Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5608_Библиотеки_им_академика_М_И_Перельмана.pdf
260
section delves into the future of CRISPR therapeutics, including nascent applications, the industrialization and projected growth of the industry, safety and ethical
considerations, and its transformative potential for human health.
Keywords CRISPR · Gene editing · Genome engineering · Base editing · Primeb
editing · Guide RNA · Cell therapy · Gene therapy
R. Kishton et al.
10.1 Introduction toCRISPR Technology
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing
is undoubtedly one of the most important scientic breakthroughs of the last century, allowing scientists to precisely modify DNA and RNA.This technology has
driven the creation of a new generation of enhanced biologics for the treatment of
diseases, as well as more relevant models of these diseases for research and therapeutic development purposes. To understand its revolutionary applications in bioengineering and therapeutics, we will rst explore the basics of CRISPR technology,
how it was discovered and adapted as a tool for gene editing, its mode of action,
advantages over previous gene editors, and the different types of CRISPR systems
currently available.
10.1.1 What Is CRISPR?
CRISPR is a key component of bacterial adaptive immune systems that has been
repurposed as a powerful tool for gene editing [1]. Composed of a CRISPR-
associated (Cas) nuclease and a customizable single-guide RNA sequence (sgRNA),
the nuclease is directed by the sgRNA to a complementary target sequence and creates double-stranded breaks in the genomic DNA.These DNA breaks are repaired
using inherent DNA repair mechanisms, resulting in premature stop codons or missense mutations, or creating conditions for inserting novel DNA payloads to precise
genomic locations. The rst CRISPR system adapted for gene editing used a Cas9
nuclease isolated from the bacteria Streptococcus pyogenes (SpCas9) [1]; this
remains the most well-described and commonly used system.
10.1.2 Discovery andAdaptation ofCRISPR
CRISPR was rst discovered in the early 1990s by Dr. Francisco Mojica, a molecular microbiologist at Spain’s University of Alicante. Dr. Mojica identied arrays of
regularly spaced repeats in the DNA of a halophilic archaea [2], and later in other
species of archaea and bacteria. After identifying a DNA sequence in the CRISPR
10 CRISPR Technology andIts Application inTherapeutics
Fig. 10.1 The CRISPR-Cas9 systemThe CRISPR-Cas9 system comprises a guide RNA (gRNA)
and Cas9 nuclease, which together form a ribonucleoprotein (RNP) complex (Fig.10.1). The presence of a specic protospacer adjacent motif (PAM) in the genomic DNA is required for the Cas9
to bind to the target sequence. The Cas9 nuclease then makes a double-strand break in the DNA
(denoted by the scissors). Endogenous repair mechanisms triggered by the double-strand break
may result in gene knockout via frameshift mutation or knock-in of a desired sequence if a DNA
template is present
261
array of Escherichia. coli identical to DNA from a virus that commonly infects
these bacteria, he hypothesized that CRISPR loci were a component of the bacterial
immune system [3]. This theory was conrmed in 2007 by Dr. Philippe Horvath by
integrating a viral resistance sequence from the invading phage into its CRISPR loci
[4] (Fig.10.1).
In 2012, Dr. Emmanuelle Charpentier of Berlin’s Max Planck Unit for the
Science of Pathogens and Dr. Jennifer Doudna of the University of California,
Berkeley, published a landmark paper in the journal Science, in which they described
the molecular mechanism of the CRISPR-Cas9 system in the pathogenic bacteria
Streptococcus pyogenes [1]. They explained that activity of the Cas9 nuclease is
directed by a dual-RNA structure of a CRISPR RNA (crRNA) and a trans-activating
crRNA (tracrRNA) which base-pair together through partial complementary
sequences. By engineering the dual-RNA structure as a single RNA chimera,
Doudna and Charpentier demonstrated that spCas9 can be reprogrammed and
directed to specic sites [1]. They postulated that CRISPR-Cas9 could be readily
adapted as a tool for gene editing, and their work resulted in the pair winning the
2020 Nobel Prize for Chemistry. Shortly after, Dr. Feng Zhang made a key contribution by demonstrating that CRISPR-Cas9 could be used to edit DNA in
262
immortalized mammalian cell lines [5]. The translation of CRISPR-Cas9 editing
from cell lines to primary cells initially proved challenging; however, a 2015 paper
described the use of chemically modied, synthetic sgRNAs to efciently edit primary human T cells and CD34+ hematopoietic stem and progenitor cells [6].
