indel-mediated frameshift

mutations, which also typically introduce premature stop codons downstream of the target

site, will disrupt gene expression. Alternatively, by simultaneously targeting two sites in a

gene, a deletion can be generated between the DSBs

11,56-58

, including megabase-size

deletions

(Figure 2a). A systematic exploration of Cas9-mediated deletion efficiencies

showed an inverse correlation between deletion size and its frequency

. In addition to

studies in cells, strategies have been developed for facilitating heritable genomic deletions in

organisms such as zebrafish

and mice

62-64

. The wide spectrum of possible Cas9-mediated

genomic deletions is accelerating the investigation of genes and genetic elements.

Gene insertions—Inserting a DNA sequence encoding an epitope tag or a fluorescent

protein into protein-coding genes to monitor endogenously-expressed proteins is a valuable

strategy for studying protein function in native cellular settings. Cas9-mediated and NHEJ-

mediated gene tagging strategies have been developed based on the integration of linear

DNA fragments at nuclease cleavage sites. In homology-independent targeted integration

(HITI), a tag is flanked with gRNA target sites, so that Cas9 can simultaneously release it

from a plasmid and cleave a recipient genomic target adjacent to the gene of interest

(Figure 2a). Generic plasmid-based systems to create endogenous amino-terminal

carboxy-terminal

66,67

gene–tag fusions using non-target-specific universal donor sequences

have also been developed. Large-scale gene tagging is now possible due to the simplicity of

these modular Cas9-mediated systems. HITI utilizes NHEJ for DSB repair, creating two

problems: generation of indels and donor integration in random orientation. To overcome

these obstacles, donor sequences can be flanked with homology arms. To circumvent the

need for molecular cloning of target-specific donor sequences, single-strand DNA (ssDNA)

were used to tag endogenous human genes with GFP-coding sequences

(Figure 2b). Mice

with multiple precise single point mutations were generated using multiplexed HDR in

mouse embryonic stem cells

. Cancer modeling in mice can also be achieved by HDR-

mediated insertion of missense gain-of-function mutations

. To generate conditional

knockout mice at high efficiency by inserting large regulatable cassettes, RNPs were co-

delivered with long ssDNA donors containing short homology arms

. Recently, an HDR-

dependent strategy termed CORRECT (consecutive re-guide or re-Cas steps to erase

CRISPR–Cas-blocked targets) was developed for producing scarless targeted knock-in of

disease-relevant mutations

. By making variations in the donor template, edited cell lines,

including human pluripotent stem cells, can be generated with pathogenic mutations and

with additional, silent mutations that block subsequent target-site recognition by the

nucleases and formation of NHEJ-mediated indels

(Figure 2b).

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Translocations—During cancer development, oncogenic fusion genes are frequently

created through chromosomal translocations. Translocations can be mediated by illegitimate

NHEJ of DSBs located at two non-homologous chromosomes. To generate models for

studying the oncogenic properties of fusion proteins, simultaneous Cas9-mediated cleavage

at two genomic loci has been used to engineer cancer-relevant translocations in human

cells

74,75

. Cas9-induced chromosomal rearrangements leading to oncogenic gene fusions

have been recapitulated also in mice

. These genetically engineered models are important

for understanding tumorigenesis and for developing therapeutic strategies against oncogenic

fusion proteins.

Single-base editing—The most common genetic variants associated with human disease

are point mutations. An ability to edit single nucleotide bases is important for creating

genetic disease models and developing corrective therapeutics. Targeted HDR-mediated

single-base editing can be achieved by co-delivering Cas9 with a homologous donor

sequence that contains the edited nucleotide of choice

. However, such strategies remain

inefficient, particularly in post-mitotic cells with decreased HDR activity. Additionally, the

need to create DSBs to induce efficient HDR carries the possibility of off-target

mutagenesis, and even on-target activation of DNA repair pathways can have adverse

consequences on cell viability

77,78

For improved single-base editing, tools have been developed that utilize Cas9 nickase

(nCas9) or catalytically deficient Cas9 (dCas9) for site-specific targeting without generating

DSBs. For direct conversion of single nucleotides, dCas9 or nCas9 have been fused to

cytidine deaminases. Fusion with deaminases such as rat APOBEC1 and lamprey cytidine

deaminase 1 can achieve targeted C→T (or G→A) nucleotide conversions within a 5-bp

activity window located within the spacer sequence

79,80

(Figure 2c). Cellular DNA repair

responses can antagonize this process and restore edited bases, therefore a uracil glycosylase

inhibitor was also used to prevent base excision repair and increase the efficiency of base

editing

79-81

. A third generation editor (BE3) containing APOBEC1 fused to a 16-residues

XTEN linker, nCas9 and a uracil glycosylase inhibitor (APOBEC1–XTEN–dCas9(A840H)-

UGI) can achieve permanent conversion of 15–75% of a target nucleotide in mammalian

cells

. Furthermore, BE3 has accomplished base editing

in vivo

through RNP-mediated

protein delivery to mouse and zebrafish embryos

82,83

, AAV-mediated delivery

in utero

mice

, and injection of mRNA and gRNA to human embryos

85,86

. In adult mice, BE3 was

used to introduce site-specific nonsense mutations into the

Pcsk9

gene, which resulted in

lowered cholesterol levels

Continued development of BE3 has resulted in improved single-base editing. For example,

improved Cas9-mediated targeting specificity has been achieved by combining BE3 with a

high-fidelity Cas9

. For optimization of base editing, lengthening of the linker between the

fused proteins and adding a second copy of the uracil glycosylase inhibitor has led to fourth

generation base editors engineered from SpCas9 (BE4) and SaCas9 (SaBE4)

. The base-

targeting range has continued to expand following fusion of APOBEC1 to catalytically

inactive dLbCas12a, which recognizes a T-rich PAM and has a 6-bp activity window

Recently, enAsCas12a was used for enhanced base-editing activity

. To narrow the editing

window at targets with potential C→T bystander alterations, base editors have been

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developed with human APOBEC3A for use in human cells

90,91

and plants

. Specifically,

eA3A-BE3, an engineered APOBEC3A domain (eA3A) fused to BE3, preferentially

deaminates cytidines according to a TCR>TCY>VCN hierarchy

. Additionally,

APOBEC3A-mediated base editing can be achieved in regions with high DNA methylation

levels and CpG dinucleotide content

Recently, the base editor toolbox has been expanded to adenine base editors (ABEs), which

can perform targeted A→G (or T→C) nucleotide conversions

. A seventh-generation ABE

with the highest reported editing efficiencies and on-target activities was developed using

directed evolution and protein engineering of a tRNA adenosine deaminase

. Optimized

and enhanced cytidine and adenine base editors include BE4max, AncBE4max and

ABEmax

Unbiased analyses of base-editing specificity is particularly difficult given the prevalence of

single-base substitutions in the human genome and the frequency of sequencing errors. An

early analysis of base editor specificity revealed off-target sites that are different from what

was detected in cells treated with Cas9 alone

. More recently, widespread gRNA-

independent off-target activity was reported for cytosine base editors in both plants and

mice,

96,97

indicating the existence of base-editing activity that is independent of Cas9–DNA

interactions. Therefore, future efforts will likely focus on strategies to restrict base editing

activity to intended targeted sites.

With the abundance of known point mutations associated with genetic disease, single base

editors can be used to make animal models with nonsense mutations or single amino acid

substitutions. Moreover, the therapeutic potential of base editors for correction or knockout

of clinically relevant human diseases is being explored.

