Clustered Regularly Interspaced Short Palindromic Repeats and Clustered Regularly Interspaced Short Palindromic Repeats–Associated Protein 9 System: Factors Affecting Precision Gene Editing Efficiency and Optimization Strategies

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) system is a powerful genomic DNA editing tool. The increased applications of gene editing tools, including the CRISPR-Cas system, have contributed to recent advances in biological fields, such as genetic disease therapy, disease-associated gene screening and detection, and cancer therapy. However, the major limiting factor for the wide application of gene editing tools is gene editing efficiency. This review summarizes the recent advances in factors affecting the gene editing efficiency of the CRISPR-Cas9 system and the CRISPR-Cas9 system optimization strategies. The homology-directed repair efficiency-related signal pathways and the form and delivery method of the CRISPR-Cas9 system are the major factors that influence the repair efficiency of gene editing tools. Based on these influencing factors, several strategies have been developed to improve the repair efficiency of gene editing tools. This review provides novel insights for improving the repair efficiency of the CRISPR-Cas9 gene editing system, which may enable the development and improvement of gene editing tools.

INTRODUCTION

In 2020, Charpentier and Doudna were awarded the Nobel Prize in Chemistry for developing an RNA-guided gene editing system, called clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) protein system. The CRISPR-Cas gene editing tools enable the correction of disease-related gene mutations and have advanced the field of life sciences into a new era.¹ In the prokaryotic adaptive immune system, the CRISPR-Cas9 system protects the host by degrading foreign genetic elements using an RNA-guided endonuclease (Cas effector) and consequently inhibits phage infection and interindividual plasmid transfer.² Single-guide RNA (sgRNA) guides Cas9 protein to DNA target sites and induces double-strand breaks (DSBs) at the first three bases of the protospacer adjacent motif (PAM) on target DNA.³ Subsequently, DSBs are typically repaired through the nonhomologous end joining (NHEJ) pathway, resulting in the formation of indels in the target DNA.

In brief, two NHEJ repair factors (XLF and XRCC4) capture the damaged terminals of broken double-stranded DNA (dsDNA) and combine them in a highly stable, but mobile complex.⁴ The terminal linkages of DSB were then directly linked and repaired by DNA ligases in the NHEJ repair pathway.⁵ Although the homology-directed repair (HDR) is a homology-mediated dsDNA repair without nucleotide deletions or additions at the broken DNA terminals, it can accurately repair DSBs.^6,7 In general, the terminals of DSB will be encapsulated by various HDR factors when DSB occurs in the S or G2 phase of the cell cycle, and the homologous sequences will be presented in the vicinity of the DSB, which will then invade the homologous dsDNA to form an exchange intermediate, such as the displacement loop (D-loop) structure.⁸

Of interest is that the key step in the HDR is the BRCA2-mediated ligation of the RAD51 enzyme to the excised broken DNA.⁹ A difference between NHEJ and HDR-mediated DNA repairs is that the HDR-mediated DNA repairs occur during the meiotic prophase or the S and G2 phases of the cell cycle, whereas the NHEJ produces DSBs in the absence of sister chromatid monomers.¹⁰

The ability of CRISPR-Cas9 to induce and subsequently repair DSBs has been utilized to knock out or silence target genes.¹¹ Thus, the CRISPR-Cas system can also be used to knockout or repair mutated human genes, indicating its potential application in the treatment of genetic diseases. At present, the CRISPR-Cas9 gene editing system has been extensively studied and applied in different fields.

CRISPR-Cas9 GENE EDITING TOOL

Bacteria and archaea can recognize and resist foreign invading viruses and plasmids through the CRISPR system.² In brief, the CRISPR array is transcribed into a long precursor CRISPR RNA (pre-crRNA). The transactivating crRNA (tracrRNA) base pairs with the repeat sequences in pre-crRNA, promoting RNase III-mediated cleavage and Cas9 binding. The processed crRNA:tracrRNA duplex then directs the bound Cas9 to DNA sequences matching the crRNA guide sequence (protospacer) and flanked by a PAM.¹² Subsequently, double-strand DNA begins unwinding and induces RNA-DNA heteroduplex formation. Finally, Cas9 cleaves the dsDNA and induces the formation of a blunt-ended DSB near the specific PAM sequences³ (Fig. 1a).

Figure 1.