R. Kishton et al.
10.1.3 Advantages ofCRISPR Over Other Gene Editors
The development of CRISPR as an easily customizable tool for gene editing was,
quite simply, a game changer in the eld of engineering biology. In the early days
of genome engineering, scientists relied on the use of restriction enzymes, errorprone polymerase chain reactions, and mutagenesis to induce alterations in the
genetic code of cells. Several tools were subsequently developed for more precise
gene editing, including meganucleases, zinc nger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs) [7–9]. Due to the simplicity, precision, and relatively low cost of CRISPR, these previous methods are used less
commonly [10]. Compared to other editing methods, which are laborious to customize or have a limited range of targets, CRISPR only requires the presence of a
specic sequence (called PAM, specic protospacer adjacent motif) near the desired
target site [11]. Each new target site simply requires a new sgRNA while maintaining the nuclease constant. Multiple edits within the same cell or organism are possible with relative ease just by introducing two sgRNAs [12, 13].
10.1.4 Mechanism Behind CRISPR Gene Editing
The Cas nuclease searches the genome for a sequence known as the protospacer
adjacent motif (PAM), typically found 3–4 nucleotides downstream of a potential
target site [1]. PAM requirements differ widely among CRISPR systems in terms of
their sequence and stringency. SpCas9, the most widely used Cas9 nuclease, recognizes a 3bp 5′-NGG-3′ PAM sequence, where N can be any nucleotide. If the PAM
sequence is found, Cas9 will search the region upstream for a sequence that is complementary to its bound guide RNA.If both PAM and target sequence are present,
Cas9 will create a double-stranded break (DSB) in the DNA [1].
Once CRISPR-Cas9 has generated a DSB in the DNA of a cell, editing can take
place by exploiting cellular DNA repair pathways–predominantly the nonhomologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms [14].
NHEJ is an error-prone DNA repair process that frequently results in either insertion or deletion mutations, together referred to as indels. This process can be
exploited to render genes nonfunctional, otherwise known as a gene knockout [15].
Conversely, HDR of a DSB can result in the insertion of new genetic material,
known as gene knock-in, if a suitable DNA donor template is provided along with
the other editing components [16] as seen in Fig.10.2.
10 CRISPR Technology andIts Application inTherapeutics
Fig. 10.2 CRISPR repair mechanismsThe two most common repair mechanisms facilitating
CRISPR-Cas9 editing are nonhomologous end joining (NHEJ) and homology-directed repair
(HDR). NHEJ results in either insertions or deletions (indels) of nucleotides to repair the
DSB.HDR repairs the DSB by incorporating the sequence encoded in the DNA donor template
into the target sequence
263
10.2 CRISPR Gene Editing: Techniques andMethods
Using CRISPR, several different techniques and methods to manipulate the genome
have been developed, including the deletion and insertion of genetic material, or the
upregulation or downregulation of gene expression.
10.2.1 Creating Gene Knockouts
Gene knockout (KO) is one of the most valuable tools in the functional genomics
toolkit because it allows researchers to effectively “delete” a particular region of the
genome, such that a functional protein is no longer produced. CRISPR-Cas9 is by
far the simplest and most cost-effective way to generate gene knockouts in many
cell types. If Cas9 creates DSBs in DNA, the cell will repair the break using NHEJ,
which is prone to errors. This frequently results in indels that alter the reading
frame, preventing a functional protein from being produced [6]. Because there are
two copies of each gene, gene knockout experiments have three possible outcomes.
The rst is known as a heterozygous knockout, sometimes referred to as a monoallelic knockout (+/−); only one allele is successfully edited, leaving the other intact.