Base editing may prove

particularly useful for multiple-gene targeting, where avoiding the formation of multiple

DSBs on different chromosomes that could generate translocations would be particularly

desirable.

High-throughput loss-of-function screens—RNA interference (RNAi) has been the

primary system for large-scale gene perturbation in mammalian cells, but the limitations of

RNAi include incomplete suppression of target genes and frequent off-target effects. These

limitations can largely be overcome by Cas9-based methods for gene knockout screening

(Box 1). Indeed, Cas9-based high-throughput screens achieve high rates of target validation.

99-102

The ease of producing large gRNA libraries coupled with efficient lentiviral delivery

platforms — for example, a genomic CRISPR–Cas9 pooled lentivirus library, which can

knockout over 18,000 human genes using 3–4 gRNAs per gene

— has made genomic

knockout screens possible in mouse

100,101,103

and human cells

99,104,105

. Such a mouse-

genome targeting library was delivered to a mouse model of tumor growth and metastasis,

and loss-of-function mutations in known tumor suppressor and novel genes were identified

in vivo

102

. For higher content readout of pooled screens, the modularity of Cas9 has been

coupled with single-cell RNA sequencing

106-108

By pairing genomic perturbation and

transcriptomic analysis within the same cell, higher order interactions can be elucidated

including the function of combinatorial interactions. Considerable advances in the

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optimization gRNA-library design are also improving the quality and throughput of these

screens

109

Beyond single gene perturbation for therapeutic targeting strategies, combinatorial studies

can be used to dissect genetic interactions. For cancer therapy, simultaneous knockout of a

synthetic-lethal gene pair can achieve cell killing via multiplexed targeting. Therefore

CRISPR–Cas-based double knockout screening has been developed for dissecting genetic

interactions and identifying synthetic-lethal drug target pairs of cancer genes

110,111

Excessive DNA damage and cell death resulting from Cas9-induced DSBs may muddle

conclusions drawn from knockout screens. Another point raised from recent Cas9 loss-of-

function screens is that not all indels result in gene knockout. To address these issues, a

DSB-independent knockout method, termed CRISPR-STOP

, was developed using

CRISPR base editors to create stop codons by single nucleotide conversion. To expand this

induction of stop codons (iSTOP) method, a database of over 3.4 million gRNAs targeting

97-99% of genes in eight eukaryotic species was compiled

112

. These Cas9-based knockout

screens have confirmed known essential genes and mediators of resistance to drugs and

toxins and provided novel genetic insights.

Although many initial applications of CRISPR–Cas-based gene editing were directed at

studying gene function, a particularly important use of this technology lies in annotating the

non-coding genome in ways that have not been previously possible. For example, the

BCL11A

gene encodes a transcription factor that controls the levels of fetal hemoglobin

113

;

modulation of

BCL11A

expression by perturbing cell type-specific enhancers could be used

as a therapeutic approach for β-haemoglobin disorders. By tiling the 10 kb of the

BCL11A

enhancer region with a gRNA library, divergence in enhancer–gene interactions was

revealed between mice and humans and crucial minimal genetic elements were revealed and

validated as targets for fetal hemoglobin reinduction

113

. This work involved the introduction

of indel mutations at noncoding sequences to identify functional gene regulatory elements,

which later led to the development of therapeutic strategies to target these sites in preclinical

models of sickle cell disease and beta thalassemia

114

. Alternatively, HDR was used to

introduce all possible nucleotide substitutions into a putative gene regulatory element to

decipher its function

115

. Numerous other high-throughput tiling approaches are being used

to identify functional elements in regulatory regions

116-123

. Finally, a genomic screening

method that targets splice sites was used to identify long non-coding RNAs (lncRNAs) that

are essential for cellular growth

124

. By using the same library to screen multiple cell lines,

cell-type-specific differences in lncRNA essentially were identified.

Molecular recording—To better understand cellular dynamics in response to external

(and internal) stimuli, CRISPR–Cas-based tools have been developed to function as

molecular recorders by tracking cellular responses in the form of nucleotide alterations. Self-

targeting gRNAs (stgRNAs) can be generated so that expression of Cas9 and the stgRNA

will result in cleavage and indel mutation accumulation at the stgRNA loci

125,126

. Thus, a

cellular response can be ‘recorded’ by linking cellular responses with the expression of the

stgRNA or Cas9. By sequencing the stgRNA locus and determining the level of accumulated

mutations, the duration or intensity of the stimulus can be measured. Alternatively, cellular

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activity can be recorded as individual nucleotide alterations using single-base editors

targeted to designated positions on plasmid or genomic DNA

127

. These CRISPR–Cas-based

molecular recording systems have been used to track cellular behavior in response to the

presence of small molecules, virus infection, light exposure and multiplexed stimuli in

bacteria and human cells

125,127

Cas9-mediated nucleotide alterations are inherited from the founder cell to its descendants,

and therefore indels can be used for cell-lineage tracing. To perform whole-organism lineage

tracing, accumulation of indel scars over multiple rounds of cell division was recorded

following Cas9 and gRNA injection into 1-cell zebrafish embryos containing a compact

DNA barcode with multiple Cas9 target sites

128

. By tracking these scars in hundreds of

thousands of cells from individual zebrafish, it was found that most organs derive from

relatively few embryonic progenitors

128

. To increase the number of traceable scars, Cas9

was targeted to its own gRNA spacer sequences. DNA repair mechanisms that form indels

within the spacer sequences results in increased scarring complexity, which provides more

information for improved phylogenetic annotation

126

. Another synthetic recording system,

termed memory by engineered mutagenesis with optical in situ readout (MEMOIR), was

developed to record and subsequently read lineage information out of single cells in situ

129

This system combines Cas9-based targeted mutagenesis with multiplexed single-molecule

RNA fluorescence hybridization (smFISH) to visualize recorded editing events for studying

lineage tracing while maintaining the relative spatial positioning of cells

129

An alternative recording strategy is based on integrating nucleotides into bacterial genomic

crRNA arrays as trackable molecular events. This mechanism utilizes the natural adaptation

process of prokaryotic CRISPR-Cas systems, in which Cas1 and Cas2 proteins capture short

fragments of invading plasmid or phage genetic material and integrate the exogenous

sequences as spacers into a crRNA array

5,130

. Since new spacers are preferentially inserted

at the 5’ end of crRNA arrays

, this mechanism can be harnessed for tracking sequential

spacer acquisition as a means of recording the temporal order of molecular events. In a

population of bacterial cells overexpressing Cas1 and Cas2, synthetic oligonucleotides can

be serially electroporated to generate stable genomic recordings of multiple molecular

events

131

. Recently, this technique has been scaled to store synthetic sequences encoding

pixel values of black and white images and a short movie into the genomes of living

bacteria

132

. These studies demonstrate the capacity of DNA to encode and store analog data.

CRISPR–Cas TARGETING RNA

Although CRISPR–Cas systems have been valuable for targeting DNA, manipulating RNA

is limited by lack of precise and efficient RNA-targeting molecular tools. RNAi and

antisense oligonucleotides can inhibit gene expression, but additional tools are needed to

expand RNA-targeting applications. Recently, development of CRISPR–Cas technology for

binding or cleaving specific RNAs has advanced RNA manipulation in living cells (Figure

3).

