PAM or PFS requirements for various CRISPR-Cas systems.
Abstract
The discovery and implementation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) systems for genome editing has revolutionized biomedical research and holds great promise for the treatment of human genetic disorders. In addition to the popular CRISPR-Cas9 and CRISPR-Cpf1 systems for genome editing, several additional Class I and Class 2 CRISPR-Cas effectors have been identified and adapted for genome editing and transcriptome modulation. Here we discuss current and emerging CRISPR-based technologies such as Cascade-Cas3, CRISPR-associated transposases (CAST), CRISPR-Cas7–11, and CRISPR-Cas13 for genome and transcriptome modification. These technologies allow for the removal or insertion of large DNA elements, the modulation of gene expression at the transcriptional level, and the editing of RNA transcripts, expanding the capabilities of current technologies.
Keywords
- CRISPR
- Cas9
- Cpf1
- Cascade
- Cas3
- Cas12k
- Cas13
- Cas7–11
1. Introduction
Since the discovery of the double helix, scientists have been searching for ways to manipulate genomes. Over the past 15 years, technological advances such as the development of targetable nucleases finally provided a means for introducing specific alterations within a genome of interest. Targetable nucleases function by introducing a DNA double strand break (DSB) at a precise location within a genome which in turn activates cellular DNA repair pathways. By hijacking these pathways, via the coadministration of DNA repair templates, a plethora of genetic modifications ranging from single nucleotide substitutions to chromosomal translocations can be engineered (Figure 1).
The first implementations of targetable nucleases included zinc finger nucleases (ZFNs) and transcription activator-like effector (TALE) nucleases (TALENs). These enzymes are formed by the combination of a non-specific DNA endonuclease called FokI and DNA binding protein domains derived from the zinc finger or TALE family of transcription factors. These enzymes function as obligate dimers and rely on protein-DNA pairing for target recognition [1, 2]. While these enzymes provide the specificity needed for engineering precise DNA alterations, their programming or reprogramming necessitates the design and synthesis of a new pair of enzymes for each new alteration. The adaptation of the Class 2 type II CRISPR-Cas9 system from
In bacteria and archaea, CRISPR-Cas systems are RNA-based immune systems that control virus and plasmid invasion [3]. CRISPR-Cas systems are taxonomically classified as Class 1 and Class 2 systems based on the number of components involved in the interference stage of the immune response. With rare exceptions, Class 1 systems, which account for approximately 90% of all CRISPR-Cas systems in prokaryotes, use multiprotein effector complexes whereas Class 2 systems use a single effector. Class 1 and Class 2 systems are further divided, based on signature genes and distinctive gene architectures, into three or more types: Type I, III and IV for Class 1 systems and type II, V and VI for Class 2 systems.
Concurrent with the implementation of CRISPR-Cas9 for genome editing, a variety of Class 1 and Class 2 systems with complementary properties to type II effector Cas9 have been identified and adapted for genome and transcriptome editing. These systems include: Class 1 type I Cascade-Cas3 systems [4, 5, 6], which are RNA-guided DNA shredding systems; Class 2 type V-K effector Cas12k-Tn7-like transposase systems [7], which are RNA-guided DNA transposition systems that allow for unidirectional insertion of large DNA cargos; and Class 2 type VI CRISPR-Cas13 [8, 9] and Class 1 Type III-E effector Cas7–11 systems [10, 11], which are RNA-guided RNA targeting systems.
In this chapter, we describe conventional as well as emerging CRISPR-Cas-based technologies for transcriptome and genome editing. We provide a simplified view of these systems and their operons. When applicable, we describe the modifications made for genome editing in mammalian cells, the sequence and structure of guide RNAs, PAM requirements, and examples of their use for genome and transcriptome editing. For simplicity, we focus on CRISPR-Cas9 from
2. Conventional CRISPR-Cas systems for genome editing
Genome editing using conventional CRISPR-Cas systems functions by introducing DNA DSBs at a precise location within a target genome. These breaks, known to be highly recombinogenic, are typically repaired via the nonhomologous end joining (NHEJ) DNA DSB repair pathway and result in the random insertion or deletion of genetic material, often referred to as indels. In actively dividing cells, homologous recombination (HR) can also occur and uses the sister chromatid as repair template, resulting in error free repair of the break. By providing an exogenous repair template, in the form of single or double stranded DNA molecules, a variety of genetic alterations can be engineered, including nucleotide substitutions, insertion of DNA elements, deletion of DNA material, inversion of DNA elements, as well as chromosomal translocations (Figure 1). Two main CRISPR-Cas systems from various species, CRISPR-Cas9 and CRISPR-Cpf1, have been adapted for genome editing and other applications.