Mechanisms of the CRISPR-CRISPR-associated protein 9 (Cas9), ZFN, and TALEN gene editing tools. (a) CRISPR-Cas9 system comprises the Cas9 effector protein and guide RNAs (gRNAs). The CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) are gRNAs that can guide Cas9 protein to the target gene sites. Fremd-DNA (foreign DNA) originates outside the organism interested or studied, it may have beneficial or harmful effects on the organism. Usually, it contains a 20 nt crRNA complementary sequence preceding the PAM sequence. PAM is recognized and cleaved by the RNP complex that comprises gRNA and Cas9 protein. The Cas9 effector-mediated DNA breakage activates the NHEJ repair pathway in cells, resulting in the formatting of target mutations owing to nucleic acid indels. (b) ZFN comprises the zinc finger DNA-binding domain and the DNA cleavage domain. The zinc finger DNA-binding domain contains three independent zinc finger repeats that can recognize three bases in the target DNA. The DNA cleavage structural domain is at the carboxyl terminus of the nonspecific nucleic acid endonuclease FokI and comprises 96 amino acid residues. FokI, a restriction endonuclease from Flavobacterium, exhibits enzymatic activity in a dimeric state. Each FokI monomer is linked to a zinc finger protein to form a ZFN. Two ZFNs recognize one target gene site and dimerize to execute the DNA cleavage step and activate a DNA recombination repair mechanism. (c) TALEN is a restriction enzyme that comprises a DNA-binding domain and a DNA cleavage domain. The DNA-binding domain, which is derived from the TALE protein of the genus Flavobacterium, can identify DNA bases through a 33–35 amino acid repeat domain. The combination of TALE and FokI (a DNA cleavage domain) forms the TALEN system. A pair of TALENs recognizes specific sequences at the upstream and downstream of the plus and minus strands of DNA. The TALEN pair binds to the target sequence and forms a dimer to execute DNA cleavage steps. CRISPR, clustered regularly interspaced short palindromic repeat; NHEJ, nonhomologous end joining; RNP, ribonucleoprotein; TALE, transcription activator-like effector; TALEN, transcription activator-like effector nuclease; ZFN, zing-finger nuclease.

The CRISPR-Cas9 gene editing system, which has both advantages and drawbacks, can be applied to target and edit different targets with specifically designed guide RNAs (gRNAs).¹³ These gRNAs can be easily generated in vitro via genetic transcription and modification. Cas9 can break the methylated DNA and introduce DSBs at multiple loci in human cells.³ Compared with other gene editing systems, such as the classical zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) gene editing tools (Fig. 1b, c), the CRISPR-Cas9 system does not require complex protein engineering. In addition to multisite gene editing, the CRISPR-Cas9 system can activate or repress the expression of genes.¹⁴ Only specific gRNAs for different target genes must be designed for the application of the CRISPR-Cas9 system.¹⁵ However, the CRISPR-Cas9 gene editing system can exert off-target effects in different cells of the same species.¹⁶

Recently, some researchers mitigated the off-target effects of the CRISPR-Cas system by modifying the gRNA structure and developing various Cas protein mutants.^17,18 Huang et al. used phage-assisted noncontinuous evolution and eVOLVER-supported phage-assisted continuous evolution to obtain a Cas9 variant, which targets most pyrimidine-rich PAM sequences in the human genome and exerts decreased off-target effects in human cell types with various N4CN PAM sequences.¹⁹ Compared with unmodified Cas9, Chen et al. fused the Cas9 construct with PRDM9 (a chromatin remodeling factor), which increased the efficiency of HDR by threefold but did not increase the incidence of off-target gene editing.²⁰ The key components of Cas9 protein were redesigned according to the resolved CRISPR-Cas9 structural features described by the research group of Bravo to generate Cas9 mutants with low off-target effects and high cleavage efficiency. The off-target efficiency of these Cas9 mutants decreased by 500-fold compared with that of the wild-type Streptococcus pyogenes Cas9.²¹

OTHER GENE EDITING TOOLS BASED ON THE CRISPR-Cas SYSTEM

In addition to the CRISPR-Cas9 gene editing system, several gene editing tools based on the CRISPR-Cas system, such as the CRISPR-Cas12 and CRISPR-Cas13 systems have been developed for a wide range of applications. In the CRISPR-Cas12 gene editing system, crRNA guides the Cas12 nuclease to the target DNA. Cas12 nuclease cleaves the target DNA near the distal PAM end of the interspace sequence to generate a cohesive terminus. The CRISPR-Cas12 gene editing system induces DSBs through a single RuvC-like nuclease functional domain but cleaves multiple crRNAs containing expression arrays through its RNase domain, enabling the simultaneous editing of multiple genes.²² After the recognition of target sites and the activation of the nuclease activity, several Cas12 homologs indiscriminately cleave single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA). This characteristic is widely used for nucleic acid detection.²³ The CRISPR-Cas13 system can target RNA²⁴ and does not permanently alter the genome.