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RCas9

Cas9 can be made to cleave ssDNA targets by providing a PAM-presenting oligonucleotide

(PAMmer) that anneals to ssDNA

133

. Similarly, a PAMmer can be provided to direct Cas9 to

ssRNA targets

134

(Figure 3a). To specifically target RNA while avoiding DNA, PAMmers

can be designed for RNA sequences that lack PAMs at the corresponding genomic DNA

sites. This RNA-targeting Cas9 system, termed RCas9, only requires the design and

synthesis of a matching gRNA and complementary PAMmer

134

. By targeting dCas9 to

RNA, RCas9 can be utilized as a programmable RNA-binding protein for RNA recognition

(Figure 3b). This modular tool permits detection of endogenous RNA without the need to

genetically encode affinity tags on transcripts. RCas9 binding to specific mRNAs has been

utilized for their visualization and tracking into stress granules [G] in living cells

135

. Further

development of this technology may provide a useful tool for RNA visualization of mRNAs

of low abundance orconcentration.

Catalytically active RCas9 can stimulate site-specific cleavage of ssRNA

134

. Thus, RCas9

can be used to control cellular processes at the transcript level. Therapeutic strategies to

block the expression of toxic RNA can utilize genome editing through DNA targeting,

however this involves a risk of causing permanent off-target DNA edits. By contrast, the

diagnostic and therapeutic potential for RCas9 has been demonstrated by visualizing and

eliminating toxic RNA species associated with microsatellite-repeat expansion[G]

diseases

136

(Figure 3c). Specific RNA targeting and elimination were observed in patient

cells

ex vivo

, but

in vivo

efficacy remains to be demonstrated. Although the development of

RNA-targeting therapies is hindered by the need for continuously targeting newly

synthesized transcripts, AAV delivery is known to support long-term transgene

expression

137

, and truncated versions of RCas9 have been generated that are compatible

with the limited AAV packaging capacity

136

Cas9 orthologs

Although in nature Cas9 is thought to preferentially target phage and DNA in bacteria, Cas9

orthologs have the capacity to also target RNA. SaCas9 and

Campylobacter jejuni

Cas9

(CjCas9) can directly cleave ssRNA in a PAM-independent manner

138

(Figure 3a). When

targeted to RNA, SaCas9 repressed gene expression in

Escherichia coli

138

and for CjCas9,

crRNA-dependent but PAM-independent binding and cleavage of endogenous RNAs was

shown

139

Francisella novicida

Cas9 (FnCas9) was originally shown to target bacterial mRNA and alter

gene expression

140

, and has been repurposed to target the RNA genome of hepatitis C virus

(HCV) in eukaryotic cells

141

. This positive-sense ssRNA virus has a cytosolic life cycle and

its RNA does not undergo reverse transcription and genomic integration. By targeting the 5’

or 3’ untranslated regions of the HCV genome, FnCas9 inhibited both viral protein

production and replication (Figure 3c). Unlike RCas9, RNA targeting by FnCas9 is PAM-

independent and thus does not require PAMmers

141

. In future, FnCas9 could also potentially

be used to target negative-sense ssRNA viruses such as those belonging to the filoviridae,

paramyxoviridae, or orthomyxoviridae families. Additional studies are needed to clarify the

potential physiological consequences of RNA targeting by Cas9 in eukaryotic cells.

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Cas13

CRISPR–Cas systems containing naturally RNA-targeting endonucleases have been recently

discovered. In bacteria, Cas13a (formerly known as C2c2) is an RNA-guided RNA-targeting

nuclease. This class 2 type VI CRISPR protein is activated upon recognition of ssRNA

targets

142

(Figure 3a). Similar to a PAM sequence, some type VI CRISPR proteins require

recognition of a PFS

142

, however Cas13a from

Leptotrichia wadei

(LwaCas13a)

143

and

Cas13b from

Prevotella sp. P5-125

(PspCas13b)

144

do not. Following target binding, Cas13a

cuts at uracil bases anywhere in its vicinity, and this ‘collateral’ cleavage extends also to

nearby, untargeted RNAs. Cas13a has been programmed to cleave specific mRNAs in both

bacteria and eukaryotic cells

142,143

. Unexpectedly, collateral cleavage by activated Cas13a

was not observed in eukaryotic cells, but the mechanism for this difference remains

unknown

143

. A catalytically inactive Cas13a variant, dCas13a, maintains the ability to bind

targeted RNA and was used for live cell imaging of RNA

143

. Similar to RCas9, dCas13a has

been targeted to mRNA to visualize the formation of stress granules

135,143

The collateral cleavage observed following programmed mRNA targeting in bacteria cells

has also been demonstrated

in vitro

with purified Cas13a protein

143,145

. This promiscuous

RNase activity, which is induced upon target recognition has been utilized as a molecular

detection platform termed SHERLOCK (specific high sensitivity enzymatic reporter

unlocking)

146

. Following detection of target RNA, Cas13a is activated for collateral-RNA

cleavage-mediated release of a reporter signal. On the basis of this method, a diagnostic test

was developed to detect viral RNA of specific strains of Zika and Dengue viruses

146

Additionally, amplified DNA can be converted to RNA for subsequent Cas13-mediated

detection

146

. Following conversion to RNA, SHERLOCK can be used to detect species-

specific bacterial pathogens, discriminate between single-nucleotide polymorphisms in the

human genome, and identify cell-free, mutated tumor DNA. Further development has

resulted in the improved SHERLOCKV2 molecular detection platform, which can perform

quantitative detection, has increased sensitivity, and can be used to detect simultaneously up

to four targets

147

. Recently, Cas12a has also been repurposed as a detection tool. Following

targeted activation by dsDNA, Cas12a nonspecifically cleaves ssDNA

148

. By providing a

quenched ssDNA reporter, the collateral cleavage of Cas12a can be used to detect viral DNA

in patient samples

146,148

A single-base RNA editing application has been developed by fusing dCas13 to adenosine

deaminase acting on RNA (ADAR) enzymes (Figure 3b). This system, termed REPAIR

(RNA editing for programmable A to I replacement), can make directed adenosine-to-

inosine edits in eukaryotic cells

144

. In translation and splicing, inosine is functionally

equivalent to guanine

149,150

. To broaden the base conversions achievable by REPAIR,

dCas13 could be fused with other RNA editing domains such as that of APOBEC for

potential cytidine-to-uridine editing. Additional applications for site-specific binding of

dCas13a include studying RNA–protein interactions, visualizing RNA trafficking and

localization with fluorescently tagged dCas13a or modulating the function or translation of

transcripts with dCas13a fused to different effectors. The application of this RNA editing

tool for treating genetic diseases remains to be explored.

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Scanning of bacterial genome sequences has led to the identification of a class 2 type VI-D

CRISPR effector, termed Cas13d. Similarly to Cas13a, Cas13d-mediated cleavage promotes

collateral RNA cleavage in bacteria

151

but not when expressed in mammalian cells

152

. RNA

recognition by Cas13d is PFS-independent. dCas13d lacks target-RNA cleavage activity but

retains

rRNA array processing activity, and notably, its smaller size makes packaging into vectors

like AAV possible for

in vivo

applications

151,152

. These characteristics have been utilized to

deliver dCas13d and a crRNA array targeting

cis

elements in pre-mRNAs to manipulate

alternative splicing in a neuronal model of frontotemporal dementia

152

(Figure 3b).

