analysis Abstract This review paper will discuss various CRISPR-Cas9 mediated experiments and methods in hopes of future development within the sci

Abstract

This review paper will discuss various CRISPR-Cas9 mediated experiments and methods

in hopes of future development within the scientific community. This review paper will target

the potential of altering the genomes of human cells and crops. We will discuss possible cancer

and neurological disorder treatments that utilize the CRISPR-Cas9 system. Lastly, we will

discuss the potential of altering the genome of crops and its potential effects. Researchers in

science have the potential to utilize CRISPR/Cas9 to modify the genomes of large ranges of

organisms in ease, ranging from cells to crops, in hopes of solving many scientific mysteries of

cancers, neurodegenerative disorders, and overpopulation. With the threat of global

overpopulation and a global climate that has increasingly become unstable, researchers are

looking for ways to improve the plant genome to meet consumer demand. The development of

the CRISPR/Cas9-based genome editing tool has revolutionized the field of agriculture.

Introduction

CRISPR-Cas9 System

The prokaryotic CRISPR-Cas9 system stands for “clustered regularly interspaced short

palindromic repeats, CRISPR-associated protein 9” (Zhan, 2019). CRISPR is found initially in

Escherichia coli and Cas9 is derived from Streptococcus pyogenes. It is a system that is continually

revolutionizing genetic engineering. The CRISPR mechanism uses two RNAs: CRISPR targeting

(crRNA) and trans-activating RNA (tracrRNA). These distinct RNAs activate and guide the Cas

proteins to bind viral DNA sequences to the correct desired sequence to be then cleaved (Zhan,

2019). Cas9 is a vital type II Cas protein generated from the Streptococcus pyogenes (SpCas9). It

is a critical component for using CRISPR/Cas9 as a system applicable for genome editing; it also

differentiates it from other Cas proteins like Cas5e, Cas6e, Cas7e, Cas8e, Cas11e, Cas12, and

Cas13 (Cong, 2013). The Streptococcus pyogenes type II CRISPR locus consists of four genes,

that included the Cas9 nuclease as well as two noncoding CRISPR RNAs (crRNAs), trans-

activating crRNA (pre-tracrRNA), and the precursor crRNA (pre-crRNA). This method is

thoroughly explained in Figure 1 below. 1

The CRISPR method was established on the natural system bacteria utilize to protect

themselves from viral infections (Khalaf, 2020). When bacteria detect the presence of viral DNA,

they produce two separate short RNA sequences. One will match the invading virus, and both will

bind to Cas9, and the single guided RNA (sgRNA) recognizes a specific nucleotide sequence.

Ultimately, the sgRNA complexed with the Cas9 nuclease cuts the DNA. So, researchers can edit

the genome by modifying a leading strand or inserting a foreign DNA sequence (Khalaf, 2020).

Figure 1. Overview of the CRISPR/Cas9 mechanism of action.

The RNA-guided CRISPR–Cas9 with the DNA endonuclease is localized to a specific

target DNA sequence (20 base pairs) in the genome by the sgRNA (single guided RNA) sequence

(Khalaf, 2020). SgRNA is a synthetic fusion between the CRISPR targeting RNAs (crRNAs) and

the trans-activating crRNA (tracrRNA). Then, the CRISPR/Cas9 joined with sgRNA will base pair

with a specific target sequence adjacent to a (PAM) sequence (Khalaf, 2020). A PAM sequence

(protospacer adjacent motif sequence) is usually NGG or a weaker NAG. PAM has positioned 20

base pairs (bp) downstream from the target sequence. The PAM sequence is deemed critical for

modification, but it should not be a part of the sgRNA sequence. This specific part of the

mechanism is demonstrated in Figure 2 below. 1

The Streptococcus pyogenes type II CRISPR system can facilitate specific loss-of-function

mutations in eukaryotic cells (Chen, 2018). When the Cas nuclease is targeted to specific locations

in the genome, the Cas9 facilitates the cleavage and results in double-stranded breaks (DSBs).

