CRISPR/Cas9-gRNA system-mediated gene editing/gene knockout study and gene editing therapy

Gene editing, also called genome editing, is a group of technologies to change the sequence of DNA in the genome. Several approaches to genome editing have been developed, including Zinc Finger, TALEN, and CRISPR/Cas9. Compared with the other gene editing tools, CRISPR/Cas9 system is faster, cheaper, more accurate, and more efficient, showing unprecedented advantages in gene therapy.

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Content of CRISPR/Cas9-gRNA system

Introduction and principles of CRISPR gene editing system

CRISPR (clustered regularly interspaced short palindromic repeats) is a family of DNA sequences derived from viruses that have previously infected the prokaryotes and has the ability to recognize invasive homologous DNA sequences from similar viruses, then directing Cas9 (CRISPR-associated 9) nuclease to specifically cleave them during subsequent infections, thus playing a key role in the antiviral defense system of prokaryotes [4]. Cas9 nuclease is an enzyme guided by CRISPR sequences to recognize and cleave specific nucleic acids which are complementary to the CRISPR sequence [5]. CRISPR sequences together with Cas9 enzymes make up the basis of a CRISPR/Cas9 technology which is a versatile genome-editing platform within organisms.

By 2010, three CRISPR/Cas9 systems have been identified in bacteria: type I, II and III. Derived from Streptococcus pyogenes, type II CRISPR system is relatively simple and has been eventually adapted for genome editing in mammalian cells [1]. This simpler CRISPR/Cas9 system contains four components, i.e. Cas9 endonuclease, trans-activating CRISPR RNA (tracrRNA) and two small RNA molecules named CRISPR RNA (crRNA) [2]. In this system, the mature crRNA base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure, guiding Cas9 nuclease to mediate the recognition and cleavage of target nucleic acids [3-5]. At sites complementary to the crRNA-guide sequence, the HNH nuclease domain of Cas9 cleaves the complementary strand, whereas the RuvC-like domain of Cas9 cleaves the non-complementary strand. To target nucleic acids with more precision and expand the application of CRISPR/Cas9 system, great efforts have been paid to engineer the Cas9 enzyme or alter PAM specificities. To date, a wide range of Cas9 derivatives with novel PAM specificities have been generated using the site-depletion assay [6], and the initial two-RNA structure, ie. CRISPR RNAs (crRNAs) base-paired to trans-activating crRNA (tracrRNA), have been programmed into a “single-guide RNA (sgRNA)” (dual-tracrRNA:crRNA) by fusing the 3′ end of crRNA to the 5′ end of tracrRNA [7]. By manipulating the nucleotide sequence of the sgRNA, the artificial CRISPR/Cas9 system could be programmed to target any DNA sequence for cleavage [7].

Figure 1. Cas9 can be programmed using a single engineered RNA molecule covering tracrRNA and crRNA features. Left, Cas9 is directed by a dual RNA structure formed by activating tracrRNA and targeting crRNA to cleave site-specifically–targeted dsDNA in type II CRISPR/Cas systems. Right, a chimeric RNA generated through fusion of crRNA to tracrRNA [7].

1) CRISPR/Cas9 gene editing system

a. Cas9: I) NHEJ mediated gene knockout; II) HDR mediated gene knockin

Guided by small guide RNA (sgRNA), Cas9 nuclease cleaves double-strand DNA, the inducing site-specific genomic double-strand breaks (DSBs). In mammalian cells, there are 2 manners with which DSBs are repaired: non-homologous end joining (NHEJ) or homology directed repair (HDR). (I) Frequently, NHEJ happens, enabling the introduction of small deletions or insertions between the ends of DSBs and resulting in frameshift mutations, and thus mediates gene knockout [8,9]. (II) With the existence of repair template, such as exogenous donor vectors, HDR takes place, introducing precise gene modifications, such as reporter insertions, and thus mediate gene knockin [10,11]. Both NHEJ mediated gene knockout HDR mediated gene knockin have been widely used in the field of gene therapy [12]. However, HDR (0.5%-20%) is much less frequent than NHEJ (20%-60%) [13], and the former only occurs during S and G2 phase while the latter can occur at any time of cell cycle [14-16]. To improve the efficiency of HDR, people have screened so many small molecules, finding that small molecules inhibitor Scr7, targeting DNA ligase IV, one key enzyme in the NHEJ pathway, significantly facilitates the HDR pathway (5%-58.3%) [17]. So far, NHEJ is widely used in the mediation of gene knockout [8,18] and correction of pathogenic gene mutation [12,19,20], while HDR is applied for knockin schemes, such as knockin cell line generation [21] or reporter/tag knockin [22].

