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_5366_Библиотеки_им_академика_М_И_Перельмана.pdf
270
R. Kishton et al.
10.4.2 Delivery Methods
There are three main categories of CRISPR delivery vehicles: viral, chemical, and
mechanical. The nal application and therapeutic conditions will inuence the
cargo format as well as the choice of vehicle for delivery. Compared to ex vivo
therapies, in which cells are edited outside the body, invivo therapies present a
much more challenging delivery scenario with minimal room for errors owing to
patient safety requirements [33]. Moreover, invivo gene therapies typically require
editing components to be targeted to specic organs or tissues, providing an additional delivery challenge.
Viral vectors work on the basis of infecting target cells or tissue to introduce the
editing components [34]. These vectors include lentiviruses (LVs; Retroviridae),
adenoviruses (AdVs; Adenoviridae), and adeno-associated viruses (AAVs;
Parvoviridae). These viral vectors act as chassis for the editing components to enter
the cell and contain elements encoding for the nuclease and guide sequences,
referred to as cargo. LVs and AdVs are large and can therefore carry considerable
cargo; however, they have several undesirable effects, including integration into the
host genome, adverse immune responses, and off-target effects. In contrast, AAVs
are nonpathogenic and nonintegrating viruses in humans, causing very mild host
immune responses. These safety advantages make them one of the most common
delivery vehicles for invivo therapeutic use. However, they cannot package DNA
above 4.7kb, limiting their application in knock-ins. Strategies to overcome this
challenge include using smaller Cas9 variants such as SaCas9 to minimize the overall cargo load and splitting the CRISPR components between two vectors.
Engineered virus-like particles (eVLPs) have also recently been described as a
highly efcient delivery vehicle for CRISPR RNP cargo [35].
Chemical delivery vehicles for CRISPR editing components include liposomes,
lipid nanoparticles (LNPs), lipo- and poly-plexes, gold and other inorganic nanoparticles, and cell-penetrating peptides [36]. Nanoparticles such as LNPs are increasing
in popularity and have been adapted for use in other non-CRISPR therapeutics, such
as the delivery of mRNA vaccines. Mechanical vehicles for CRISPR delivery
include microinjection into cells, electroporation, nucleofection, and microuidics.
However, these are not compatible with invivo therapies and therefore are exclusively used for exvivo gene-edited cell therapies and invitro experiments.
10.5 CRISPR Therapeutics: Gene Therapies
andGene- Edited Cell Therapies
The development of CRISPR-based therapeutics has ushered in a new era of personalized medicine, including both gene therapy and gene-edited cell therapy. In the
decade since CRISPR was rst adapted as a gene editor, the eld has made
10 CRISPR Technology andIts Application inTherapeutics
astonishing progress, with the rst exvivo CRISPR gene-edited cell therapy beginning clinical trials in 2016, and the rst invivo gene therapy beginning trials in 2019.
271
10.5.1 What Are Cell andGene Therapies?
Cell and gene therapies are a broad category of medical therapeutics based on the
transfer of either whole cells (cell therapy) or genetic material (gene therapy) to
patients to treat disease. Gene therapies are performed invivo and involve either
introducing new genetic material to patients or disrupting genes that bear pathogenic mutations [37]. There are currently over 5000 known diseases caused by
mutations in a single gene [38]. These monogenic disorders can theoretically be
treated by either correcting the pathogenic mutation, known as gene correction therapy, or delivering a functional copy of part or all of the gene to alleviate the condition, known as gene replacement therapy [39].
Cell therapies engineered using CRISPR or other technologies are known as
gene-edited cell therapies. Gene-edited cell therapies include ex-vivo biologics that
can be either autologous (patient-derived) or allogeneic (derived from a healthy
donor) [40]. They aim to replace diseased cells, compensate for a lack of a particular
cell type, or generate a desired functionality through engineered physiological
activity. Many different types of gene-edited cell therapies have been developed,
and they can be categorized by the types of cells used, for example, hematopoietic
stem and progenitor cells (HSPCs), or primary immune cells like T cells. Geneedited cell therapies are becoming increasingly common for the treatment of many
different medical conditions, including cancer, infectious diseases, and genetic
diseases.