Furthermore, the RNA editing effect of the CRISPR-Cas13 system can be adjusted in a dose-dependent manner and is reversible, which is a major advantage for the treatment of acquired metabolic diseases.²⁵ However, the CRISPR-Cas13 RNA gene editing system exhibits a general collateral activity in mammalian cells.²⁶ The CRISPR-Cas13 RNA gene editing system may randomly cleave nontarget ssRNAs in the presence of both crRNA and target RNA, which can lead to severe off-target effects²⁷ and major toxic effects in animal cells or individuals.²⁸

More than half of human genetic diseases are caused by point mutations.²⁹ Thus, the correction of mutated genes with gene editing tools is a good therapeutic strategy for genetic diseases. Various CRISPR-Cas9–based single-base gene editing systems have been developed, such as the adenine base editors (ABEs) and cytosine base editors (CBEs) (Fig. 2). The ABEs and CBEs can directly interact with genomic DNA or cellular RNA to produce precise point mutations without inducing DNA DSBs.^30,31 In addition, ABEs and CBEs do not require DNA donors as templates for cell-dependent HDR.³² Thus, the target sequences of ABEs or CBEs are edited without the introduction of any small indels.

Figure 2.

Schematic of the single-base editors (ABEs and CBEs). (a) Based on the activity of the modified adenine deaminase and Cas9 at the DNA target site, ABEs usually generate a precise point mutation via deamination of a certain range of adenine at the target site, which results in an A–G substitution. The A–G substitution was achieved through DNA repairing and replication but without a DSB generation. (b) The CBEs use cytosine deaminase and Cas9 and deaminate the cytosine at genomic targets in a certain range. Furthermore, UGI proteins in the CBE system are incorporated to inhibit the repairing of uracil bases in the target genome, resulting in a precise point mutation through C–T substitution. The C–T substitution was also achieved through DNA repairing and replication but without DSB generation. ABEs, adenine base editors; CBE, cytosine base editor; DSB, double-stranded break; UGI, uracil glycosylase inhibitor.

The CRISPR-Cas gene editing technology has potential therapeutic applications in genetic diseases. The application of gene editing tools in genetic disease therapy is limited by their low repair efficiency. Precise targeting of target genes and mitigating off-target effects are critical for gene editing.³³ The ABEs and CBEs based on the CRISPR-Cas gene editing system were mainly used to correct single-base mutations in genetic diseases.³⁴ CRISPR-Cas9, Cas12, and Cas13 were widely used in genetic correction and detection. Significantly, Cas9 and Cas12 can target targeting DNA and cleave its 5′ PAM side sites, Cas12 also cleaves the 3′ PAM side sites of target DNA.³⁵ Both Cas9 and Cas12 can cleave dsDNA or ssDNA. Target sequences matched by Cas12 is usually smaller than 17 base pair (bp), it usually forms to an R-loop, which makes base-pair hybridization between the crRNA and the target DNA strand.³⁶

The Cas9 matches sgRNA sequences ranging in size between 20 and 24 nt.³⁶ The Cas13 can target ssRNA, and the size of matched sgRNA sequence to the Cas13 ranges from 22 to 28 nt.^37,38 The CRISPR-Cas13 system is mainly used for RNA gene editing.³⁹ The CRISPR-Cas12 gene editing system nonspecifically cleaves ssDNA and is usually applied for DNA detection.⁴⁰ The CRISPR-Cas9 gene editing tool is adapted to edit GC-rich PAMs, which are abundant in the mammalian genome⁴¹ and can correct both single-base and multibase mutations. Thus, the CRISPR-Cas9 gene editing system is widely used in mammalian systems for genetic disease therapy. The gene repair efficiency and targeting accuracy are the major limiting factors of the CRISPR-Cas9 gene editing system. For example, the CRISPR-Cas9–mediated HDR efficiency is only ∼15% upon transfection of sgRNA, DNA templates, and CRISPR-Cas9 into HEK293T cells via electroporation in vitro.⁴²

This gene editing efficiency is not optimal for clinical genetic disease therapy. The research group of Liang et al. suggested that the CRISPR-Cas9 gene editing system exerts off-target effects in human trinuclear zygotes.⁴³ Unexpected mutations resulting from the off-target effects of the CRISPR-Cas9 system limit its applications in genetic disease treatment.⁴⁴ In the following sections, recent advances in factors affecting the gene editing efficiency of the CRISPR-Cas9 system and CRISPR-Cas9 system optimization strategies have been reviewed.