2.1 CRISPR-Cas9
The most frequently used CRISPR-Cas system for genome editing in mammalian cells has been derived from the Class 2 type II CRISPR-Cas system from
System | Bacterial strain | PAM or PFS |
---|---|---|
Cas9 | Streptococcus pyogenes | NGG |
S. pyogenes (VQR) | NGAG | |
S. pyogenes (VRER) | NGCG | |
Streptococcus mutans | NGG | |
Staphylococcus aureus | NNGGGT NNGAAT NNGAGT | |
Streptococcus thermophilus (CRISPR3) | NGGNG | |
S. thermophilus (CRISPR1) | NNAAAAW | |
Campylobacter jejuni | NNNNACA | |
Neisseria meningitidis | NNNNGATT | |
Pasteurella multocida | GNNNCNNA | |
Francisella novicida | NG | |
Treponema denticola | NAAAAN | |
Cpf1 | Franciselle novicida | TTN |
Acidaminococcus sp. BV3L6 | TTTN | |
Moraxella bovoculi 237 | (T/C)(T/C)N | |
Cascade-Cas3 | AAG | |
Escherichia coli | ARG | |
Pseudomonas aeruginosa | AAG | |
CRISPR-Cas12k | Scytonema hofmannii (ShCAST) | NGTN |
Anabaena cylindrica (AcCAST) | NGTN | |
Cascade-Tn6677 | Vibrio Clolerae (Tn6677) | CC |
CRISPR-Cas13 | Leptotrichia shahii | A,U,C (not G) |
CRISPR- Cas7–11 | Scalindua brodae | N/A |
CRISPR-SpCas9 systems have also been developed to introduce a limited number of nucleotide substitutions without introducing DNA DSBs. These systems, called Base Editors and Prime Editors, make use of catalytically impaired SpCas9 fused to various base modifying enzymes like a cytidine deaminase (CD), an uracil DNA N-glycosylase (eUNG), or a modified dimeric tRNA adenine deaminase (TadA*), which catalyze base transversion or base conversion within a precise window upstream of the PAM sequence (Figure 2D-K). The selection of a base editor depends on several criteria including the desired edit, the availability of PAM sequences within the target sequence, the position of the target nucleotide relative to the PAM sequence, the possibility of engineering undesired bystander mutations, and the need to minimize off-target editing [18]. The most recent versions of these systems are BE4max, a cytidine base editor which catalyzes the conversion of a cytidine into a thymine (C- > T) (Figure 2D); BE7.10, an adenine base editor which catalyzes the conversion of adenine to guanine (A- > G) (Figure 2E); CGBE1, a base editor that catalyzes cytidine to guanine (C- > G) base transversion (Figure 2F) [19, 20, 21].
Prime editing, on the other hand, makes use of a catalytically impaired SpCas9 fused to a reverse transcriptase (RT) and a prime editing guide RNA (pegRNA) (Figure 2G) [22]. The pegRNA not only provides target specificity and scaffolding, but also contains sequences that are complementary to the target site and substitutions encoding the desired edits. Following excision of the target strand, a 3′ flap is exposed and the pegRNA complexes with the exposed 3′ flap and serves as primer site for the RT, which extends the 3′ flap and incorporates the desired nucleotide substitutions. Stabilization of the locus is performed by the endogenous endonuclease FEN1 which removes the 5′ flap and allows the hybridization of the edited 3′ flap, resulting in the incorporation of edited bases and conversion of the unmodified allele via the DNA mismatch repair (MMR) pathway.
In addition to providing the framework for various genome editing technologies, CRISPR-SpCas9 has been morphed into DNA and RNA imaging devices [23, 24], epigenetic modifiers [25] as well as transcriptional modulators [26, 27, 28] via the fusion between catalytically inactive but sgRNA competent SpCas9 and transcriptional activators, transcriptional repressors, epigenetic modifiers, fluorescent proteins, and others (Figure 2H-K).