10.5.2 Role ofCRISPR inCell andGene Therapy
The simplicity, accuracy, and wide range of potential targets of the CRISPR system
make it ideal as a therapeutic agent for many different diseases. CRISPR’s adoption
in the eld of cell and gene therapy has been rapid. The rst clinical trial of an
exvivo CRISPR gene-edited cell therapy began in 2016, only 4 years after the original CRISPR paper was published, followed by another in 2018. The rst trial of a
CRISPR-based invivo gene therapy began in 2019. The popularity and rapid clinical translation of CRISPR gene therapies and gene-edited cell therapies have only
increased, with new trials beginning regularly. While currently no CRISPR therapies have been approved by the Federal Drug Administration (FDA), it is likely that
many cell and gene therapies will be CRISPR-based in the near future.
272
R. Kishton et al.
10.6 CRISPR inCancer Therapy
Cancer comprises a diverse, heterogeneous group of diseases, making it difcult to
model and treat [41]. Cancers are driven by mutations in DNA, and various types of
cancer are often characterized by commonly occurring signatures of mutations in
oncogenes and tumor suppressor genes [41]. Heterogeneity in the drivers and features of tumors is observed between tumors arising from various tissue types and is
also seen within tumors of similar tissue origin. Further, there can be considerable
heterogeneity within a single tumor mass due in part to the variety of cell types that
are contained within the complex tumor microenvironment [42]. The genetic complexity of cancer inuences not only how treatable the disease is but also how it
spreads, the potential for drug resistance, and the likelihood of recurrence. Metastatic
cancer, which has spread to multiple tissues and organs, presents challenges because
each tumor may exhibit a heterogeneous mutational prole, potentially resulting in
differential biological features and responses to treatment [42, 43]. Additionally,
cancer cells have signicant capacity to either avoid detection by or otherwise suppress the immune system [44].
CRISPR technology has quickly accelerated cancer research, both in terms of
disease modeling and therapeutic strategies [45]. In therapeutic settings, CRISPR
gene editing enables scientists to create better, safer, and more affordable geneedited cell therapies. These therapies offer the ability to treat cancer in a highly
specic manner and are highly desirable over traditional treatments like chemotherapy and radiotherapy, which are toxic to healthy cells.
Cellular immunotherapies that have been broadly used in the clinic as cancer
treatments include the infusion of expanded tumor-inltrating lymphocytes, T cell
receptor (TCR) engineered T cells, and chimeric antigen receptor (CAR) engineered
T cells [46]. These are often referred to as adoptive cell therapies, and each of these
therapies generally involves harvesting T cells from patients and promoting or
imbuing antitumor specicity. Following exvivo expansion to a large number of
cells, the T cell products are infused into patients [47]. Cellular immunotherapies
for the treatment of hematological cancers have generally shown more clinical efcacy to date than those used to treat solid tumors. CRISPR-mediated gene editing in
T cell therapeutic products is now being explored as an avenue to generate cell
therapies that are safer and more efcacious than previous iterations.
10.6.1 Tumor-Inltrating Lymphocyte Therapy
Tumor-inltrating lymphocytes (TIL) are T cells that are localized within a tumor,
with a fraction of TIL exhibiting the capacity to recognize tumor antigens and kill
tumor cells. The degree of TIL inltration in tumors can have positive prognostic
10 CRISPR Technology andIts Application inTherapeutics
273
value in several types of cancer, and TIL can be expanded exvivo and reinfused in
patients as an autologous cancer therapy. The infusion of expanded TIL in lymphodepleted patients in conjunction with interleukin-2 (IL-2) support can be clinically effective, with high rates of response reported in patients with metastatic
melanoma. Despite the efcacy of TIL in certain settings, the expansion and efcacy of TIL can be limited in cancers that evade T cell surveillance and recognition
[48]. Additionally, the antitumor activity of TIL may not be sufcient to mediate
durable clinical responses in many patients. Consequently, CRISPR gene editing of
TIL has been explored as a pathway to enhance T cell expansion and function, such
as through the knockout of T cell intrinsic genes that inhibit antitumor function
[49, 50].