CORRELATION BETWEEN HDR EFFICIENCY AND GENE REPAIR EFFICIENCY OF THE CRISPR-Cas9 SYSTEM

Cas9-generated DSB can be repaired through the NHEJ and HDR pathways. HDR is the most common pathway used by the CRISPR-Cas9 gene editing system to introduce specific changes to target DNA sequences. In cases where the endogenous or exogenous DNA repair template is homologous to a certain region of target DNA around the DSB sites (also known as the donor DNA), the DNA DSB is repaired by related repair factors through the HDR pathway. This enables the precise insertion, deletion, and alteration of bases during the exchange of new gene fragments with target DNA (Fig. 3). Previous studies have suggested that inefficient HDR during gene editing may be a limiting factor for CRISPR-Cas9 gene editing efficiency.⁴⁵ The enhancement of HDR efficiency can improve the gene editing efficiency of the CRISPR-Cas9 system.⁴⁶

Figure 3.

Schematic of HDR mechanism. The target DNA is cleaved by the CRISPR-CRISPR-associated (Cas) system to induce a DSB. After DSB formation at the target DNA, an exogenous ssDNA with a homologous arm is integrated into the target DNA via DNA repairing factors through the HDR pathway. HDR, homology-directed repair; ssDNA, single-stranded DNA.

HDR efficiency is limited by the NHEJ pathway in some cell types. The NHEJ pathway predominantly repairs DSBs and can suppress the HDR pathway through various mechanisms.⁴⁷ The cell cycle phase markedly affects the HDR pathway. The NHEJ repair pathway is highly active throughout the cell cycle, whereas the HDR pathway is restricted only to the S/G2 phases. DSB repair via the NHEJ repair pathway is faster than that via the HDR pathway.⁴⁸ The knockdown of Ku70/80 heterodimer or DNA ligase IV, which are two key factors involved in the NHEJ pathways, can improve HDR efficiency.^49,50 The small molecule compounds M3814 and trichostatin A (TSA), which are both NHEJ inhibitors, affect HDR efficiency to varying degrees. The application of M3814 markedly improved HDR efficiency after Cas9-mediated gene editing in different cell types.

Moreover, the combination of M3814 and TSA increased HDR efficiency by three times compared with the DMSO control.⁴⁵ Similarly, treatment with the DNA ligase IV inhibitor Scr7 increased HDR efficiency in cells.⁴⁹ In addition, the overexpression of RAD51/52/54, BRCA1/2, or HDR-related genes improved HDR efficiency.⁵¹ The synchronization of cells at the G2/M cycle using CCND1 and nocodazole compounds increases HDR efficiency in induced pluripotent stem cells.⁵² Therefore, the overexpression of HDR pathway-related genes, the knockout or suppression of NHEJ pathway-related genes, or the synchronization of cell cycle can improve HDR efficiency and the gene editing efficiency of the CRISPR-Cas9 system.⁵³

EFFECTS OF DIFFERENT DONOR FORMS ON GENE EDITING EFFICIENCY OF THE CRISPR-Cas9 SYSTEM

Different DNA donor forms significantly affect the HDR and gene editing efficiencies. Common sources of donor DNA include plasmids, dsDNA, ssDNA, and chemically modified DNA. For example, one study reported that transfecting mammalian cells with asymmetric homology arm donor DNA, Cas9, and nickase mutants for gene editing improved HDR efficiency to ∼60%.⁵⁴ Compared with that of vectors harboring nonlinear donors, the HDR efficiency of ZFN-containing multiple tandem donor DNAs was ∼30-fold higher in 293T cells.⁵⁵ After the cleavage of the target gene by the CRISPR-Cas9 system, a double cleavage donor with two flank sgRNA and PAM sequences is released. The HDR efficiency of this double cleavage donor was two- to fivefold higher than that of the cyclic plasmid donor.⁵² Linear donors or changing donor sources can improve HDR efficiency during cellular gene editing events.