GENE REGULATION by CRISPR–Cas

Beyond gene editing through the formation of DNA breaks, site-specific gene regulation is

possible by engineering Cas9 as a DNA recognition complex rather than a targeted

nuclease

153

. Mutations in the RuvC (D10A) and HNH (H840A) nuclease domains destroy

the catalytic activity of Cas9 while maintaining its RNA-guided DNA targeting

capacity

10,154

. The CRISPR–Cas toolbox has been expanded by fusing this dCas9 with

diverse effectors such as transcription repressors or activators, epigenetic modifiers, and

fluorophores (Figure 4).

Transcription regulators

The modularity of dCas9 is exemplified by the ability to tether protein effectors to dCas9 or

to the gRNA and still maintain dCas9-mediated DNA targeting. Thus, a versatile DNA-

targeting platform can be combined with various protein effectors for a broad range of

applications.

CRISPRi—Binding of dCas9 to DNA elements may repress transcription by sterically

hindering the RNA polymerase machinery

154

. dCas9-mediated steric interference, termed

CRISPR interference (CRISPRi), works efficiently in prokaryotic cells but is less effective

in eukaryotic cells

154-156

. To enhance the repressive capacity of CRISPR in eukaryotic cells,

dCas9 has been tethered to transcription repressor domains such as that of Krϋppel-

associated box (KRAB)

156

, which is found in many natural zinc-finger transcription

factors

157

. KRAB is known to induce heterochromatin formation, and changes in chromatin

structure often accompany dCas9–KRAB-targeted transcription repression

158

. dCas9–

KRAB is a robust tool in mammalian cells that can effectively silence single genes and

noncoding RNAs by targeting promoter regions, 5’ untranslated regions and proximal and

distal enhancer elements

156,159-161

(Figure 4a). For improved repressive capabilities, dCas9

was fused to a bipartite repressor consisting of the transcription repression domains of

KRAB and of methyl-CpG-binding protein 2

162

. The versatility of dCas9–KRAB is

highlighted by its capacity to repress transcription by targeting both genes and gene-

regulatory regions

161

CRISPRa—dCas9 can also be fused to activator effectors for programmed transcription

activation, termed CRISPR activation (CRISPRa). In eukaryotes, both reporter genes and

endogenous genes can be activated by dCas9 fused to the transcription activation domains of

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the NF-κB transactivating subunit (p65) or to VP64 (four repeats of the herpes simplex

VP16 activation domain)

156,163-165

. Synergistic gene activation has frequently been

observed with these synthetic transcription factors by targeting multiple gRNAs to a

promoter region

163,164

. In addition, synergy can be achieved by combining different

activator domains

166-170

. Multiplexed activation of endogenous genes can also be used for

cellular reprogramming

171

. For example, direct conversion of primary mouse embryonic

fibroblasts to induced neuronal cells was achieved following activation of lineage-specific

transcription factors by targeting VP64–dCas9–VP64 (dCas9 fused to VP64 at each of its

termini) to the endogenous

Brn2, Ascl1 and Myt1l

genes

172

(Figure 4a), and similar

approaches have been applied to reprogramming cells into pluripotency

173

or to myogenic

cells

167

Epigenome editing—Targeted epigenetic modifications, such as acetylation and

methylation of histones and methylation of DNA, can be achieved using dCas9-based

tools

153

. For example, the fusion of dCas9 to the catalytic core of the human histone

acetyltransferase p300 was targeted to promoters and enhancers for catalyzing the

acetylation of histone H3 Lys 27, leading to robust gene activation

174

. DNA demethylation

was achieved using dCas9 fusions with the catalytic domain of methylcytosine dioxygenase

TET1. Targeting dCas9–TET1 to the

BRCA1

promoter resulted in transcription up-

regulation

175

. As a potential therapy, dCas9–TET1 was used to de-methylate the CGG-

expansion mutation in the 5’ untranslated region of the gene

FMR1

and reverse its silencing,

which is associated with Fragile X syndrome

176

(Figure 4a). Importantly,

FMR1

expression

was maintained following engraftment of edited cells into mouse brains.

For heritable transcriptional silencing, dCas9–KRAB can be used in combination with DNA

methyltransferases (DNMTs). Stable silencing of the β2-microglobulin promoter–enhancer

was achieved in up to 78% of K562 cells by the transient expression of dCas9 fused to the

KRAB domain and to the catalytic domains of DNMT3A and DNMT3L, along with seven

gRNAs

177

. Combined with the robustness of dCas9-mediated targeting, the plethora of

potential epigenetic effectors provides many applications for epigenetic studies.

Dynamic control of Cas9 function

Inducible systems function by requiring particular stimuli for gene activation. Based on the

type of stimulus, various strategies have been developed for generating inducible Cas9-based

systems that permit temporal control of Cas9-mediated gene targeting (Figure 4b).

Chemical induction—Chemical compounds can activate Cas9 expression through

inducible promoters. This may be desirable to precisely time gene knockout in certain cell

types, rather than use constitutive knockout cell lines. Doxycycline-inducible expression of

Cas9 has been used in human pluripotent stem cells

178,179

and in adult mice

180

. However,

doxycycline-independent mutagenesis has been observed in the transfected cells, suggesting

the expression of Cas9 is leaky in some of these systems

180

Inducible dCas9-based systems also offer versatility in epigenome engineering. A

doxycycline-inducible CRISPRi system enabled efficient, tunable and reversible disease

modeling in induced pluripotent stem cell (iPSC)-derived cardiomyocytes

181

. Chemically

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inducible CRISPRa systems have been developed using conditionally stabilized dCas9–

activators

182

, or split dCas9–activators that dimerize following chemical induction

183,184

(Figure 4b). Beneficial uses of inducible split dCas9–activators include minimizing leaky

dCas9 expression and targeting multiple genes for multiplexed temporal regulation

184

Optogenetics—Light-inducible dCas9 systems enable precise dynamic regulation of

endogenous genes and the possibility of spatial control. For example, light-inducible

dimerization of the plant-derived cytochrome proteins CRY2 and CIB1 has been used to

create photoactivatable dCas9–p65

185

and dCas9–VP64

186

(Figure 4b). An anti-CRISPR

protein [G] was engineered for light-mediated spatiotemporal control of genome and

epigenome editing in human cells by pairing a photosensor from

Avena sativa

with a SpCas9

inhibitor

187

. A second-generation optogenetic split-protein system was developed and

targeted to upregulate the expression of the gene neurogenic differentiation 1 to induce

neuronal differentiation in iPSCs

188

. For more complex regulation, multiple chemical- and

light-inducible systems have been used to dynamically manipulate the activation or

repression of multiple genes

189

(Figure 4b). These light-inducible systems hold promise for

modeling development and disease with reversible and temporal control of gene expression.

Other genomic dCas9 applications

CRISPR-dCas9 gene regulation systems are proving immensely valuable for elucidating the

function of transcribed genes. Another important application of these tools lies in

understanding the function of the noncoding genome. Cas9 and dCas9-engineered effectors

provide an opportunity to explore these genomic regions, for which there are no other tools

for direct perturbation.