CRISPR/Cas9 mediated genome engineering can then repair DNA double-strand breaks (DSBs)

in two pathways: the non-homologous end joining (NHEJ) pathway or the homology-directed

repair (HDR) pathway (Sánchez-Rivera, 2015). More specifically, when NHEJ repairs the

homologous ends, it will result in either an insertion or deletion mutation that can cause a loss of

function mutation in the DSBs. These indels then can generate major frameshift mutations that

could induce the production of premature stop codons (Sánchez-Rivera, 2015). Repair via the

NHEJ pathway is very error-prone, while the HDR+ donor pathway can perform precise DNA

modifications within the genome (Sánchez-Rivera, 2015). The basic format of the mechanism can

be seen thoroughly in Figure 1 above. 1

Figure 2. This is a figure from the research article: Applications of the CRISPR-Cas9 system in
Cancer Biology that was published by Francisco Sánchez-Rivera and Tyler Jacks in 2015. It
explains the two repairing pathways that DNA double stranded breaks (DSBs) can be repaired.
First being the non-homologous end joining (HEJI) pathway or the homologous-directed repair
(HDR) pathway. Cas9-mediated induction of a DSB in the DNA target sequence leads to indel
mutations via NHEJ or precise gene modification via HDR.1

Cancer is a rising incidence worldwide, carrying the highest disease-caused mortality in

the human population. Innovations within genomic engineering are constantly being accelerated

utilizing CRISPR/Cas9 system technology (Zhan, 2019). Genomic engineering concerning the

CRISPR/Cas9 mechanism has enabled researchers to modify the genomic sequence of cells in

organisms and introduce epigenetic and transcriptional modifications. Experimental approaches

using this technology can potentially change the field of cancer genetics. Utilizing the

CRISPR/Cas9 mechanism to genetically modify the sequence of cells could possibly save millions

of lives (Zhan, 2019).1

Analysis

1. Utilization of CRISPR/Cas9 in Cancer Genomics

CRISPR/Cas9 Delivery in Mammalian Cells

For in vivo and in vitro genome editing, there are approximately three formats of Cas9 and

sgRNA delivery. The first format consists of the plasmid, the second mRNA, and the third

ribonucleoprotein (Xu, 2019). The first method delivers a DNA plasmid that encodes Cas9 proteins

and sgRNA. The second option is delivering mRNA (messenger RNA) and sgRNA. Then the

delivered mRNA can be converted into Cas9 nucleases by translation. The final form for

CRISPR/Cas9 delivery is through a Cas9 protein complexed with sgRNA, which will produce

ribonucleoproteins (RNPs). This third method takes measures that account for safety and low off-

target effects (Xu, 2019). The formats of in vivo and in vitro genome editing are demonstrated in

Figure 4 below. 1

Figure 4. Schematic illustration of different configurations of Cas9/gRNA elements and
intracellular delivery mediated by the non‐ viral vectors. For in vitro and in vivo genome editing,
there are typically three formats of Cas9 and sgRNA delivery, namely plasmid, mRNA, and
ribonucleoprotein (Xu,2019).

Tumor Immunotherapy Background

Immunotherapy is a common and effective form of cancer treatment, following other

standardized treatment methods (Lu, 2020). Clinical applications of CRISPR-Cas9 in the human

genome against cancerous cells in tumors require a knockout of the programmed cell death protein

1 gene (PDCD1) ex vivo. The PD1 (programmed cell death protein-1) has T-cell receptors that

regulate immune tolerance and decrease autoimmune reactions. The expression of PD1 in the

genome activates T cells. So, when PD1 is highly expressed, then that must mean there are T cells

activated specifically on cells that possess the ability to proliferate abnormally, like tumor cells

(Han, 2020). PD1 can play several opposing roles in the human body; the first is to reduce the

regulation of harmful immune responses, ultimately maintaining our immune tolerance. However,

PD-1 can negatively interfere with immune responses by dilating malignant cells (Han, 2020).