In 2018, one study reported that the 5′ modification of the dsDNA donor preserved its monomeric conformation, maximally enhanced HDR efficiency, and facilitated target gene replacement and gene labeling.⁵⁶ The fusion of Cas9 to a single avidin domain to bind with a biotinylated donor DNA increased the HDR efficiency to up to 90% during gene editing.⁵⁷ The chemically modified donor DNA exhibits increased intracellular stability, HDR efficiency, and DSB repair ability.⁵⁸

The impact of the proximity of CRISPR components to donor DNA also influences HDR efficiency. Such as, it has been demonstrated that when Cas9 proteins genetically edit the target fragment to produce DSBs, the higher concentration of ssDNA was presented in the vicinity of the DNA incision, the higher the probability of HDR repair favors.^58,59 Besides, a Cas9–avidin–biotin ssDNA (CAB) system that consists of Cas9 fused with avidin, sgRNA and biotin-modified single-strand donor DNA (biotin-ssDNA) was developed by Ma et al. and applied to gene editing in mouse zygotes. By utilizing the high affinity between avidin and biotin, biotin-ssDNA would be enriched near the cleavage sites, which also improved the precision of HDR insertion by threefold compared with the traditional CRISPR-Cas9 system.⁶⁰

In addition, Lee et al. have demonstrated that the gRNA and donor DNA can be directly conjugated together into one molecule, and they also showed that the HDR efficiency of this gRNA–donor DNA conjugate improved three times than that of transfecting cells with cationic polymers, unconjugated gRNA and donor DNA.⁵⁸ Moreover, according to evidences from Farbiak et al., they showed that when the CRISPR-Cas9 system was used to edit the same gene site by liposomal encapsulation of three nucleic acids (Cas9 mRNA, sgRNA, ssDNA) in different weight ratios, with the percentage of sgRNA increasing, the HDR efficiency also improved.⁶¹

The length of the homologous arm at both ends of donors and the donor insert segment sizes affect the efficiency of gene integration and editing. Yang et al. reported that HDR donors with a length of 90 nt exhibit excellent gene editing efficiency.⁶² Jian-Ping Zhang et al. constructed donors with different homologous arm lengths for CRISPR-Cas9 gene editing events and reported that a linear donor is more efficient than a circular donor for HDR-mediated gene integration. The HDR efficiency was ∼6–10% for a linear donor whose left and right homologous arms had a length of 50 bp and increased by twofold when the length of the left and right homologous arms was increased from 50 to 100–150 bp. Additionally, the HDR efficiency increased to 26% when the length of the double-cut donor homologous arms was increased to 900 bp.⁵² Thus, different donor DNA forms or the lengths of donor homologous arms markedly affect HDR efficiency, as well as the repair efficiency of the CRISPR-Cas9 gene editing system.⁶³

EFFECT OF THE CELLULAR DELIVERY MODE OF CRISPR-Cas9 GENE EDITING SYSTEM ON GENE EDITING EFFICIENCY

The CRISPR-Cas9 gene editing system can be delivered into cells in the form of plasmids, mRNAs, or proteins. The delivery form of the CRISPR-Cas9 gene editing system into cells also affects its gene editing efficiency. For example, the transfection efficiency of linear DNA (ssDNA and dsDNA) is higher than that of plasmid donor DNA at the same transfection dosage.⁵² The preparation of plasmid DNA of the CRISPR-Cas 9 gene editing system is affordable, and this plasmid DNA can be easily delivered into cells. However, the plasmid DNA can overexpress Cas9 protein in cells for a prolonged duration, resulting in off-target effects.⁶⁴ The cellular delivery of the CRISPR-Cas9 gene editing system in the mRNA form enables the direct translation of Cas9. Subsequently, the translated Cas9 can rapidly edit genes without the need for DNA transcription. Therefore, the CRISPR-Cas9 gene editing system delivered in the mRNA form can efficiently and rapidly edit the target genes.⁶⁵

However, mRNA is susceptible to degradation and is difficult to prepare and preserve in practical applications. The chemical modification of mRNA enhances its stability. Thus, the delivery of the CRISPR-Cas9 gene editing system in the chemically modified mRNA form, which has high efficiency and decreased off-target effects, will have wide applications in gene editing.⁶⁶ The target genes can be immediately edited after the cellular delivery of the CRISPR-Cas9 gene editing system in the ribonucleoprotein (RNP) form.⁶⁷ The gene editing duration of the CRISPR-Cas9 delivered in the RNP form is markedly shorter than that delivered in the DNA and mRNA forms.⁶⁸ In addition, the off-target effect of the CRISPR-Cas9 delivered in the RNP form is lower than that delivered in the plasmid DNA and mRNA forms.⁶⁹