Annotating the non-coding genome

With the inception of dCas9–effector tools, CRISPRi and CRISPRa methods are also being

developed for high-throughput screening to annotate the non-coding genome

170,190-194

(Box

1). Epigenome editing with these methods permits efficient perturbation of regulatory

elements without mutating the DNA, and CRISPRa-based methods enable gain-of-function

studies. CRISPRi and CRISPRa have been combined in parallel screens to target DNaseI

hypersensitive sites [G] that surround genes of interest

193

. This unique approach identified

regulatory elements that may be dependent on the direction of dCas9-based transcription

perturbation. Collectively, these dCas9-based methods enable the elucidation of the roles of

regulatory sequences in their native genomic contexts and allow the screening of lncRNAs

whose function might not be altered by introduction of indels with Cas9 nucleases

195,196

Chromatin interactions

Chromatin structure modulates genome function, however elucidating the molecular basis of

this modulation has been limited by an inadequate availability of methods to study

chromatin–protein interactions. To identify proteins that interact with specific genome loci,

the chromatin can be immunoprecipitated using an antibody against a dCas9–tag fusion

protein, which is co-expressed with a gRNA that targets the desired DNA sequence. This

method, named engineered DNA-binding molecule-mediated chromatin

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immunoprecipitation (enChIP) is then followed by mass spectrometry to identify the locus-

associated proteins

197

. enChIP was used for biochemical analysis of transcription and

epigenetic regulation at specific genomic loci in living cells

198

. Alternatively, dCas9 has

been tethered to APEX2, which is an engineered peroxidase that promiscuously labels

nearby proteins with biotin

199,200

. dCas9–APEX2 can be used to biotinylate proteins in the

vicinity of a targeted genomic locus; these proteins can then be identified following affinity

purification and mass spectrometry (Figure 4c)

200

Regulation of gene expression is also influenced by the formation of long-range chromatin

interactions, often referred to as chromatin looping. To better understand the role of

chromatin interactions, dCas9-based methods have been developed for precisely modifying

chromatin looping. Biotinylated dCas9 has been used to identify chromatin-associated

proteins and study long-range chromatin interactions

201

. Chromatin loop reorganization with

CRISPR–dCas9 (CLOuD9) can selectively and reversibly establish chromatin loops and

modulate the expression of associated genes

202

(Figure 4c). As an alternative to the

chemically induced CLOuD9 system, a light-inducible dCas9 system was developed for

directing rearrangement of chromatin looping on faster time scales

203

. A chemically

inducible and reversible system termed CRISPR-GO can control spatial genome

organization within the cell

204

. CRISPR-GO enables studying chromatin interactions within

nuclear compartments to help elucidate their function. CRISPRi tools such as dCas9–KRAB

have also been used to disrupt anchored looping interactions that coordinate changes in gene

expression

205

. These studies have helped to confirm the roles of interactions between loci in

the maintenance of gene expression. Chromatin restructuring facilitated by these

technologies will be greatly beneficial for studying the dynamic roles of genome architecture

in gene regulation.

Imaging loci

Methods to image specific DNA sequences are useful for studying the spatial organization of

the genome. Fluorescence in situ hybridization techniques have been valuable for this

purpose, however they require cell fixation. For live cell imaging, EGFP-tagged dCas9 and

structurally optimized gRNAs have been targeted to repetitive elements and to coding

genes

206

. By targeting a large number of loci, labeling of an entire chromosome was made

possible for live cell imaging

207

. Depending on chromosome length, painting entire human

chromosomes could require about 100–800 gRNAs.

To expand these tools to multicolor genome imaging, orthogonal dCas9s have been tagged

with different fluorescent proteins

208,209

. Other dCas9-based multicolor, live cell imaging

methods have focused on engineering gRNA scaffolds [G]. By adapting gRNA scaffolds to

bind sets of fluorescent proteins, up to 6 targeted chromosomal loci were visualized

simultaneously

210

. Additionally, gRNA aptamer [G] insertions have been engineered that

concurrently bind two different fluorescent protein tags

211

. This dual-color approach is

tolerant to photobleaching [G], which makes it useful for long-term imaging of genomic

loci.

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BIOMEDICAL APPLICATIONS OF CRISPR TOOLS

CRISPR–Cas-based gene editing and epigenome engineering tools have revolutionized our

ability to manipulate the genomic functions. Importantly, these tools are now being applied

in gene therapy and in enhancing cell therapy.

Pre-clinical gene therapy

Genome editing technologies have transformed the gene therapy paradigm from delivery of

an exogenous transgene to editing human genome sequences. The therapeutic potential of

making precise, targeted genome modifications includes a wide variety of diseases and

disorders, but potential limitations must be overcome as CRISPR–Cas-based technologies

advance to the clinic (Box 2). Although the most obvious therapeutic applications of genome

editing are correcting mutations that cause genetic diseases, a variety of editing strategies

exist that manipulate genes involved in more common, complex disease. For example, by

targeting SpCas9 to the mouse cholesterol homeostasis gene

Pcsk9

through adenovirus

delivery, a reduction in low-density lipoprotein cholesterol was demonstrated following gene

disruption and silencing

in vivo

212

. For preclinical assessment of somatic genome editing

applications, this work has been expanded to successfully target the human

PCSK9

gene in

mice engrafted with human hepatocytes

213

. Similar approaches have been explored using

epigenetic silencing of

Pcsk9

by viral delivery of dCas9–KRAB.

214

Therapeutic genome editing strategies are currently being explored for ocular diseases such

as retinitis pigmentosa, which can result in blindness. Cas9-mediated disruption of the gene

Nrl

by indel formation preserved the function of cone photoreceptors in three different

mouse models of retinal degradation

215

. A HITI-mediated Cas9 insertion repaired the 1.9kb

deletion in the kinase gene

Mertk

in a retinitis pigmentosa rat model and restored MERTK

function

. The gene therapeutics were delivered to the eye using AAV vectors. Importantly,

AAV is the most frequently used gene-therapy delivery vehicle due to its effective and safe

track record and wide range of tissue targeting. AAV delivery to skeletal and cardiac muscle

can be used for treatment of neuromuscular disorders such as Duchenne muscular dystrophy

(DMD). In most individuals with DMD, a hotspot of various deletions exists that disturbs

the open reading frame of the

DMD

gene, which encodes dystrophin

216

. Restoration of the

reading frame

in vivo

has been achieved in several studies following AAV-mediated delivery

of CRISPR–Cas9 to excise additional exons through NHEJ around the inherited

deletion

217-219

, including a mutation correction that has been sustained for at least one year

after CRISPR–Cas9 administration

220,221

. These deletion-based editing approaches resulted

in the expression of a truncated but partially functional dystrophin. Importantly, progress has

been made in advancing these approaches to testing in large-animal models of DMD

222

. In

order to avoid the generation of DSBs, Cas9-mediated single-base editing of splice site

donors and acceptors has also been explored in these models

223,224

In addition to viral delivery methods, lipid nanoparticles (LNPs) can be utilized to deliver

Cas9

in vivo

. Recently, LNPs containing SpCas9 mRNA and a chemically modified gRNA

targeting the mouse

Ttr

gene were delivered to mice

225

. Following a single administration, a

reduction of TTR serum protein levels was observed and levels of

in vivo

genome editing

required for therapeutic benefit were achieved. The clinical significance of CRISPR–Cas-

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based therapeutics relies on coupling genome editing developments with continued

advancements in delivery methods

226

. In particular, transient, non-viral-mediated delivery

strategies may be useful in addressing concerns about long-term expression of immunogenic