The second is called a homozygous knockout, otherwise known as a complete or
biallelic knockout (−/−); both alleles are edited and no protein can be produced.
264
The third and nal outcome is that both alleles are edited to include frameshift
mutations but each allele has different mutations—this is also referred to as a biallelic knockout.
The likelihood of generating a functional knockout increases when two or more
guides target different regions of the same gene. Cas9 will create multiple DSBs and
can therefore create large-scale deletions or multiple indels. This strategy is known
as multiguide knockout and increases the chances of generating complete, functional gene knockout [17]. Additionally, CRISPR can be used to generate double or
multiple gene knockouts through the introduction of multiple sgRNA sequences to
the cell. Gene knockouts can be controlled spatially and temporally; constitutive
knockouts delete the target gene in an entire organism, whereas conditional knockouts can delete the target gene in specic tissues or at a certain time point, for
example, a specic developmental stage [18]. Cells and animal models generated
using CRISPR are now used for research in labs around the world for studying gene
function and disease modeling.
R. Kishton et al.
10.2.2 Creating Gene Knock-Ins
A gene knock-in (KI) involves the insertion of new genetic material into the genome
of a cell or organism. Unlike knockouts, which rely on the cell’s NHEJ-based repair
mechanism to create indels, knock-ins take advantage of the other main DNA repair
mechanism, HDR. A DNA knock-in template is designed to include homology
regions on either side of the cut genomic DNA sequence, allowing for the targeted
insertion of the desired genetic material [19]. Like knockouts, knock-ins can be
either biallelic (heterozygous) or monoallelic (homozygous).
One of the most impactful therapeutic applications of KIs in therapy is in correcting pathogenic mutations for the treatment of genetic diseases. However, they
are signicantly more challenging to achieve than knockouts for two key reasons:
rst, HDR-based repair is less frequent than NHEJ in many cell types [20]; and
second, providing a donor template for repair adds to the challenges of delivering
editing reagents into cells. Due to the low knock-in editing efciencies, a variety of
techniques have been developed to increase the likelihood of success, including cell
cycle synchronization [20] or use of chemical compounds to favor HDR over NHEJ.
10.2.3 CRISPR Activation andCRISPR Inhibition
In addition to inserting and deleting DNA sequences, CRISPR has been used to
upregulate or downregulate gene expression using a catalytically dead Cas9 (dCas9)
[21]. Because dCas9 cannot induce DSBs, it is used in these systems to specically
bind to DNA.CRISPR activation (CRISPRa) uses dCas9 fused to transcriptional
effectors such as VP64 or p65, directing them to promoter or enhancer regions of
10 CRISPR Technology andIts Application inTherapeutics
265
Fig. 10.3 Mechanisms of CRISPR interference and activationCRISPRi and CRISPRa methods
use catalytically dead Cas9 (dCas9) to alter the gene expression of targets by directing a transcriptional regulator protein near the gene transcriptional start to (a) inhibit or (b) activate its expression
DNA upstream of the target gene, resulting in increased transcription, and therefore
translation, of the gene. In contrast, CRISPR interference (CRISPRi) can either
simply use dCas9 to bind to DNA to interfere with transcription or fuse dCas9 to the
Kruppel-associated box (KRAB) repressor domain, directing it to the promoter
region to reduce transcription [22]. The latter method provides more consistent and
reliable gene silencing than dCas9 alone. Like knockouts and knock-ins, these systems, as seen in Fig.10.3, can be used to elucidate gene function by modulating
their expression and examining the effects on the phenotype of cells and organisms.
10.2.4 CRISPR Screens
CRISPR screens involve large-scale genetic manipulations in cells to examine the
impact of gene modications on cell phenotypes and attributes, therefore determining the function of various genes [23]. CRISPR screens can be loss-of-function,
whereby genes are knocked out or downregulated by CRISPR or CRISPRi, or gainof- function, whereby genes are upregulated using CRISPRa. Loss-of-function
266
R. Kishton et al.
screens are useful tools in functional genomics, particularly as they allow researchers to identify and validate targets for drug discovery.