EFFECTS OF DIFFERENT TRANSFECTION METHODS FOR CELLULAR CRISPR-Cas9 SYSTEM DELIVERY ON GENE EDITING EFFICIENCY

The CRISPR-Cas9 gene editing system can be introduced into eukaryotic cells via physical, chemical, or biological transfection.⁷⁰ Different transfection methods for cellular delivery of CRISPR-Cas9 and their gene editing efficiencies are given in Table 1.

Table 1.

Different transfection methods for cellular delivery of clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) protein 9 system

		Composition	Immunogenicity Pathogenicity	Cargo	Diameter	Charge	Packing Capacity	Gene Editing Efficiency	Refs.
Physical methods	Microinjection	NA	NA	RNP mRNA Plasmid	NA	NA	Unlimited	40.0–93.7%; can damage the zygotes	^31,71 –73
Physical methods	Electroporation	NA	NA	RNP	NA	NA	NA	50–90%; high efficiency but not suitable for electroporation-sensitive cells	⁷⁴
Chemical methods	Lipid nanoparticle	Lipid, cholesterol, PEG, and DOPE	Low	DNA RNA Protein	150–200 nm	Positive electricity	NA	60–98%; safe with no cytotoxicity	^75 –77
	Calcium phosphate coprecipitation	Calcium phosphate	Low	RNP Plasmid	NA	NA	NA	Approximately 20%; dual-targeted delivery systems based on polymer/inorganic hybrid nanoparticles exhibit improved gene editing efficiency	^67,78
	Nanopolymer	Dendrimer nanocarrier	Low	DNA RNA Protein	300 nm	Can bind both cations and anions	NA	Approximately 40%; Dendritic macromolecules can bind to negatively charged proteins	⁷⁹
	Gold nanoparticles	Arginine, cationic polymer	Low	DNA RNA Protein	500 nm	Positive electricity	NA	Approximately 30%; delivery via membrane fusion	⁸⁰
	Graphene oxide	GO-PEG-PEI	Low	Protein sgRNA	220 nm	Positive electricity	NA	Approximately 39%; maintains high stability and suppresses sgRNA degradation	^81,82
	Metal-organic frameworks	Organic–inorganic hybrid crystalline porous materials	Medium	Protein	NA	Positive electricity	NA	Approximately 35% (independent of the size of the protein and its relative molecular mass)	⁸³
Biological method	Adenovirus	Double-stranded DNA	High	RNA	100 nm	NA	8 kb	Approximately 80%; high packaging capacity and does not integrate with host genome but is affected by immunogenicity	^84 –86
	Adeno-associated virus	Single-stranded DNA	Low	DNA	25 nm	NA	4.7 kb	34–94%; limited loading capacity and is affected by immunogenicity	^87 –90
	Lentivirus	Single-stranded DNA	Low	DNA	130 nm	NA	8 kb	32.5–70%; has an excellent ability to infect both dividing and nondividing cells but may be genotoxic	^91 –94
	Cell-penetrating peptides	Polypeptide	Low	Cell-penetrating protein sgRNA	NA	Positive electricity	NA	30–98%; can target neuronal genes and edit primary cells with a high efficiency and a low toxicity	^95 –97
	DNA nanoclews	Single-stranded DNA	Low	RNP	NA	Positive electricity	NA	Approximately 30%; highest editing efficiency when DNA nanoclews sequences and sgRNA sequences are partially complementary	^67,98

DOPE, 1, 2-dioleyl-sn-glycero-3-phosphoethanlamine; NA, not applicable; PEG, polyethylene glycol; RNP, ribonucleoprotein; sgRNA, single-guide RNA.