Cas proteins and integration of DNA vectors into the genome

221,227

Translation to the clinic

The most clinically advanced gene editing strategies rely on

ex vivo

cell manipulation that

provides therapeutic effects following the administration of the cells back to the donor. In

particular, engineered autologous T cells have been successful in adoptive T-cell

immunotherapy

228

. Gene editing approaches have been used for enhancing the properties of

these engineered cells. For example, the insertion of transgenes encoding programmable

chimeric antigen receptors [G] (CARs) into the endogenous T cell receptor alpha constant

gene, rather than overexpression of CARs from viral vectors, prevents the exhaustion of T

cells from overstimulation

229,230

. Another important therapeutic application of genome

editing is in knocking out components of the human leukocyte antigen system to generate

universal cell donors [G],

231

which would address the practical and economic challenges of

patient-specific autologous cell therapies. Researchers have also targeted programmed cell

death protein 1 (

PD-1

) to block inhibitory signals that prevent T-cell recognition of tumor

cells

230,232,233

. In fact, autologous T cells that were treated

ex vivo

with Cas9 to knock out

PD-1 were infused back into individuals with cancer, in the first use of CRISPR–Cas gene

editing in a human clinical trial in the United States or Europe (NCT03399448)

234,235

. Also

currently underway are the first human trial of CRISPR–Cas to treat a genetic disease, β-

thalassemia (NCT03655678), and the first trial of

in vivo

genome editing by CRISPR–Cas

in retina to treat a rare form of blindness (NCT03872479).

227

Importantly, these CRISPR–

Cas-based clinical trials build on a foundation of several genome editing clinical trials using

zinc finger nucleases

236

. Collectively, these clinical trials will establish the therapeutic

potential of recently developed genome engineering tools.

CONCLUSIONS AND FUTURE DIRECTIONS

Repurposing CRISPR–Cas systems for use in eukaryotic cells has revolutionized the

genome engineering field. Even with the extensive use of type II CRISPR-Cas systems,

continued discovery and development of CRISPR systems from prokaryotic species has

resulted in new, beneficial technologies, such as Cas13a-based RNA targeting tools. Fusing

dCas9 to the plethora of effectors will continue to expand possibilities for targeted

epigenetic modulation.

The ease of gRNA-library generation for large-scale Cas9 targeting coupled with

advancements in next-generation sequencing has made genome-wide genetic and epigenetic

screens readily available. Perturbation at this magnitude will advance our understanding of

biological mechanisms and aid the discovery of new therapeutic targets. Additionally, the

multiplexed targeting potential of CRISPR–Cas systems will enable more complex and

sophisticated manipulation of cellular processes.

As CRISPR–Cas-based therapeutics enter clinical testing, they hold great potential for

correcting genetic diseases and enhancing cell therapies. Preclinical results are promising

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but safety and efficacy need to be monitored closely during these studies. A potential risk of

using gene editing methods is the introduction of off-target changes to genome sequence,

and thus enhancing methods for detecting rare mutations and quantifying their potential

risks will be important for future clinical advancement.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgements

This work was supported by an Allen Distinguished Investigator Award from the Paul G. Allen Frontiers Group,

Open Philanthropy, US National Institutes of Health (NIH) grants R01DA036865, R01AR069085, R21NS103007,

R33DA041878, P30AR066527, R41GM119914, R41AI136755, U01HG007900, UM1HG009428, UG3TR002142,

and the National Science Foundation grants EFRI-1830957 and DMR-1709527. A.P-O. is supported by a Pfizer-NC

Biotech Distinguished Postdoctoral Fellowship.

Glossary

Spacer

The interchangeable portion of the guide RNA that is complementary to the targeted

sequence

Directed evolution

Method to generate and select for nucleic-acid or protein variants with desirable properties

crRNA arrays

In bacterial genomes, series of spacers flanked by repeats, which are transcribed as a single

pre-crRNA array and subsequently processed into individual crRNAs

Indels

Small insertions or deletions of nucleotides at repair sites of DNA double-strand breaks

Stress granules

Denote a type of cytosolic membraneless bodies with high concentrations of RNA and/or

proteins, which form in different cell stress conditions

Microsatellite-repeat expansion

Repetitive DNA sequences that can expand between generations and encode RNAs that are

toxic to cells and cause neurological disorders

Anti-CRISPR protein

A protein that interacts with and inhibits CRISPR–Cas activity

DNaseI hypersensitive sites

Chromatin regions accessible to the enzyme DNase I; generally denote gene-activity-

permissive chromatin

gRNA scaffolds

The backbone (invariable) portions of gRNAs, which are recognized by Cas proteins

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gRNA aptamer

RNA structures added to the gRNA scaffold, which can bind specific effector molecules

Photobleaching

Reduction in the intensity of fluorescence emission owing to the imaging of a sample over

time

Seed region

PAM-proximal nucleotides in the target sequence, where spacer mismatches are less

tolerated for on-target gRNA binding

Chimeric antigen receptors

T cell receptors engineered to recognize a specific antigen

Universal cell donors

Cells engineered to avoid recognition by a recipient immune system

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Box 1 ∣

Genome-wide pooled screens using CRISPR–Cas-based tools.

The simplicity of targeting CRISPR–Cas tools to the genome has facilitated high-

throughput genetic screening. Genome-scale targeting of Cas9 is possible with synthesis

of a guide RNA (gRNA) library. The breadth of the gRNA library can be customized, for

example, loss-of-function screens may use saturation mutagenesis and target only exons

of human genes

99,104,105

, and screens to annotate the noncoding genome may target sites

of accessible chromatin

193

or transcription factor motifs

123

. The gRNA libraries are

generated by synthesizing pools of oligonucleotides, cloning them into plasmids and

producing a lentivirus library that encodes the gRNAs (see the figure, part a). A Cas9-

expressing cell line can be generated prior to gRNA delivery, or cells can be co-

transduced with Cas9 and the gRNA library. Fusion of Cas9 nickase (nCas9) or

catalytically deficient Cas9 (dCas9) to different effector proteins can enable genome

editing (for example, by the cytidine deaminase APOBEC1) or epigenome and gene

regulation (for example, histone acetylation by p300, DNA demethylation by TET

dioxygenases or transcription repression by KRAB domains; see the figure, part b). To

screen for functional elements, gRNAs that elicit the phenotype of interest must be

enriched or depleted. For example, positive selection can identify elements that function

in drug resistance

237

and negative selection can identify elements involved in synthetic

lethality

238

(see the figure, part c). Alternatively, gene regulatory elements can be

identified by selecting cells with altered gene expression either through direct

immunofluorescence staining or through tagging an endogenous gene with a reporter

193

By selecting cells with low or high reporter expression, factors that affect gene

expression can be identified (see the figure, part c). Following selection, next-generation

sequencing and bioinformatics are used to compare the unselected gRNA library with the

selected gRNA library and identify enriched and depleted gRNAs and thus specific

genomic loci (see the figure, d). A wide range of applications are possible with CRISPR-

based screens. Interrogation of gene function can identify genes involved in cell survival

and proliferation or cancer genes

100,105,238,239

; drug targets can be identified based on

resistance or sensitivity to drugs, toxins or pathogens

238,240

. Targeted screens are also

mapping the function of the noncoding genome by perturbing enhancer sequences

113

modulating particular sets of genes – for example, targeted activation of all transcription

factor genes to identify factors involved in stem cell differentiation

241

. Although pooled

CRISPR-based screens have so far used Cas9-based tools, in the future other Cas proteins

could be used for other functions or for orthogonal screening.

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Box 2 ∣

Potential limitations of CRISPR–Cas medical applications.