CRISPR screening can be conducted in two formats, pooled and arrayed. Pooled
screens commonly involve packaging sgRNA-containing plasmids into lentiviral
particles (one per vector) and then combining each virus together equally. This
pooled lentiviral library is then transduced into host cells. The stable expression of
guide (along with Cas9) facilitates the knockouts of targeted genes. Because the
edits occur across all targets in a single tube of cells, it is difcult to link the phenotype of each individual cell with the underlying genetic perturbation. Pooled screens
are thus only compatible with binary assays that physically separate edited cells
exhibiting a phenotype of interest from those that do not. The enrichment or depletion of particular sgRNAs following selection relays information about the involvement of these genes in a particular phenotype and indicates targets that can be
investigated further. Arrayed screens involve targeting each gene separately across
wells of a multi-well plate (Fig.10.4). This format is a newer technology that is
more versatile in both methodology and analysis than pooled screens. Library delivery may be accomplished through transfection or transduction. Because gene targets are separated across wells, phenotypes do not need to be selected for and
sequencing, and data deconvolution is not required to associate phenotypes with
genotypes. Arrayed screens are compatible with both binary and multiparametric assays.
10.3 CRISPR Evolution andDerivative Technologies
Since the discovery of the original CRISPR-Cas9 system, researchers have identied many new CRISPR systems, expanding the range of components that can be
delivered for therapeutic purposes. Known CRISPR systems are currently divided
into two classes, six types, and 33 subtypes [24]. Class 1 is made up of multi-subunit
effector proteins, further divided into types I (subtypes A-G), III (subtypes A-F),
and IV (subtypes A-C). Class 2 comprises single effector proteins and is divided
into types II (subtypes A-C, which includes the famous spCas9), V (subtypes A-I,
K), and VI (subtypes A-D) [24]. These systems differ widely in size, complexity,
targeting, and cleaving ability. This includes Cas9 nucleases from other species that
are smaller than SpCas9, as well as other nucleases that recognize different PAM
sequences, target RNA rather than DNA, target single-stranded DNA rather than
double-stranded, or work at different temperatures. Several naturally occurring
class 2 Cas nucleases are now offered by commercial vendors like Synthego for
creating engineered cells. Staphylococcus aureus Cas9 (SaCas9) is now a popular
nuclease for therapeutic editing because its corresponding DNA sequence is 1kb
smaller than SpCas9 and therefore easier to deliver invivo [26]. Cas12a is also
increasing in popularity because it creates staggered or “sticky-end” DSBs, making
10 CRISPR Technology andIts Application inTherapeutics
267
Fig. 10.4 Example of a CRISPR arrayed screening workowIn a knockout CRISPR arrayed
screening, a gRNA targeting each gene is introduced to each well of a multi-well plate. After editing the cells, applying treatment (such as a drug) enables the identication of which genes (when
rendered inoperative) that cause the cells to have heightened or diminished sensitivity to the drug.
This process can be used to identify potential drug targets
268
R. Kishton et al.
it more suitable for knock-in experiments relying on HDR for the repair [27], as
compared to SpCas9, which creates blunt-end DSBs. Cas12a can also be used for
multiplexed editing because it can process multiple guides simultaneously [28].
Additionally, many new Cas9 nucleases have been engineered for specic purposes: high-delity Cas9 variants, which have increased specicity [29]; Cas9 nickases, which cut only a single strand of the DNA double helix [43]; Cas9 nucleases
with different PAM recognition sites or specicities, which have an expanded range
of targets [30]; and catalytically dead Cas9, which does not cut DNA but retains its
recognition and binding capacity [31]. The original SpCas9 system remains the
most widely used, largely because it is the most well-studied and therefore has a
more predictable safety prole for therapeutic use. However, several naturally
occurring and engineered nucleases are also approaching clinical use. For example,
the EDIT-301 exvivo gene-edited cell therapy (NCT04853576) developed by Editas
Medicine aims to treat sickle cell disease (SCD) using Cas12a (see Sec. 10.7.1).