2.2 CRISPR-Cpf1
Another popular system for genome editing in mammalian cells has been derived from the Class 2 type V CRISPR-Cpf1 system from
3. Emerging CRISPR-Cas systems for genome and transcriptome modification
Concomitant with the development of CRISPR-Cas9 and CRISPR-Cpf1 for genome editing, other CRISPR-Cas operons have been adapted for genome and transcriptome modifications. These emerging technologies provide a means to engineer large genomic deletions, large DNA insertions into safe harbor loci, which remains somewhat challenging using conventional CRISPR-Cas systems, or for the modulation of gene expression. These systems include Cascade-Cas3 from
3.1 Cascade-Cas3
The Type I CRISPR-Cas system from
Several other Cascase-Cas3 systems have been developed for genome editing in bacteria and human cell lines. These include Type I-E Cascade-Cas3 from
3.2 CRISPR-associated transposases
Insertion of large DNA elements using CRISPR-SpCas9 or CRISPR-FnCpf1 technologies has remained a major challenge. One emerging technology that may resolve this issue is CAST, which functions by recruiting Tn7-like transposase components to a specific location within the genome of a cell via guide RNA-target complementarity recognized by a naturally occurring inactive Cas12k variant. The Class 2 Type V-K Tn7-like CRISPR system from
3.3 RNA interference systems
Whereas the vast majority of CRISPR-Cas systems have evolved to protect against invading DNA species, Type VI CRISPR-Cas13 and the newly identified Type III-E CRISPR-Cas7–11 effectors are RNA-guided RNA interfering systems. These systems have been used to silence gene expression at the transcriptional level and have been modified to edit RNA transcripts. Several Type VI and Type III-E systems have been described. For simplicity, we present two of these systems: Type VI CRISPR-Cas13 from
Type VI CRISPR-Cas13a from
Not only have Type VI CRISPR-Cas13 systems been developed for the degradation of RNA species, but the fusion of a nuclease-dead Cas13b from
The Type III-E effector system Cas7–11 from
4. Discussion
Whereas the vast majority of genetic manipulations can be performed using conventional CRISPR-SpCas9 technology, there are some inherent limitations that may be alleviated by emerging technologies. These include the possibility of introducing DNA DSBs at off-target sites; the possibility of inserting undesired mutations at on-target sites; the requirement for specific PAM sequences, which may somewhat limit the number of target possibilities; the scope of editing; and the delivery of these reagents, particularly for manipulations in vivo or for therapeutic interventions. Table 2 explores the similarities and differences between the systems described in this chapter as well as their advantages and limitations.
System | Source | Mechanism | Advantages | Limitations |
---|---|---|---|---|
CRISPR-Cas9 | Cas9 pairs with a sgRNA to introduce a DNA DSB 3 nt upstream of the PAM. DSBs are resolved by NHEJ or HR | A well-established, conventional system; can be used to engineer a plethora of genetic modifications, ranging from nucleotide substitutions to chromosomal translocations; a single effector system which may be easier to use as opposed to multiprotein effector systems | Off-target editing of genomic DNA may occur; uses a long sgRNA; uses a large endonuclease which may be difficult to package in viral delivery systems; introduces DNA DSBs which may have deleterious effects if not resolved properly, particularly for therapeutic applications | |
CRISPR-Cpf1 | Cpf1 pairs with a crRNA to introduce scattered DNA breaks at position 18 of the non-target strand and 23 of the target strand, leaving a 5′ overhang. DSBs are resolved by NHEJ or HR | Can be used to engineer a plethora of genetic modifications, ranging from nucleotide substitutions to chromosomal translocations; uses a different PAM than SpCas9, extending the range of possible target sites; a single effector system which may be easier to use as opposed to multiprotein effector systems; uses a short crRNA that may be easier to synthesize and deliver | Off-target editing of genomic DNA may occur; uses a large endonuclease (although smaller than SpCas9) which may be difficult to package in viral delivery systems; introduces DNA DSBs which may have deleterious effects if not resolved properly, particularly for therapeutic applications | |
Cascade-Cas3 | Cascade pairs with a crRNA to recruit Cas3, a highly processive DNA helicase-nuclease, which nicks the non-paired DNA strand and unidirectionally shreds the target DNA upstream of the PAM sequence in a 3′ to 5′ orientation | Can be used to generate large deletions; uses a short crRNA that may be easier to synthesize and deliver | Off-target editing of genomic DNA may occur; large multiprotein effector which can be difficult to package for viral delivery; limited in the scope of editing; no control over the length of the deletions | |