Despite the advances in CRISPR–Cas-based genome engineering technologies, some

challenges remain for translating these tools to the clinic:

•

Adeno-associated virus (AAV), which is the most frequently used gene-

therapy delivery vehicle, provides limited packaging capacity of genetic

information. This restriction has led to continued development and

in vivo

testing of smaller Cas9 orthologs such as

Staphylococcus aureus

Cas9

(SaCas9)

and

Campylobacter jejuni

Cas9

. Nevertheless, prolonged

expression of Cas9 from AAV vectors and integration of AAV vectors into

DSBs remain undesirable consequences of AAV delivery.

221

•

Off-target effects, which remain a major concern, can be reduced with

preliminary guide RNA selection and optimization. For example, VIVO

(verification of in vivo off-targets)

242

can be used with CIRCLE-seq

(cleavage effects by sequencing)

243

to screen off-targets using the genomic

DNA from the specific patient or organism. More-sensitive methods are

necessary to detect possible off-target editing and to understand the possible

implications of any unintended genome changes.

•

Immunogenicity of Cas proteins is another potential obstacle to their clinical

application. Immune responses to Cas9 following its delivery into mouse

models is well-documented

221,244

, but the implications of this for therapeutic

approaches are still unclear. Recently, pre-existing adaptive immunity to

Streptococcus pyogenes

Cas9 and SaCas9 has been detected in human blood

samples

245-247

. In the case of intracellular expression of virus-delivered Cas9,

T cell responses may be worrisome if they are reactive to Cas9 peptides

displayed by treated cells. More studies are needed to decipher the

implications for clinical use of pre-existing immunity. The high prevalence of

exposure of the human population to

S. pyogenes

and

S. aureus

is an

additional motivation for ongoing testing of novel Cas9 orthologs. Other

approaches to limit immunogenicity include the reengineering of

immunogenic epitopes of Cas proteins, the use of transient

immunosuppressive drugs during treatment, or

ex vivo

cell modification.

•

A potential limitation is the observation that CRISPR–Cas-mediated gene

editing is more efficient in cells that have lost the function of the tumor

suppressor

p53

77,78

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Figure 1. Overview of the main CRISPR–Cas gene editing tools.

a ∣ CRISPR-associated endonuclease 9 (Cas9) proteins rely on RNA guidance for targeting

specificity. In engineered CRISPR–Cas9 systems, Cas9 interacts with the backbone of the

guide RNA (gRNA). Complementary pairing of the spacer portion of the gRNA to a DNA

target sequence positioned next to a 5’ protospacer adjacent motif (PAM) results in

generation of a blunt DNA double-strand break by the two Cas9 nuclease domains, RuvC

and HNH

11-13

. b ∣ Cas12a nucleases recognize DNA target sequences with complementarity

to the crRNA spacer positioned next to a 3’ PAM. Target recognition results in generation of

a staggered DNA double-strand break by a RuvC domain and a putative nuclease (Nuc)

domain

. c ∣ Cascade is a multimeric complex that targets DNA that has complementarity to

the spacer portion of a crRNA and that is positioned next to a 3’ PAM

37-42

. Following target

recognition, Cascade recruits Cas3 to generate a single-strand nick, which is followed by 3’

to 5’ degradation of the targeted DNA

37,39,44,45

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Figure 2. Genome editing strategies.

Nucleases generate targeted DNA double-strand breaks (DSBs), which can be repaired by

different repair pathways. a ∣ Non-homologous end joining (NHEJ)-mediated repair is error-

prone and induces small insertion or deletion mutations (indels). Large, targeted deletions

can be produced through repair between two DSBs produced by simultaneously targeting

nucleases to two genomic sites. Alternatively, homology-independent targeted integrations

(HITI) can be directed to a single cut site by providing donor DNA that is independently

targeted for cutting

. b ∣ The homology-directed repair (HDR) pathway can be utilized for

genome editing by providing either double-strand or single-strand oligodeoxynucleotide

(ssODN) donor templates that contain homology arms to the cut target site. Single

nucleotide alterations or insertion of larger sequences can be mediated by introducing

variations into the donor template, which may also consist of plasmid DNA, viral DNA

248

long single-stranded DNA

. Following HDR, silent mutations — also referred to as

blocking mutations (B) — that prevent subsequent target site recognition by the nucleases

and formation of NHEJ-mediated indels, can be incorporated into the donor template along

with the intended alterations

. c ∣ For single nucleotide C→T (or G→A) conversion, Cas9

nickase has been fused to cytidine deaminases such as APOBEC1

. For increased base

editing efficiency, two uracil glycosylase inhibitors (UGI) have been fused to a base editor

for preventing cellular base excision repair

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Figure 3. RNA targeting tools and their applications.

a ∣

Streptococcus pyogenes

Cas9 was repurposed to target RNA (RCas9) by providing it a

matching guide RNA (gRNA) and a complementary PAM-presenting oligonucleotide

(PAMmer)

134

. Cas9 orthologs such as

Staphylococcus aureus

Cas9 and

Campylobacter

jejuni

Cas9, can target RNA in the absence of a PAMmer, thereby demonstrating PAM-

independent RNA cleavage

138

. Cas13 proteins are RNA-guided RNA-targeting nucleases,

some requiring recognition of a protospacer flanking sequence (PFS), that generate cuts

along target and non-target RNA molecules using two HEPN domains, which are

nucleotide-binding domains with RNA cutting activity

142

. b ∣ Similar to catalytically

deficient Cas9 (dCas9), dCas13 maintains the capacity to bind the targeted RNA. For RNA

visualization and tracking purposes, a fluorescent protein can be fused to the dCas protein

and co-localize with an array of crRNAs or gRNAs

135,143

. Adenosine deaminase RNA

specific (ADAR) enzymes can be fused to dCas for RNA A→I base editing to correct

disease-relevant mutations. To promote alternative splicing, dCas13 can be targeted to bind

splicing regulating

cis

elements

152

. c ∣ Cas13 can be used for targeted RNA degradation in

eukaryotic cells for applications such as targeting viral RNA or toxic RNAs that contain

microsatellite repeat expansions

136

Francisella novicida

Cas9 has been repurposed in

eukaryotic cells to target the RNA genome of hepatitis C virus

141

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Figure 4. Targeted gene regulation and other applications.

a ∣ For transcription repression, catalytically-deficient Cas9 (dCas9) alone or dCas9 fused to

effectors such as the transcription repression domain of Krϋppel-associated box domain

(KRAB)

157

can be targeted to promoters, 5’ untranslated region (5’ UTR)

enhancers

156,159-161

. Transcription activation can be targeted by fusing dCas9 to

transcription activation domains such as VP64: VP64–dCas9–VP64 activated the expression

of the neuronal transcription-factor genes

Brn2, Ascl1 and Myt1l

and thus directed the

conversion of primary mouse embryonic fibroblasts into neuronal cells

172

. Similarly, dCas9

was fused to the catalytic domain of methylcytosine dioxygenase TET1 and targeted to the

FMR1

gene, to reverse the hypermethylation and silencing of the gene, which is associated

with Fragile X syndrome

176

. b ∣ Inducible Cas9-based systems allow dynamic control of

gene targeting. For example, chemical induction by rapamycin of the dimerization of split

dCas9 fused to the rapamycin-binding domains of FKBP and FRB activates target-gene

expression. Alternatively, light-inducible dimerization of the cytochrome proteins CRY2 and

CIBN can be used in photoactivatable systems. Combinations of inducible dCas9-ortholog-

based systems can be used for dynamic manipulation of multiple targets simultaneously. For

example, dimerization of

Streptococcus pyogenes

dCas9 (dSpCas9)–KRAB by the addition

of abscisic acid (ABA) can repress one gene while dimerization of

Staphylococcus aureus

dCas9 (dSaCas9)–VP64–p65–Rta (VPR) by the addition of gibberellin can lead to activation

of another gene

189

. c ∣ CRISPR–dCas9 tools can monitor or manipulate chromatin

interactions that regulate gene expression. The fusion of dCas9 to the peroxidase APEX2

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can be used to biotinylate proteins in the vicinity of a targeted genomic locus; the proteins

are then identified using mass spectrometry

200

. Distal loci can be brought into proximity

using chromatin loop reorganization with CRISPR–dCas9 (CLOuD9). In the CLOuD9

system, dSpCas9 and dSaCas9 are fused to the dimerizing, ABA-binding proteins PYL1 and

ABI1

202

. ABA induces targeted protein dimerization and chromatin looping, which can be

reversed following its removal to restore the endogenous chromatin conformation.