When PD-1 is bound to its ligand PD-L, it creates programmed cell death- ligand 1 (PD-L1), which

enables T cells from killing other cells, including cancer cells. In normal conditions, PD-L1

activates unhealthy cells’ apoptosis (programmed cell death), while it acts as a pro-tumorigenic

factor (Dong,2018). So, by knocking out the PDCD1 gene, the PD-1 expression on T cells

decreases, and the antitumor ability of receptor T is significantly improved (Lu,2020).1

The clinical trials discussed in this review genetically modified the genome of the cell ex

vivo and measured the rate of apoptosis in cancer cells post knockout. A knockout is a common

laboratory technique where scientists genetically modify organisms to lack a specific gene or genes

to measure the effect of the gene on an organism. (Lu, 2021). Many researchers believe that

conventional gene knockout technology has very complex systems, can be costly, expensive, and

ineffective because it produces many off-targets (Lu,2020). In contrast, CRISPR-Cas9 mediated

gene editing is more precise and easier to use. 1

Clinical Trial NCT02793856

The first clinical trial (NCT02793856) that utilized CRISPR for cancer therapy was

conducted at West China Hospital Sichuan University in 2016. This interventional phase I clinical

trial consisted of twelve participants aged 31- 68, with PD-L1 positive advanced non-small lung

cancer (NSCLC). Patients enrolled in this trial underwent multiple lines of standardized treatment

and showed no improvements. The study’s primary objective was to determine the safety and

tolerability of autologous PD-1 T cells after editing them with CRISPR-Cas9 in patients with

NSCLC. Their secondary objective was to investigate the antitumor activity of CRISPR-Cas9

edited PD-1 T cells and the rate of apoptosis. It is important to note that patients enrolled in this

trial had a life expectancy that was more significant than three months but less than most cancer

patients. 1

Patients were divided into three treatment groups where each group consisted of 2

treatment cycles, and each cycle was approximately 28 days in length. It is notable to mention that

patients were screened for PD-L1 expression positivity prior to determining the number of PD-1

edited T cells infused each cycle. The effectiveness of the infusions was studied by assessing the

tumors at baseline, the 8th week, the 12th week, and once every eight weeks during the treatment

and follow-up as per the RECIST guidelines. This is a standard method of measuring a cancer

patient’s response to treatment based on the size of the tumor by CT or MRI.1

Method of Delivery

Peripheral blood lymphocytes were selected carefully from patients with solid tumors and

were taken to the laboratory, where they were expanded ex vivo. Then the immune checkpoint

gene PD1 was knocked out with the aid of the CRISPR-Cas9 mechanism. In order to knock out

the PD-1 gene in the human T cells, researchers designed a pair of sgRNAs: sgRNA1 and sgRNA2

(Lu, 2020). The S. pyogenes Cas9 (SpCas9) was utilized to construct a sgRNA vector (which

consisted of the pair of sgRNAs’). Which specifically targeted exon 2 on the PD-1 gene, and this

sgRNA design can be seen in Figure 6 below. Then the Cas9 endonuclease and sgRNA plasmids

were cotransfected into T cells using the electroporation technique (Lu, 2020). This physical

transfection method utilizes electrical impulses to create pores in cell membranes to increase

permeability, allowing substances to pass into cells. After electroporation, flow cytometry was

used to ensure decreased expression of the PD-1 gene. To further confirm their findings, two

rounds of PCR amplification were performed on the cells to confirm the results. Once the

lymphocytes possessed decreased expression of the PD-1 gene, they were infused back into the

patient. Where they induced an immunological response against the tumor cells.1

Figure 5. A CRISPR/cas9 ex vivo knockout is shown: Peripheral blood lymphocytes are collected
from the patient with tumor (A) and CRISPR/ Cas9 mediated knockout of the immune checkpoint
gene PD1 is performed in T- cells (B–C). The PD1-knockout T-cells are expanded ex vivo and
then transfused back to the patient (D), where they are supposed to induce immunological response
against tumor cells (Zhan, 2018).