Figure 2. A schematic model of CRISPR system-induced non-homologous end joining (NHEJ) or homology directed repair (HDR) [23,24].

b. Cas9 nickase: off-target and D10A

Although CRISPR/Cas9 system is much cheaper, more efficient and flexible, high efficiency of off-target mutations or unwanted DNA cleavages have limited the application of this system. Mutation exploration revealed that Cas9 nickase carrying the mutation of D10A (D10A Cas9) loses the nuclease activity in RuvC domain, but does not affect the nuclease activity of HNH domain. In combination with paired gRNAs, D10A Cas9 cuts target DNA and generates two single-strand breaks with higher specificity and little off-target mutations, which is confirmed by exome sequencing analysis [25,26]. By comparing gene-disrupting efficiency of Cas9 paired nickases with that of nuclease, the T7E1 assay and deep sequencing revealed comparable on-target efficiency, even sometimes higher on-target efficiency [27]. Moreover, FACS enrichment of paired D10A Cas9 nickases will significantly facilitate efficient gene editing with minimized off-target effects [28]. Further modifications showed that hRad51–Cas9 nickase, generated by hRad51 mutants fusion with Cas9 (D10A) nickase (RDN), mediates HDR without double-stranded breaks, which results in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease [29].

Figure 3. A schematic model of paired Cas9 nickase-mediated chromosomal deletions. Newly synthesized DNA strands are shown in red [25].

c. dCas9 application: I) dCas9 activation system: MPH, VP64; II) dCas9 repression system

The nuclease-deactivated Cas9 variant, dCas9, was generated by introducing two mutations (D10A and H840A) (Figure. 2A), resulting in no catalytic activity in both RuvC domain and HNH domain [30]. With great DNA binding activity, dCas9 has been widely applied to regulate gene expression in combination with transcription activator or repressor [14,31] (Table 3). (I) dCas9 activation system: transcription activator domains (VP64, p65, MPH) or epigenetic activation modifications (p300, CBP etc.), have been fused to dCas9 to activate gene expression [32-35]; (II) dCas9 repression system: transcription repressor domains (KRAB) or epigenetic repression modifications (LSD1, DNMT3A etc.), have been fused to down-regulate the expression of target genes [34,36-38]. Besides the direct effector fusion manner, functional scaffolds (such as SunTag) can be incorporated into dCas9/sgRNA complex to recruit effectors to affect transcription [36,39]. Spatiotemporal control in dCas9/sgRNA-induced effector recruitment was also achieved upon chemical or light induction [40,41].

Figure 4. A modular RNA-guided genome regulation platform: CRISPR/dCas9 system. Targeted by sgRNA, dCas9 can mediate transcription activation or repression via genetically fusion with specific effectors [42].
Table 1. Effectors in dCas9 systems
Effector Transcription regulation
Activator VP64 transcription activation
Activator VP48 transcription activation
Activator VP120 transcription activation
Activator p65 transcription activation
Activator MPH (MS2-P65-HSF1) transcription activation
Activator P65-HSF1 transcription activation
Activator VP64-p65-Rta transcription activation
Repressor KRAB transcription repression
Repressor SID4x transcription repression
Repressor Mxi1 transcription repression
Histone acetylation p300 transcription activation
Histone acetylation CBP transcription activation
Histone demethylation LSD1 transcription repression
DNA methylation DNMT3A transcription repression
DNA demethylation TET1 transcription activation

2) Other CRISPR system: Cas13, Cpf1, etc.

The traditional CRISPR/Cas9 system requires specific PAM sequence, which might be an obstacle to perform gene editing in some cases. Moreover, the off-target effect and cytotoxicity also limit the application of CRISPR/Cas9 system. To overcome these difficulties, researchers successfully screened several other tools to perfect the gene editing system, such as CRISPR/Cas13 and CRISPR/Cpf1. etc.

a. Cas13

Cas13 family, containing Cas13a (also known as C2c2), Cas13b and Cas13c [43], belongs to class 2 type VI and have two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) endoRNase domains to mediate precise RNA cleavage in targets with protospacer flanking sites [44,45]. Cas13 enzymes are programmable and have been applied for nucleic acid detection as well as RNA knockdown and transcript tracking [46,47]. Combined with base editor, such as ADAR2 (adenosine deaminase acting on RNA type 2), catalytically inactive Cas13 (dCas13) have been used as guidance with no strict sequence constraints to direct RNA base editing, which is a promising platform with great applicability for both basic research and clinical therapy [48].