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Table 1.

Cas9 variants with altered PAM and targeting specificities

Name Included mutations PAM (5’ to 3’) Notes

SpCas9

Native

Streptococcus pyogenes

Cas9

NGG

249

1368 amino acids

VRER SpCas9 D1135V, G1218R, R1335E, T1337R

NGCG

Altered PAM variant; Bacterial-selection-based

screening

VQR SpCas9 D1135V, R1335Q, T1337R

NGAN or NGNG

Altered PAM variant; Bacterial-selection-based

screening

EQR SpCas9 D1135E, R1335Q, T1337R

NGAG

Altered PAM variant; Bacterial-selection-based

screening

xCas9-3.7 A262T, R324L, S409I, E480K, E543D, M694I,

E1219V

NG, GAA, GAT

Altered PAM variant; Phage-assisted continuous

evolution

eSpCas9 (1.0) K810A, K1003A, R1060A NGG Enhanced specificity; Structure-guided protein

engineering

eSpCas9 (1.1) K810A, K1003A, R1060A NGG Enhanced specificity; Structure-guided protein

engineering

Cas9-HF1 N497A, R661A, Q695A, Q926A NGG

Enhanced specificity

HypaCas9 N692A, M694A, Q695A, H698A NGG

Enhanced specificity

evoCas9 M495V, Y515N, K526E, R661Q NGG

Enhanced specificity; yeast-based screening

HiFi Cas9 R691A NGG Enhanced specificity for ribonucleoprotein

delivery

ScCas9 Native

Streptococcus canis

Cas9

NNG

250

1375 amino acids

StCas9 Native

Streptococcus thermophilus

Cas9

NNAGAAW

11,25

1121 amino acids

NmCas9 Native

Neisseria meningitidis

Cas9

NNNNGATT

26-28

1082 amino acids

SaCas9 Native

Staphylococcus aureus

Cas9

NNGRRT

1053 amino acids

CjCas9 Native

Campylobacter jejuni

Cas9

NNNVRYM

984 amino acids

CasX Deltaproteobacteria and Planctomycetes phyla

TTCN

980 amino acids

AAV, adeno-associated virus

ABA, abscisic acid

ABE, adenine base editor

ABI1, dimerizing ABA-binding protein

ADAR, adenosine deaminase acting on RNA

APEX2, engineered apurinic/apyrimidinic endodeoxyribonuclease 2 peroxidase

APOBEC1, apolipoprotein B mRNA editing enzyme complex-1

BE3, third generation base editor

BLESS, breaks labeling, enrichment on streptavidin and next-generation sequencing

Cas9-HF1, high fidelity Cas9 variant

CIB1, plant-derived cytochrome protein

CIRCLE-seq, circularization for in vitro reporting of cleavage effects by sequencing

CLOuD9, chromatin loop reorganization with CRISPR–dCas9

CORRECT, consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets

CRISPR, clustered regularly interspaced short palindromic repeats

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CRISPRa, CRISPR activation

CRISPRi, CRISPR interference

crRNA, CRISPR RNA

CRY2, plant-derived cytochrome protein

dCas9, catalytically deficient Cas9

Digenome-seq,

in vitro

Cas9-digested whole-genome sequencing

DNA, deoxyribonucleic acid

DNMT, DNA methyltransferase

DSB, double-strand break

dsDNA, double-stranded DNA

enAsCas12a, enhanced AsCas12a variant

enCHIP, engineered DNA-binding molecule-mediated chromatin immunoprecipitation

EQR, Cas9 variant with triple mutations

eSpCas9, enhanced specificity SpCas9 variant

evoCas9, evolved high fidelity Cas9 variant

FKBP, rapamycin-binding domain

FRB, rapamycin-binding domain

GFP, green fluorescent protein

gRNA, guide RNA

GUIDE-seq, genome-wide, unbiased identification of DSBs enabled by sequencing

HCV, hepatitis C virus

HDR, homologous-directed repair

HEPN, higher eukaryotes and prokaryotes nucleotide binding domain

HiFi Cas9, high fidelity Cas9 variant

HITI, homology-independent targeted integration

HNH, Cas9 nuclease domain

HTGTS, high-throughput, genome-wide, translocation sequencing

HypaCas9, hyper-accurate Cas9 variant

Indels, insertions or deletions

iPSC, induced pluripotent stem cell

lncRNA, long non-coding RNA

KRAB, Krϋppel-associated box

LNP, lipid Nanoparticle

MEMOIR, mutagenesis with optical in situ readout

mRNA, messenger RNA

nCas9, Cas9 nickase

NHEJ, non-homologous end joining

p65, NF-

B transactivating subunit

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PAM, protospacer adjacent motif

PAMmer, PAM-presenting oligonucleotide

PFS, protospacer flanking sequence

PYL1, dimerizing ABA-binding protein

REPAIR, RNA editing for programmable A to I replacement

RNA, ribonucleic acid

RNAi, RNA interference

RNP, ribonucleoprotein

RuvC, Cas9 nuclease domain

SHERLOCK, specific high sensitivity enzymatic reporter unlocking

smFISH, single-molecule RNA fluorescence hybridization

ssDNA, single-stranded DNA

ssODN, single-stranded oligodeoxynucleotide

stgRNA, self-targeting gRNA

TET1, ten-eleven translocation methylcytosine dioxygenase 1

tracrRNA, trans-activating crRNA

tRNA, transfer RNA

UGI, uracil glycosylase inhibitor

VIVO, verification of in vivo off-targets

VP64, four repeats of the herpes simplex VP16 activation domain

VQR, Cas9 variant with triple mutations

VRER, Cas9 variant with quadruple mutations

xCas9, expanded PAM SpCas9 variant

AsCas12a –

Acidaminococcus

spp. Cas12a

CjCas9 –

Campylobacter jejuni

Cas9

FnCas9 –

Francisella novicida

Cas9

LbCas12a –

Lachnospiraceae

spp. Cas12a

LwaCas13a –

Leptotrichia wadei

Cas13a

NmCas9 –

Neisseria meningitidis

Cas9

PaCsy4 –

Pseudomonas aeruginosa Csy4

PspCas13b –

Prevotella sp. P5-125

Cas13b

SaCas9 –

Staphylococcus aureus

Cas9

ScCas9 –

Streptococcus canis

Cas9

SpCas9 –

Streptococcus pyogenes

Cas9

StCas9 –

Streptococcus thermophilus

Cas9

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