10.6.2 TCR Engineered T Cell Therapy
T cell receptor (TCR) engineered T cell therapy uses T cells that are modied to
express TCRs conferring the capacity for the specic recognition and destruction of
tumor cells [51]. TCRs mediate T cell recognition of tumor cells through the major
histocompatibility complex (MHC)/human leukocyte antigen (HLA) complex [52]
presentation of antigenic peptides. The presented peptides can originate from either
intracellular or membrane-bound proteins. TCR engineered T cells are produced by
the engineered expression of exogenous TCRs with a known capacity to recognize
tumor antigens in therapeutic cell products. The exogenous TCR has historically
been introduced into T cells through viral transduction, but recent work has demonstrated that CRISPR-mediated knock in of a therapeutic TCR is a viable strategy to
produce clinical products. As seen with TIL therapy, TCR engineered T cells can
mediate clinical responses in select patients, but widespread efcacy is lacking.
This is due to a number of factors, including tumor downregulation of HLA expression and mispairing of engineered TCR protein components with endogenous TCR
proteins. Mispairing of the transgenic TCR with the endogenous TCR is a particularly concerning issue, as it can result in inefcient antigen recognition or even
autoimmunity and graft-versus-host disease [52].
CRISPR-Cas9 editing can be used to knock out the endogenous TCR as well as
knock-in the transgenic TCR, eliminating mispairing and toxicity issues [47].
Recent research used CRISPR-Cas9 to knock out the endogenous TRAC (T cell
Receptor Alpha Chain) locus in primary T cells and knock in a TCR with specicity
to epitopes of Wilm’s tumor antigen 1, which is overexpressed in many types of
cancer [53]. The resulting TCR cell therapy displayed potent activity against both
hematological malignancies and solid tumors in mouse models, including acute
myeloid leukemia, acute lymphoblastic leukemia, and glioblastoma, with no observable off-target editing [53].
274
R. Kishton et al.
10.6.3 Chimeric Antigen Receptor T Cell Therapy
Chimeric antigen receptor T (CAR-T) cell therapy involves engineering T cells to
express chimeric receptors on their surface to mediate recognition and killing of
cancer cells [54]. Unlike TCRs, which are naturally occurring and recognize MHC/
HLA, chimeric antigen receptors are engineered proteins that recognize specic
antigens expressed on the surface of cancer cells.
Similar to other cell immunotherapies, CAR-T is often inhibited by the cancer
cells overexpressing immune checkpoint inhibitors, such as programmed cell death
protein 1 ligand (PD-L1) on the cell surface. PD-L1 binds to the PD-1 receptor
expressed on the surface of T cells, inhibiting immune cells [55]. Overcoming the
PD-L1 inhibition pathway provides a promising avenue for next-generation cell
therapies via a CRISPR knockout of PD-1 [55]. CRISPR can also be used for targeted knock-in of the CAR, which is then continuously expressed on the cell surface. Results of a Phase I trial of mesothelin-specic CAR-T cells with PD-1 and
TCR gene knockouts have established the safety and feasibility of this method in
human subjects [56].
Allogeneic CAR-T cells, although advantageous to manufacture relative to
autologous cell products, are often unable to persist in hosts. This is due to recognition and rejection by the host immune system or the presence of cytotoxic drugs.
CRISPR has been employed in attempts to circumvent this issue by engineering
universal allogeneic CAR-T therapy; by knocking out key genes involved in recognition by the host immune system, the cells can theoretically be used for any patient.
Unlike previous technologies, CRISPR-Cas9 can be used to perform these complex
edits; currently, several clinical trials are underway, in which multi-knockout allogeneic CAR-T cells are being tested in human patients with various types of cancer
[57, 58]. By generating CAR-T cells from iPSCs rather than from healthy donors,
large stocks of “off-the-shelf” CAR-T cells are ready to treat patients with minimal
wait times.
10.6.4 In Vivo CRISPR Editing ofSolid Tumors
In recent years, CRISPR has also been adapted for invivo editing of cancers by
targeting cancer-specic mutations [59] or otherwise disrupting oncogenes. CRISPR
can be used to disrupt the expression of key proteins that are overexpressed in cancer cells, such as Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) [60]. NRF2
regulates hundreds of other genes, and its overexpression in cancer cells can result
in resistance to chemotherapy. However, CRISPR-based knockout of the NRF2 protein can restore chemosensitivity of the cancer cells. This strategy is being investigated in preclinical studies by researchers at the ChristianaCare Gene Editing
Institute [60]. Other recent research in tumor editing includes improving delivery
systems to efciently target tumors invivo. For example, one key study from Tel
10 CRISPR Technology andIts Application inTherapeutics
Aviv University developed targeted lipid nanoparticles (LNPs) for the delivery of
CRISPR-Cas9 components [61] to target the KO of the PLK1 gene, resulting in high
(~80%) editing efciencies, potent inhibition of tumor growth, and improved survival rates [61].