Figure 5. C2c2 (Cas13a) is a crRNA-guided RNase to cleavage of exogenous RNAs. Guided by crRNA, C2c2 mediates RNA CLEAVAGE without the need of sequence complementarity to crRNA guide sequence [44].

b. Cpf1

Distinct with Cas9 enzyme, Cpf1 is from class 2 type V CRISPR system (Prevotella and Francisella 1) and mediates DNA cleavage with three features. 1) The guiding sequence is processed into mature crRNA with no requirement of the tracrRNA. 2) Guided by mature crRNA, Cpf1 efficiently cut the DNA with a short T-rich protospacer-adjacent motif (PAM), not the G-rich PAM in Cas9 system. 3) Cpf1-crRNA system generates a staggered DNA double-stranded break with a 4 or 5-nt 5’ overhang [49]. These features greatly expand the application of CRISPR system [50].

Figure 6. Cpf1 is a RNA-guided DNA nuclease cleaving target DNA with a T-rich protospacer-adjacent motif via a staggered DNA double-stranded break.

CRISPR/Cas9 delivery system

CRISPR/Cas9 system can be delivered in vitro by the non-viral reagents (such as polymer, liposome, nano-particle, electroporation) or viral vectors (such as lentivirus, adenovirus and AAV) and in vivo by adenovirus or AAV vectors.

1)In vitro non-viral vectors CRISPR-Cas9 delivery system

In vitro non-viral vectors CRISPR/Cas9 delivery system can be mediated by non-viral reagents, such as polymer [51], liposome [52], nano-particle [53], electroporation [54]. The characteristics of these methods are summarized in the following table 2. For more detailed information about the transfection reagents, you can refer to the website:

Table 2 – Comparison between polymer, liposome, nano-particle.









membrane fusion/impale cells

 transient increase in the permeability of cell membrane






Time to peak expression





Sutainable time

transient expression

transient expression

transient expression

transient expression

Cell Type

a number of cell types

 adherent and suspension cell lines

Almost all the cells

not suitable for sensitive cell types

Particle diametre

75 to 520 nm




Animal experiment

low effiency

low effiency

target delivery

target delivery






Immune Response






low effiency (<10%)

depend on cell type

high efficiency

high efficiency





most expensive (electroporation device)

2) In intro viral vectors CRISPR-Cas9 delivery system

a. lentiviral

With the ability to mediate efficient transfection and long-term expression of exogenous genes in both dividing and non-dividing cells, lentiviral is a good vector to deliver CRISPR system [55,56]. More useful information of lentivirus can be found on this website:

b. adenoviral

Based on human adenovirus type 5 (Ad5), recombinant adenovirus (Ad) a replication-defective adenoviral vector system, is widely used for CRISPR-Cas9 system in most cell types [57-59]. Genemedi got a rich experience in adenovirus packaging, you could find more information on

3) In vivo CRISPR-Cas9 delivery system-AAV-SaCas9

With mild immunogenicity, adeno-associated viruses (AAV) have superior biosafety rating and broad range of infectivity and mediate long-term and stable expression of CRISPR system in vivo. Considering the loading capacity of AAV (4.7kb), SaCas9 variant has been identified, referred to as KKH SaCas9, displaying robust genome editing activities at target sites with NNNRRT PAMs and showing comparable off-target effects between wild-type and KKH SaCas9 [60]. Combining CRISPR/SaCas9 system and AAV vector, AAV-SaCas9 has been widely applied for gene editing in vivo [61-63]. Genemedi is good at AAV production, please find more information on this website:

Table 3 – Comparison between Retrovirus, Lentivirus, Adenovirus and AAV vectors.







ss RNA

ss RNA

ds DNA

ss DNA






Packaging Capacity





Time to peak expression




cell: 7 days; animals: 2 weeks

Sutainable time

about 3 weeks

stable expression

transient expression

> 6 months

Cell Type

Most Dividing

Most Dividing/Non-Dividing Cells

Most Dividing/Non-Dividing Cells

Most Dividing/Non-Dividing Cells


10^7 TU/ml

10^8 TU/ml

10^11 PFU/ml

10^12  v.g./ml

Animal experiment


low efficiency

lowest efficiency

most suitable

Immune Response





Protocol of CRISPR /Cas9 mediated gene knockout in vitro and in vivo

Genemedi has launched a comprehensive AAV packaging service combined with CRISPR/Cas9, the versatile genome-editing platform. The followings are some protocols of CRISPR /Cas9 mediated gene knockout in vitro and in vivo.