CRISPR-CAST | Cas12k and Tn7-like transposase components pair with a sgRNA to insert large DNA cargo unidirectionally in a 5′ LE to 3′ RE orientation 60–66 nt downstream of the PAM. A 5 bp integration site is duplicated and found 5′ and 3′ of the cargo integration | Can be used to insert large DNA elements up to ~10Kb | Off-target editing of genomic DNA may occur; insertion results in the integration of flanking sequences and duplication of the integration site; a multiprotein effector which can be difficult to package for viral delivery; uses a long sgRNA; limited in the scope of editing; demonstrated efficacy only in bacterial systems | |
CRISPR-Cas13 | C2c2, a HEPN containing protein, pairs with a crRNA to cleave ssRNA transcripts at exposed uracil residues | Targets RNA rather than DNA, can be used to silence gene expression at the translational level instead of permanently altering the DNA; a single effector system which may be easier to use as opposed to multiprotein effector systems; uses a short crRNA that may be easier to synthesize and deliver | Off-target editing of ssRNA transcripts may occur; cleavage of ssRNA transcripts occurs at exposed uracil residues, which are difficult to predict; a large effector protein which can be difficult to package for viral delivery; demonstrated efficacy only in bacterial systems | |
Cas7–11 | A protein (gRAMP) with Cas7-like and Cas11-like domains with intrinsic endoribonuclease activity pairs with a crRNA to cleave ssRNA transcripts at positions 3 and 9 of the spacer | Targets RNA rather than DNA, can be used to silence gene expression at the translational level instead of permanently altering the DNA; cleavage of ssRNA transcripts occurs at positions 3 and 9 of the spacer, which are easily predictable compared to CRISPR-Cas13 systems; a single effector system which may be easier to use as opposed to multiprotein effector systems; uses a short crRNA that may be easier to synthesize and deliver | Off-target editing of ssRNA transcripts may occur; a large effector protein which can be difficult to package for viral delivery; demonstrated efficacy only in bacterial systems |
4.1 Off-target mutations
One major limitation associated with the use of CRIPSR-SpCas9 technology is the potential for inserting genetic changes at sites other than the intended ones, also referred to as off-target sites. Off-target cleavage may occur due to the lack of SpCas9 specificity, which stems from the tolerance of the endonuclease for RNA–DNA mismatches, RNA bulges, or DNA bulges [12, 13]. Although there are still no simple and definitive guidelines defining SpCas9 specificity, the number and the position of mismatches relative to the PAM sequence are important. Whereas a single mismatch within the first 13 nucleotides upstream of the PAM sequence can abrogate SpCas9 activity, up to seven mismatches at the 5′ end of the guide sequence can be tolerated [12, 13]. To avoid off-target modifications, various strategies have been established. These include the development of bioinformatic tools to identify highly specific target sequences; the modification of SpCas9 to improve specificity or the duration of its action within cells; and the development of delivery formats and methods to limit the duration of SpCas9 activity [14]. Emerging RNA-guided RNA modifying systems may also help resolves this issue. These systems can modulate gene expression by targeting RNA transcripts rather than modifying genes at the DNA level [9, 11].
To identify highly selective guide sequences, various guide selection applications have been developed. These include Cas-Designer, quick guide-RNA designer for CRISPR/Cas derived RNA guided nucleases (http://www.rgenome.net/); CRISPR Design (http://crispr.mit.edu/);E-CRISP (http://www.e-crisp.org/E-CRISP/); ZiFit (http://zifit.partners.org/ZiFiT/). During the implementation of CRISPR-SpCas9 technology for mouse genome editing, our laboratory also developed a stringent guide selection procedure which makes use of Cas-Designer and Cas-Offinder from CRISPR RGEN Tools (http://www.rgenome.net/). For more details about this guide selection procedure, we recommend reading.
To control the duration of SpCas9 activity within cells, genetically encoded inducible systems have been developed. One of these makes use of split SpCas9 fused to the Magnet Photoactivatable System. Fusion of the split SpCas9 is achieved by illuminating cells with blue light. The fused split SpCas9 is then able to bind a sgRNA and cleave its target site. A second system involves self-cleavable CRIPSR systems, where sequences encoding SpCas9 are targeted by a sgRNA in order to promote its degradation upon expression of the endonuclease. Duration of SpCas9 activity can also be controlled by the delivery of RNA transcripts encoding the various components of the CRISPR-SpCas9 system or via delivery of the ribonucleoprotein complex comprising both the sgRNA and the endonuclease.