275
10.7 CRISPR Treatments forGenetic Disease
There are currently over 7000 known monogenic disorders that are ideal targets for
CRISPR-mediated gene therapy [38]. CRISPR therapies for monogenic diseases
can take several different forms based on the type of mutation(s) which cause the
disease. For example, if a disease is caused by a gain-of-function mutation in a particular gene, CRISPR knockout gene therapy can be used to delete the gene or the
specic regions which bear the pathogenic mutations. Alternatively, if the disorder
is caused by loss-of-function mutations in a gene that cause it to produce an abnormal protein or no protein at all, CRISPR knock-in can be used to correct the mutations, which is also referred to as gene correction therapy. Disorders in which a
particular gene is overexpressed or underexpressed can also be therapeutically targeted using CRISPRi and CRISPRa, respectively. Many monogenic disorders are
caused by single nucleotide polymorphisms (SNPs), making them possible to correct using base- and prime-editing techniques which do not induce DSBs. Depending
on the target organ or tissue, these methods can be performed invivo as gene therapies, or exvivo as gene-edited cell therapies. In this section, we cover these different
therapeutic options with examples from proof-of-concept and preclinical studies.
10.7.1 CRISPR Gene Correction forMonogenic Diseases
SCD is perhaps one of the best-known monogenic disorders and was the rst genetic
disease for which researchers attempted to generate a CRISPR cure. SCD is caused
by a SNP mutation in the ß-globin gene (HBB), which normally produces adult
hemoglobin (hemoglobin A) and oxygenates the blood [62]. The traditional treatments are blood transfusions or a bone marrow transplant from a healthy donor.
CRISPR has offered several promising avenues for treating this debilitating condition via autologous gene-edited HPSC therapy.
The rst and currently most popular method is sometimes referred to as gene
switching or gene substitution. Fetal hemoglobin (hemoglobin F) is produced until
~6months of age in humans, at which time it is switched off by activation of the
BCL11A transcription factor and replaced by hemoglobin A.Hemoglobin F is an
adequate substitute for hemoglobin A [63]. Therefore, by performing CRISPRCas9 knockout of BCL11A, hemoglobin F production can be restored. This approach
has been championed by several biotechnology companies, including CRISPR
Therapeutics and Vertex Pharmaceuticals, who jointly ran the rst clinical trial
276
R. Kishton et al.
based on this approach using their CTX-001 therapy [64]. Alternative approaches
are being developed by Editas Medicine [65], using the Cas12a nuclease, and Beam
Therapeutics, using base editing [66].
Therapies for SCD based on upregulating hemoglobin F can also be used to treat
a related condition called beta-thalassemia. While they have similar symptoms and
limited treatment options, beta-thalassemia is caused by multiple mutations in HBB
rather than just one SNP. This makes it more difcult to cure with gene therapy
because repairing many mutations is more challenging than correcting a single one.
The ability to treat both diseases with a single therapy is why many companies have
focused on the therapeutic upregulation of hemoglobin F.The CTX-001 therapy
developed by CRISPR Therapeutics and Vertex Pharmaceuticals has also been
tested on beta-thalassemia patients with similar levels of success [67].
Many genetic disorders require the correction of one or more pathogenic mutations in a particular gene. X-linked agammaglobulinemia (XLA) is an immunodeciency disorder caused by a variety of mutations in the Bruton’s tyrosine kinase
(BTK) gene [68]. Researchers at the University of California, Los Angeles created a
CRISPR gene-edited cell therapy approach for XLA, correcting the majority of
pathogenic mutations in the BTK gene in peripheral blood stem cells (PBSCs) [68].
The corrected PBSCs could therefore be used as an autologous cell therapy for
XLA, with preclinical research being continued for this strategy.