Microinjection and electroporation are the major physical transfection methods for introducing the CRISPR-Cas9 gene editing system into cells. In the microinjection method, the cells are injected with genetic materials via a microinjection needle. The microinjection method is efficient to load the CRISPR-Cas9 gene editing system into cells. However, highly trained personnel and sophisticated equipment are needed to perform microinjection, which has limited its wide application. At present, microinjection technology is widely used for the transfection of fertilized eggs in animals to generate transgenic animal models.⁹⁹ The microinjection of a base editor comprising CRISPR-Cas9–fused cytidine deaminase into mouse fertilized eggs resulted in a nonsense mutation. The gene editing efficiency was 44–57%, whereas the allele frequency was up to 100% in F0 mice. This indicated that the microinjection technology was an efficient method to generate mice with target point mutations.³¹

The electroporation transfection method is commonly used for gene editing. In the electroporation transfection method, the cell membrane is stimulated by applying pulses of electric current, which promotes the formation of temporary pores and allows the delivery of materials into cells. Electroporation-mediated transfection is not limited by the cell type and has good transfection efficiency in both walled and suspended cells. Several studies have applied electroporation transfection–mediated ex vivo gene editing, which has been widely used in the treatment of hematological malignancies.¹⁰⁰ This method has contributed to the development of stem cell therapies. However, the strong current generated during electroporation can induce cell death. Hence, this method is not suitable for electroporation-sensitive cell types.

Chemical transfection methods have been commonly used. Chemical transfection methods that mediate the delivery of the CRISPR-Cas9 gene editing system into cells include cationic liposome transfection, calcium phosphate coprecipitation, and diethylaminoethyl (DEAE)-dextran. The transfection efficiency of cationic liposome transfection and calcium phosphate coprecipitation methods for adherent cells is higher than that for suspended cells. Although these methods are widely used, they exert cytotoxic effects on cells.¹⁰¹ Therefore, the amount of chemical transfection reagent must be regulated and the new culture medium must be replaced at an optimal time when these two methods are used. DEAE-dextran, which was one of the first reagents used for mammalian cell transfection, is a cationic polymorph that binds to negatively charged nucleic acids close to the cell membrane and is easily taken up by transfected cells.¹⁰²

DEAE-dextran–mediated transfection, which requires a small amount of DNA, has been successfully used in transient expression studies but is less reliable for stable transfection. Similar to cationic liposome-mediated transfection and calcium phosphate–mediated transfection, DEAE-dextran–mediated transfection can exert toxic effects on cells.¹⁰³ In addition, the serum must be removed when DEAE-dextran–mediated transfection is used. The lipid nanoparticle (LNP) delivery method can help to realize the instantaneous expression of the Cas9 effector. The immunogenicity of LNP is lower than that of virus expression vectors. In addition, LNP can be incorporated into the CRISPR-Cas9 system and improve the in vivo delivery safety of the system.¹⁰⁴ Several chemical transfection methods have been developed for delivering the CRISPR-Cas9 gene editing system into cells and are used in different cell types. However, the chemical transfection method–mediated gene editing is associated with decreased transfection efficiency compared with other transfection methods (Table 1).

Biological methods for the cellular delivery of the CRISPR-Cas9 gene editing system include adenovirus (Ad)-mediated, lentivirus (LV)-mediated, and adeno-associated virus (AAV)–mediated transfection. These viral transfection methods are associated with high transfection efficiency. In addition, the viral transfection of gene editing tools increases gene editing efficiency. For example, the lentiviral CRISPR-Cas9 system successfully knocked out melanoma cell adhesion molecule (MUC18) in human primary nasal airway epithelial cells with high efficiency (>90%).¹⁰⁵ One study demonstrated that AAV6 donor can effectively edit hematopoietic stem and progenitor cells upon electroporation of Cas9/sgRNA RNP, achieving up to 94% monoallelic editing frequency at the gene encoding the hemoglobin subunit β (HBB) locus.⁹⁰ The AAV-mediated transfection method is characterized by low immunogenicity, serotype dependence, and stability of transgene expression¹⁰⁶ and can be used for the cellular delivery of Cas9, sgRNA, or donor templates.

The size of the cargo that AAVs can carry is <4.7 kb, which limits their application in carrying gene editing–related proteins. AAVs can be used to transfect cells with donor DNA.¹⁰⁷ In contrast to AAV-mediated transfection, the Ad-mediated transfection method can transfect large genetic material (8–36 kb).¹⁰⁸ However, Ads are highly immunogenic.¹⁰⁹ LV is regarded as a quality delivery tool for carrying the CRISPR-Cas9 gene editing system.¹¹⁰ Lenti viruses can package up to 10 kb, and the LV-mediated transfection has the advantage of low cell cycle propensity.^94,111 For example, Cas9 protein was persistently expressed in cells by packaging the sgRNA and Cas9 protein expression genes into the same LV expression vector. Moreover, this LV expression vector has a good ability to infect both dividing and nondividing cells.¹¹²

However, LV-mediated CRISPR-Cas9 gene editing is a safety hazard because the LV genome can randomly integrate into the genomes of somatic cells, limiting its clinical applications.¹¹³ Recently, a peptide-assisted genome editing system that enables powerful and rapid editing of primary cells with minimal cytotoxicity was developed by Zhang et al.⁹⁷ Kreitz et al. developed a novel protein delivery system that can precisely deliver any protein to any human cell.¹¹⁴ Thus, peptide-mediated or protein-mediated delivery is a new research trend in gene editing tool delivery.