In addition to robust guide selection procedures and inducible/self-inactivating systems, various additional strategies have been developed to improve target specificity. These include the use of paired SpCas9 nickases, in which the RuvC-like domains are inactivated, that introduce scattered DNA DSB, guided by a pair of sgRNAs recognizing juxtaposed sequences. The requirement for recognizing these sequences doubles the length of the target sequence and thus increases target specificity. Similarly, catalytically inactive but sgRNA competent pairs of SpCas9 fused to the non-specific endonuclease FokI was shown to reduce off-target activity. Directed evolution has also been used to engineer improved SpCas9 with increased target specificity. These enzymes have been shown to increase on-target over off-target activity by several folds.
Finally, modifications to the guide RNAs themselves were also used to reduce off-target cleavage. Previous studies have shown that, counterintuitively, shortened guide sequences increase specificity without affecting on-target activity.
4.2 Unintended mutations at the target site
Engineering specific mutations using conventional CRIPSR-SpCas9 technology relies on HR. While HR and NHEJ are both active in most (but not all) dividing cells, NHEJ is usually the sole repair pathway active in postmitotic cells. DNA repair via the NHEJ pathway, as previously mentioned, results in the insertion or deletion of genetic material, and does not allow for the introduction of desired mutations. To get around this, Base Editors and Prime Editors were developed. These systems, as described above, can introduce specific mutations by directly changing the nucleotide composition at the target site, bypassing the need to activate DSB repair pathways. Stabilization of the mutation is performed by the DNA MMR pathway which is present and active in all cells. While these systems allow for the insertion of precise mutations, the window in which they operate is narrow (a few nucleotides), the insertion of mutation(s) depends on the presence of a PAM sequence, stabilization of the mutation is not always complete, and undesired collateral nucleotide substitutions may occur [18, 19, 20, 21, 31, 33]. Moreover, these systems are bulkier than SpCas9 and may not be easily packaged within viral delivery systems. Nevertheless, these systems provide an alternative to conventional CRISPR-SpCas9 systems for engineering mutations in cells that are not amenable to HR or for therapeutic intervention where introducing DNA DSBs may have deleterious effects.
4.3 Editing scope
While conventional CRISPR-Cas9 systems can be used to engineer virtually any kind of mutations in vivo, insertions or deletions of large DNA elements remain somewhat challenging. Cascade-Cas3 and CRISPR-CAST systems may provide alternatives to using conventional CRISPR-Cas9 systems. Cascade-Cas3 systems have been used to engineer large deletions in cultured cells and can potentially be applied to animal models [4, 5, 6]. Deletions range from several hundred to several thousand base pairs. The major drawback of using this technology is the apparent uncontrollable processivity of the helicase-nuclease Cas3. Consequently, deletions of various sizes must be characterized using a large number of primer pairs flanking the potential deletions. Other limitations include the difficulty of packaging multiprotein systems for viral delivery in vivo. CRISPR-CAST systems, on the other hand, allow for targeted integration of large genetic material. In
4.4 Delivery
The vast majority of CRISPR-Cas systems for genome editing make use of either multiprotein effectors or large single effectors. Although delivering these systems, together with their cognate guide RNAs, in cultured cells or zygotes for the generation of animal models does not represent a major hurdle and is routinely performed, delivering these systems in vivo, for therapeutic interventions, does represent a major challenge. More compact CRISPR-Cas9 and Cas3 systems have been identified and these may represent viable alternatives to other larger and more complex CRISPR-Cas system for therapeutic purposes [6].
4.5 PAM requirements
The requirement for CRISPR-Cas systems to recognize short genomic sequences has long been viewed as major disincentive for the use of these technologies for genome engineering. However, most PAM sequences are quite short and are likely present at a high frequency within mammalian genomes. Moreover, the development of several CRISPR-Cas9 and Cpf1 systems with distinct PAM requirements and the generation of engineered SpCas9 endonucleases with altered PAM specificities have expanded the targeting capabilities of CRISPR-Cas systems.
5. Conclusion
Since the discovery of targetable nucleases, more notably CRISPR-Cas systems, the field of genetic and genome engineering has expanded exponentially. In less than a decade, these systems have not only revolutionized how research is performed but have also allowed for a plethora of scientific discoveries and paved the way for novel human therapeutics. Emerging technologies such as Cascade-Cas3, CAST, CRISPR-Cas7–11, and CRISPR-Cas13 provide alternatives to current technologies and may fill a critical technological gap to improve the specificity and scope of genome editing. Moreover, the implementation of these tools as therapeutic agents offers the potential to treat or even cure human genetic diseases.
Acknowledgments
The author would like to thank the Indiana University School of Medicine for the financial support of the Indiana University Genome Editing Center.
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