10.7.2 CRISPR Knockout ofRepeat Hyperexpansions
Some genetic diseases are caused by multiple repeats of certain nucleotides that
form hyperexpansions and interfere with the normal production of protein from the
gene. Friedreich’s ataxia (FA) is an autosomal recessive disorder caused by mutations in the FXN gene which encodes frataxin, a mitochondrial protein responsible
for aspects of energy production and iron metabolism [67]. The FXN gene bears a
GAA trinucleotide repeat in its rst intron. Healthy individuals have ve to 30
repeats. FA patients have hyperexpansion of between 44 to 1700 repeats of this trinucleotide sequence [69]. The number of GAA repeats is correlated with the severity of FA, as well as the age of onset.
CRISPR offers the potential to cure FA by excising the pathogenic trinucleotide
hyperexpansion; researchers from the University of California, San Diego School of
Medicine developed a CRISPR method to delete large sections of the expansion in
patient-derived hematopoietic stem and progenitor cells (HSPCs). The treatment
successfully increased the production of frataxin protein in the HSPCs, which then
differentiated normally with no noticeable off-target editing or cellular toxicity
[69]. Additional therapies are being developed by CRISPR Therapeutics and
Capsida Biotherapeutics for FA [70]. As part of the collaboration, these companies
are also creating a similar treatment for amyotrophic lateral sclerosis (ALS), a large
number of cases of which are caused by a GGGGCC hexanucleotide repeat within
the C9orf72 gene [71].
10 CRISPR Technology andIts Application inTherapeutics
277
10.7.3 CRISPR InVivo Gene Therapies
Leber’s congenital amaurosis (LCA) is a retinal disease caused by loss-of-function
mutations in multiple genes affecting the rod and cone cells of the retina [72]. The
most common causes are mutations in the RPE65 and CEP290 genes, making these
genes prime targets for gene therapy. An early attempt to generate a cure for LCA
was developed by Spark Therapeutics in a therapy known as Luxterna, which simply provided a functional copy of RPE65 cDNA in an AAV vector via sub-retinal
injection [73]. However, this therapy is not a one-time cure, and the patient group
suitable for treatment with Luxterna is limited.
Editas Medicine more recently developed a CRISPR-based gene therapy known
as EDIT-101, an RNP cargo packaged in an AAV5 delivery vehicle and administered via sub-retinal injection [74]. The BRILLIANCE clinical trial of EDIT-101
was the world’s rst-ever trial of an invivo CRISPR gene therapy, using CRISPRCas9 to edit the CEP290 gene, restoring healthy expression of the CEP290 protein
and photoreceptor function. Interestingly, rather than correct the IVS26 mutation in
CEP290, EDIT-101 uses two guide RNAs to create a DSB on either side of the
mutation and delete it [75]. After repair, transcription and RNA splicing are restored,
and the protein can be translated normally. Preliminary results of the ongoing Phase
I/II trial of EDIT-101 indicate no serious toxicity or adverse events of the therapy
and evidence for clinical benet [76]. Base editing is also being explored as an
alternative therapy for LCA by researchers at the University of California, Irvine
(UCI), with a proof-of-concept study demonstrating the correction of the RPE65
mutations invivo in a mouse model of the disease [77].
10.8 CRISPR inInfectious Disease Treatment
While cancer and genetic disease may appear the most obvious targets for CRISPR
therapies, there is also signicant potential for this technology in the treatment of
infectious diseases. Pathogenic microorganisms, including viruses, bacteria, fungi,
and parasites, can all be targeted using CRISPR, and there has been signicant
progress in this eld in recent years.
10.8.1 Circumventing Antibiotic Resistance inBacteria
CRISPR’s role in protecting bacteria from invading bacteriophages is now being
used to turn the tables, targeting bacteria themselves. This can involve killing the
target pathogenic bacteria or deleting or repressing the transcription of key genes in
the bacterium, such as antibiotic resistance genes. A key strategy for the targeted killing of pathogenic bacteria is to engineer bacteriophages using CRISPR systems [78].
The key benet of these therapies is that they eliminate the need for antibiotic
278
treatments and can be used to target multi-drug resistant “superbugs” [78]. This
approach has been pioneered by Locus Biosciences in their clinical trial of LBP-EC01,
which uses CRISPR-Cas3 enhanced bacteriophages (termed crPhage™) for the therapeutic removal of Escherichia coli strains that cause urinary tract infections [79].