Figure 6. CRISPR–Cas9-mediated PD-1 gene editing in T cells. a, The pair of sgRNAs (sgRNA1
and sgRNA2) that was designed to target exon 2 of PD-1. The PAM sequences are marked with
an underscore. The Cas9 and sgRNA plasmids were cotransfected into T cells by electroporation.
b, The levels of PD-1 expression on edited T cells or unedited T cells in the first cycle were
measured by flow cytometry. c, The frequency of PD-1 editing efficiency on all infused T cells
was measured by NGS. Box-and-whisker plot shows the frequency of PD-1 editing efficiency; the
box represents the values from lower quartile to upper quartile, the center line in the box represents
the median data and the whiskers (vertical) lines outside the box represent the minimum and
maximum values. Each dot represents the editing efficiency of infused cells in an individual cycle.
n represents the number of tested samples (patient ID); n = 1 (pre-A-02, A-02, A-03, B-03 and C-
01), n = 2 (A-01, A-04, B-02 and C-03), n = 3 (pre-A-01), n = 4 (C-02), n = 9 (B-01) (Lu, 2020).

Safety and Clinical Results

The data provided from the clinical trial suggested that CRISPR-Cas9 gene-edited T cell

therapy targeting the PD-1 gene was well tolerated, safe, and feasible for patients with NSCLC

(Lu, 2020). Weeks following the infusion, most of the patient’s side effects and symptoms

decreased slightly. Most participants had no severe adverse effects while enrolled in the trial, but

11/12 patients had grade 1/ 2 treatment-related adverse events two years following the event. An

extreme adverse event is defined as one to cause death, threaten life (risk of sudden death) or cause

a permanent or significant hospital stay. 1 Figure 7 below shows the duration of treatment-related

adverse events (Lu, 2020). 1

Figure 7. Duration of treatment-related adverse events. Different colors are used to represent each
patient. Bar length represents duration of the adverse effect. All related AEs were grade 1 or 2.
Grade 2 AEs are outlined in black (Lu, 2020).

After eight weeks following the infusion, the results between patients varied. The lung

cancer size shrunk in around 20% of the participants, while the rest had no noticeable results.

These results were recorded as per the response evaluation criteria in solid tumors (RECIST) as

shown in Figure 8 below. The researchers that conducted this study emphasized that each

individual would respond to the treatment differently, apart from the unknown factors that could

arise. From the results presented in Figure 8 only three patients were found to have new lesions. 1

Figure 8. Bar graph representation of best percentage change from baseline tumor size, as
determined based on Response Evaluation Criteria in Solid Tumors (RECIST), v.1.1. The dashed
line represents the cut-off for PD (at 20% change in tumor size from baseline). An asterisk denotes
PD with new lesions. c, Swimmer plot represents the treatment duration for 11 patients. Arrow
represents that the patient was alive at the time of date cut-off (31 January 2020). Color bar
represents the PFS time; blank bar represents the survival time after PD. Hollow and solid dots
represent that patient C-02 experienced only intracranial progression and then intrapulmonary
lesion PD, respectively (Lu, 2020).

Figure 9. Kaplan-Meier estimates of survival in 12 patients. a, Overall survival. b, Progression-
free survival (Lu, 2020).

Researchers in this clinical trial are unsure if there is a direct causation or correlation of

CRISPR-Cas9 mediated PD1-knockout with survival rate post-infusions. As of January 31, 2020,

all patients showed disease progression, and 11 out of 12 died from tumor progression. It is

important to note that patients had a life expectancy of more than three months before this trial.

The overall survival in the measure of weeks is shown in Figure 9. The overall survival ranged

from 10.3 weeks to 74.9 weeks post-infusion. The remaining patient (C-02) was still receiving

therapy at the time (Han,2020). However, the progression-free survival had a median of 7.7 weeks

after the last infusion. These findings and results left researchers uncertain if there was a decrease

in the antitumor activity of CRISPR-Cas9 edited PD-1 T cells or the apoptosis rate in T cells.