For AAV CRISPR/Cas9 service, please visit:

For lentivirus CRISPR/Cas9 service, please visit:

For Adenovirus CRISPR/Cas9 service, please visit:

  CRISPR/Cas9 AAV Production-User Manual Cas9%20User%20Manual.pdf

Recombinant Adenovirus-CRISPR/Cas9 Knockout System-User Manual

Recombinant Lentivirus-CRISPR/Cas9 Knockout System-User Manual

gRNA design and validation

Cas9-gRNA design principles:

1) The PAM sequence that SpCas9 recognizes is NGG (AGG, TGG, CGG, GGG), while SaCas9 recognizes NNGRRT (NNGAAT, NNGAGT, NNGGAT, NNGGGT) or NNGRRN. For sgRNA, the length is about 21 or 22 nucleotides.

2) For sgRNA, the GC content in 40%~60% is better.

3) If the sgRNA is driven by U6 or T7 promoter, the 5’ end of sgRNA can be designed as G or GG to improve transcription efficiency, which should be considered.

4) The binding site of sgRNA should be as close to the coding region in the downstream of ATG as possible to induce frameshift mutation, the first or second exon is better.

5) SNPs should be checked in the binding site of sgRNA.

6) The distance between paired sgRNA should be taken into consideration before designing paired-gRNAs, if using Cas9 single nickase.

7) Whole genome off-target effect analysis is suggested. At least 5 bases can be allowed for the base mismatch and whether the off target is located in the gene encoding regions need to be confirmed. What’s more, base insertion or deletion in off targets should be detected.

To date, there exist so many CRISPR/Cas9 design tools, and some common tools are list in table 1.

Table 4. Common CRISPR/Cas9 design tools[64,65]




CRISPR design

Massachusetts Institute of Technology


DKFZ German Cancer Research Center

CRISPR design tool

The Broad Institute of Harvard and MIT


Harvard Medical School


University of Virginia

GPP sgRNA Designer

The Broad Institute of Harvard and MIT

1)SpCas9 gRNA design, database and validation

Based on the above principles, SpCas9-gRNA can be designed on the following website [64]: Choose the CRISPR enzyme type “SpyoCas9 (NGG)”, then input transcript IDs, Gene IDs/Symbols, raw DNA sequence or upload a list of transcript IDs, Gene IDs/Symbols, a FASTA file of DNA sequences. Click on the “submit” button, and you can get and download the gRNA sequence from the following website.

Synthesize the SpCas9 gRNA sequence and its anti-sense sequence, anneal the sequences into a double chain and clone into pLv-SpCas9 vector or pAd-SpCas9 vector, which is confirmed by Sanger sequencing.

Deliver the pLv-SpCas9 vector or pAd-SpCas9 vector containing target sgRNA sequences into cells, and verify knockout efficiency.

2)SaCas9 gRNA design, database and validation

Based on the above principles, SaCas9-gRNA can be designed on the following website [64]: Choose the CRISPR enzyme type “SaurCas9 (NNGRR)”, then input transcript IDs, Gene IDs/Symbols, raw DNA sequence or upload a list of transcript IDs, Gene IDs/Symbols, a FASTA file of DNA sequences. Click on the “submit” button, and you can get and download the gRNA sequence from the following website. 

Synthesize the SaCas9 gRNA sequence and its anti-sense sequence, anneal the sequences into a double chain and clone into pAAV-SaCas9 vector, which is confirmed by Sanger sequencing.

Deliver the pAAV-SaCas9 vector containing target sgRNA sequences into cells, and verify knockout efficiency.

3)Summary of approaches for gRNA validation

The cutting efficiency of Cas9 nuclease is mainly affected gRNA target efficiency, which can be validated by T7 endonuclease I (T7EI) assay, Sanger sequencing and Western blot.

a.T7 endonuclease I (T7EI) assay
T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA, which is a simple and cheap method to determine genome targeting efficiency of CRISPR/Cas9 system. First, extract the genomic DNA of cells whose genomes were targeted by Cas9. Second, perform PCR experiments to amplify target DNA. Third, anneal the PCR products and digest them with T7 Endonuclease I. Fourth, analyze the fragments with agarose gel to determine the efficiency of gRNA targeting.

Figure 7. Principle of T7E1 assay for knockout clones

4)How to detect the off-target?

Although CRISPR/Cas9 system is a versatile gene editing technology that is widely applied in the study of basic science and translational research, high frequency of off-target activity is really a restriction factor for its therapeutic and clinical applications. So far, several methods have been used to determine the off-targets with different advantages and disadvantages [65].