R. Kishton et al.
10.8.2 CRISPR-Edited Cell Therapies fortheTreatment
ofViral Pathogens
CRISPR technology offers the possibility of targeting a variety of viral pathogens
[80], including DNA viruses, such as human papillomavirus (HPV) and EpsteinBarr virus, as well as RNA viruses, such as SARS-CoV-2. The E6 and E7 genes of
HPV have been the subject of many recent CRISPR studies due to their roles in
causing cervical cancer, making them key targets for CRISPR-based knockout [81].
In preclinical research, CRISPR-Cas9 was used to knock out the E7 gene in a mouse
model bearing HPV16 tumors; treatment was effective at clearing the tumors from
the animals without causing liver or spleen toxicity [82].
Another promising option for the treatment of infectious diseases is CRISPRedited B cell therapy. B cells produce antibodies and are an essential component of
the adaptive immune system [83]. Vaccines are designed to prime the adaptive
immune system by encouraging B cells to recognize new pathogens and produce
antibodies for them [84]. CRISPR-edited B cells have been suggested as an alternative to vaccines because they can be used to produce large quantities of neutralizing
antibodies for pathogens via targeted gene knock-in [83, 84]. This strategy has been
adopted to treat the respiratory syncytial virus, HIV, Epstein-Barr virus, and inuenza viruses [85].
10.8.3 In Vivo CRISPR Therapy toExcise Integrated Viruses
fromtheHuman Genome
HIV is a retrovirus that infects human CD4 helper T cells, in which it reversetranscribes its RNA genome and then inserts itself into the DNA genome of the host
cells [86]. Due to its integration into the human genome and its ability to hide in
dormant, noncirculating helper T cells for long periods of time, even antiretroviral
drugs can never completely remove HIV from the body [87]. CRISPR now offers a
potential cure for HIV, cutting it out of the human genome with minimal risk of offtarget editing using a multi-guide approach successful for excising large DNA
sequences [20]. Excision Bio is currently testing their invivo EBT-101 therapy for
HIV in a Phase I/II trial [88] and is also developing similar therapies for other
viruses. This includes EBT-103 therapy for JC virus, which can cause progressive
10 CRISPR Technology andIts Application inTherapeutics
multifocal leukoencephalopathy, EBT-104 for herpes simplex virus, and EBT-107
for hepatitis B virus [89].
279
10.9 The Future ofCRISPR Therapeutics
Given the current trends around FDA approvals, the prediction is that in the near
future, approximately one-third of medical treatments will be cell and gene therapies. Given the advantages of CRISPR over previous gene editing technologies, it is
likely that many of these cell and gene therapies will be CRISPR-based. In the next
section, the critical factors that will determine the future of CRISPR technology in
medical therapeutics will be explained.
10.9.1 Industrialization ofCRISPR Editing: Increasing Safety
andLowering Costs
Many of the CRISPR cell and gene therapies currently in trials show promise.
However, the size of patient groups in clinical studies is generally limited. If a trial
is successful and the therapy in question gains FDA approval, there are often signicant obstacles in scaling up these therapeutics for widespread use in larger patient
populations [90]. This includes accessing viral vectors for delivery, the necessary
Good Manufacturing Practice (GMP)-grade reagents, and adequate production
facilities. Signicant costs are associated with cell and gene therapies, making these
medicines difcult for many patients to access. Due to the long wait times and high
costs, cell and gene therapy developers often decide whether to build out GMP
facilities for viral vector production. Commercial entities like Synthego, Editas and
Agilent manufacture GMP sgRNA. Moreover, the inherent variability of geneedited cell therapies and issues associated with invivo gene editing means that great
care must be taken in order to ensure these medicines are safe and appropriately
regulated [91].
10.9.2 Safety Considerations
An emerging concern is the safety of CRISPR therapies. Signicant efforts have
been made by the scientic community to increase the safety of CRISPR therapies,
including engineered Cas9 and other nuclease variants with increased accuracy. In
late March, the FDA recently released updated guidelines on the topic of Human
Gene Therapy Products Incorporating Human Genome Editing [92]. These guide-
lines include the identication of off-target editing activity, including the type,