Researchers’ primary concern during this clinical trial was the off-target uncertainties that could

be generated during CRISPR-Cas9 system editing. They deemed it a significant concern for future

technology clinical applications (Lu, 2020). This clinical trial utilized off-target analysis by next-

generation sequencing (NGS). As seen in Figure 10, the numbers of off-target mutations ranged

from 0 mutations to 9-11 in patient C-02. 44% of the off-target mutations were found to be

intergenic mutations. The composition of on-target mutations resulted mostly in frameshift

deletions which were necessary to carry out the intended function of the trial.1

Figure 10. Off-target analysis by NGS. a, Characteristics of off-target mutation types,
frequencies and numbers determined by NGS for the edited T cells of 12 enrolled patients before
the first cycle of infusion. Bar graph and pie graph above represent the types, the numbers and
the composition of off-target mutations, color-coded according to the legend to the right.
Intergenic (48.1%) and intronic (35.1%) mutations composed the majority proportion. Heatmap
shows the mutation number of predicted off-target sites (18 off-target sites, OT1–18) and on-
target sites for individuals. Bar graph on the right represents mean mutation frequencies of each
site among all of the patients (bars represent mean ± s.d., each dot represents individual data, n =
12). The modification ratio of on-target/off-target was 48.7. Pie graph on bottom right shows the
composition of on-target mutation. The mutation types of on-target consisted of frameshift or
non-frameshift (deletion/insertion mutation) and stop-gain mutations, while the vast majority
were deletion mutations (90.6%) (Lu,2020)

Other clinical trials

Other studies in China have also started, some finished and some withdrawn due to the lack

of funding. These studies had similar methods and were intended to be carried out very similarly.

Trial NCT03081715 was completed on advanced esophageal cancer; however, results are not yet

posted. Clinical Trials NCT02863913, NCT02867345, and NCT02867332 were all led by

Professor Wukuang Liu at Peking University but withdrawn due to lack of funding or financial

support. A summary of these studies can be seen in Table 1 below. 1

Table 1. Registered clinical trials that utilize PD-1 knockout of T cells for the treatment
of cancer and their current status. Information obtained from clinicaltrials.gov on 11-05-
2021.1

Discussion

Future Perspectives and limitations of CRISPR/Cas9 Systems in Cancer Research

In order to be able to incorporate the CRISPR/Cas 9 mechanism effectively and safely in

the cell, they need to deliver both the Cas9 enzyme and the sgRNA to the target cell (Cong,

2013). This method must provide high gene editing efficiency while causing low

immunogenicity and being able not to kill off healthy cells or negatively affect them. Using

multiple single-guide RNAs (sgRNAs) allows CRISPR/Cas9 to edit the genome in parallel. Most

cancers result from mutations that occur in multi-steps, so it is believed that the multiplexing

nature of the CRISPR/Cas9 mechanism can facilitate a good model for cancer research (Wen,

2016). For example, the NHEJ pathway is error-prone and can frequently lead to random

insertions or deletion mutations, leading to disruptive frameshift mutations that could induce or

generate premature stop codons (Sánchez-Rivera, 2015).1

CRISPR/Cas9 must undergo major trials not to create the possibility to undesirably

induce carcinogenesis and toxicity to the genome (Wen, 2016). Methods that are efficient to

deliver CRISPR/Cas9 through vectors might generate unwanted immune responses. Gene editing

is not entirely error-proof, and when gRNAs are not specific, it can lead to off-targets,

predominantly when Cas9 is expressed in high levels (Biagioni,2018). They are still evaluating

in vivo and ex vivo trials for efficacy and efficiency because the CRISPR/Cas9 delivery system

and mechanism are extraordinarily intricate and can cause more harm than good. CRISPR-Cas9

has extremely high gene efficiency, which causes an off-target which ultimately affects its

clinical application (Biagioni,2018). 1

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