Table 5. Common CRISPR/Cas9 design tools [65]





T7E1 assay


Poor sensitivity, not cost-effective


Deep sequencing


Biased, misses potential off-target sites elsewhere in the genome


In silico prediction

Predicts some off-target mutation sites

Fails to predict bona-fide off-target sites



Unbiased detection of Cas9 binding sites genome-wide

Most off-target DNA-binding sites recognized by dCas9 are not cleaved at all by Cas9 in cells



Unbiased, sensitive (0.1%), qualitative translocations, identifies breakpoint hotspots

False negatives present, limited by chromatin accessibility



Identifies translocations

False negatives present, limited by chromatin accessibility



Programmable, sensitive (1%)

Many bona-fide off-target sites cannot be captured



Sensitive (0.1% or lower), unbiased and cost-effective

Not widely used




Less precise


ChIP-seq, Chromatin immunoprecipitation followed by massively parallel DNA sequencing
GUIDE-seq, Genome-wide unbiased identification of DSBs enabled by sequencing
HTGTS, high-throughput, genome-wide, translocation sequencing
IDLV, integrase-defective lentiviral vectors
FISH, fluorescence in situ hybridization

5)How to minimize off-target?

a. Alter the sgRNA sequence. Truncation of the 3′ end of sgRNA, shortening the region complementary to the target site at the 5′ end of the sgRNA by as many as 3 nt (tru-gRNA) or addition of two guanine nucleotides to the 5′ end of the sgRNA improves target specificity [79].

b. Try to optimize the design of Cas9 and sgRNA design with design tools or directed evolution [64,80].

c. Purified Cas9 ribonucleoprotein works more immediately than plasmids containing Cas9 gene sequence and can be degraded rapidly in cells, which may help minimize the off-target frequency [81,82].

d. Strategies that restrict the transcription and translation of Cas9 may help minimize off-target effects [83].

e. Replace the wild-type Cas9 nuclease with D10 mutant Cas9 nickase and paired with two sgRNAs that each cleaves only one strand [28].

f. Improve DNA cleavage specificity using fusions of dCas9 with FokI nuclease domain (fCas9) (Figure 6) [84,85]. 

Figure 8. Architectures of Cas9 and FokI-dCas9 fusion variants. Two monomers of FokI nuclease (red) fused to dCas9-sgRNAs as shown in figure cleavage nucleic acids within the target locus. The catalytically active FokI nuclease dimer can only be assembled by adjacently bound by two FokI-dCas9 monomers to trigger dsDNA cleavage [84].

Gene editing therapy

1)Gene editing therapy vs gene replacement therapy

Gene therapy, to be brief, is delivering corrective gene materials into cells to treat or alleviate the symptom of disease [86], so far, including gene editing therapy mediated by CRISPR/Cas sytem, ZFN or TALEN etc., and gene replacement, such as lentivirus/adenovirus/AAV-mediated gene expression [20]. Gene editing therapy via CRISPR technology is to correct the pathogenic mutation into non-pathogenic ones, such as gene silence or disruption by CRISPR mediated knockout, which has been widely used in the therapy of hemophilia B [19], Huntington’s disease [87], Parkinson’s Disease [88], hematologic diseases, infectious diseases and malignant tumor [89]. Gene replacement therapy by lentivirus/adenovirus/AAV is to replace the pathogenic gene with its corrective type, which is widely applied in treating lipoprotein lipase deficiency (LPLD) [90], spinal muscular atrophy (SMA) [91], retinal dystrophy [92,93], cystic fibrosis [94,95] muscular dystrophies [96]. Combined CRISPR with virus vectors, AAV-SaCas9 or lentivirus-Cas9 has been used for gene therapy of Hemophilia A [61], Usher Syndrome [97], Intervertebral Disc Degeneration [98] etc.

Click here for Pipelines landscapes of gene replacement therapy and gene editing therapy of different companies..

2) Landscape of gene editing therapy: companies and pipelines

As a powerful and versatile platform, gene editing therapy has been led into a new era. Besides basic research, great progress has been made in the clinical studied based on CRISPR. Several companies have been operated to develop transformative genomic medicines for the treatment a range of genetically addressable diseases. Founded by Zhangfeng, Jennifer A. Doudna, George Church, J. Keith Joung and David R. Liu in 2013, Editas Medicine, Inc. develops several gene medicines for disease therapy, such as EDIT-101 for the treatment of Leber Congenital Amaurosis type 10, which has entered into the phase I/II dose escalation study. Founded in Swiss in 2013, CRISPR Therapeutics has developed gene medicine CTX001 for the therapy of anemia, showing promising data in the phase I/II clinical study. Operated by Zhangfeng, David R. Liu and J. Keith Joung in 2018, Beam Therapeutics focuses on treating genetic diseases caused by point mutations with CRISPR-mediated single base editing technique. Click here for Pipelines landscapes of gene replacement therapy and gene editing therapy of different companies..


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