10.3.1 Derivative Technologies
A myriad of technologies has been derived from CRISPR that perform genomic
modications without generating DSBs. They deploy variants of Cas9 that will nick
(nCas9) or bind to (dCas9) the DNA.One notable example is base editing, which
generates a base substitution. Base editors comprise Cas9 variants (nCas9 or dCas9)
fused to enzymes that either convert adenosine to inosine which ultimately results
in an A-T to G-C substitution (adenine base editors or ABEs) [25] or cytidine to
uracil, generating a C-G to T-A substitution (cytosine base editors or CBEs) [26]
Since their development, multiple iterations of these base editors have been created
to improve the activity as well as minimize off-target effects.
Prime editing is another derivative of CRISPR, using nCas9 fused to an engineered reverse transcriptase [27]. This system requires the use of a specic type of
guide RNA known as a prime editing guide RNA (pegRNA), which contains both a
guide sequence and a template encoding for the desired substitution(s). After nCas9
creates a single-stranded break, it is repaired via reverse-transcription of the provided template encoded in the pegRNA.
Another novel technology called Programmable Addition via Site-specic
Targeting Elements (PASTE) has been developed to allow for very large insertions
of DNA [29]. PASTE editing works via a dual system of prime editors and serine
integrase proteins. In a rst step, the prime editor knocks in the integrase landing
sequence in the desired target location. Next, the integrase performs targeted DNA
insertion of the desired sequence in the previously generated landing pad.
An additional nascent application of CRISPR is in epigenome editing (eGE),
which uses dCas9 fused to epigenetic enzymes or their catalytic domains, such as
DNA methyltransferases (DNMT) or ten-eleven translocation dioxygenases (TETs)
to regulate the expression of genes without inducing DSBs [22].
10 CRISPR Technology andIts Application inTherapeutics
Another level of modifying protein expression without DSBs is targeting messenger RNA (mRNA). Many species of bacteria possess CRISPR systems that are
capable of targeting RNA, such as those found in Class 2, type VI CRISPR-Cas13
systems [32]. Unfortunately, the rst reported RNA targeting enzyme (Cas13) presents collateral activity; after cleaving the target transcript, it will degrade other transcripts present in the cell nonspecically. This has led to the search for other
RNA-targeting enzymes that can efciently edit RNA without collateral activity,
such as Cas7–11 [33].
269
10.4 CRISPR Component Format andDelivery Methods
For editing to occur via a nuclease, a guide RNA and knock-in template (for knockins) must be delivered into cells. These components can be delivered as a number of
biological constructs such as DNA (plasmid), RNA, or protein. The delivery of the
components into the cell can be through viral, chemical, or mechanical methods.
The choice of biological construct and delivery method is dependent on the nal
application and therapeutic conditions, such as delivery for in vivo vs. ex vivo
therapies.
10.4.1 Gene Editing Components: Cas Nucleases
andCargo Formats
Introduction of the Cas nuclease and sgRNA to cells can take three possible forms:
(i) DNA plasmid(s), (ii) mRNA transcripts for translation, or (iii) a ribonucleoprotein (RNP) complex [32]. DNA plasmids were commonly used in the early years of
genetic engineering; however, several factors have hamstrung their therapeutic
value. These include variable editing efciencies, cytotoxicity issues, potential random incorporation into the host genome, and difculties choosing appropriate promoters for nuclease protein expression. The use of DNA plasmids results in delayed
editing owing to the time required for the transcription and translation of the editing
components, and the long-term expression of Cas9 can result in off-target editing.
RNPs have rapidly become a very popular form of CRISPR cargo for therapeutic
purposes for several key reasons. RNP cargos result in rapid, precise editing because
the components are immediately active, and reduce the risk of toxicity and offtarget editing events. Moreover, RNPs are amenable to delivery using almost all of
the currently available vehicles. Therapeutic gene knock-in adds another layer of
complexity to delivery. In this case, an additional editing component of a DNA
donor template for HDR must be provided along with the Cas nuclease and sgRNA.