In summary, the CRISPR-Cas9 gene editing system can be delivered to cells through different methods, which are associated with differential gene editing efficiency. The optimal mode of CRISPR-Cas9 system delivery is determined based on the advantages of different transfection methods and the application.

DISCUSSION

This review demonstrated that the components of the CRISPR-Cas9 system, HDR efficiency–related factors, and the delivery forms and modes of the CRISPR-Cas9 gene editing system into cells affect the gene editing efficiency of the CRISPR-Cas9 system. Optimization strategies to improve the gene editing efficiency of the CRISPR-Cas9 system were also discussed. This may guide in improving the gene editing efficiency and developing new gene editing tools.

For example, DSBs of target DNA are generally repaired through the NHEJ pathway, which can generate indels, especially in nondividing cells. Unrepaired DSBs may cause unpredictable gene safety risks, resulting in unnecessary genomic changes, such as chromosomal translocations and large fragment deletions, and activation of the p53 pathway. Thus, improving the HDR efficiency of the CRISPR-Cas9 gene editing system has potential applications in the precise treatment of genetic diseases and precise DNA repair. This review discussed the factors affecting HDR efficiency and CRISPR-Cas optimization strategies, which may enable the development of precise gene editing tools and genetic disease therapy.

Viral vector-mediated transfer of the CRISPR-Cas gene editing system has markedly improved gene editing efficiency. The commonly used viral vectors include AAVs, LVs, and Ads. More interesting, the integration-deficient lentiviral vector (IDLV) was obtained by mutation of the D64V gene of integrase.¹¹⁵ The development of IDLV is highly anticipated. It can overcome the genotoxicity associated with LV when used for CRISPR-Cas9 delivery.¹¹⁶ At present, a research has reported that IDLV has significant lower levels of indel formation and other off-target effects compared with that of integrase-competent lentiviral vectors, both in vitro and in vivo.¹¹⁶ Moreover, Michael Chavez et al. utilized an IDLV to encode the homology arm and the payload (large segments of DNA) on both sides of the “cleavage site” of DSBs, which has achieved stable expression of a large transgene by integrase-deficient LV knock-in, leading to HDR at an endogenous essential gene locus.¹¹⁷

Thus, the advantages of IDLV-mediated CRISPR-Cas9 delivery would make it to be a more and more popular tool in the future study of gene therapy. However, a single optimal viral vector that can deliver different components of the CRISPR-Cas gene editing system is not available. The advantages and disadvantages of different viral vectors vary. Hence, the immunogenicity, integrability, and extra-target effects must be considered when viruses are used as vectors to deliver gene editing components. Delivery of the CRISPR-Cas via the nonviral vectors LNPs and polymer nanoparticles is associated with some advantages for gene therapy. The transfection of target-editing plasmids and donor DNA using multiple transfection methods can improve the integration efficiency after gene editing.¹¹⁸ Therefore, an appropriate delivery mode must be selected to ensure that the CRISPR-Cas gene editing components are efficiently delivered to the most relevant cell populations, which will improve the gene editing efficiency of the CRISPR-Cas9 system under different application conditions and in different cell types.

The gene editing efficiency can be improved through several other methods. For example, base editing proteins can be optimized for specific therapeutic targets. The optimized protein can maximize the recruitment of gene editing products to the target site and minimize off-target effects, improving gene editing efficiency.¹¹⁹ In addition, reducing the size of the effector protein will enable the incorporation of other regulatory elements in the delivery system, which can potentially increase gene editing efficiency. A small Cas9 system (named Nme2-Cas9) that was optimized and modified through the protein-directed evolution system by Huang et al. successfully overcame the limitations of traditional PAM (N4CC). This system could effectively recognize the PAM of N4YN and achieved efficient gene editing, which markedly expanded the application scope of accurate editing tools.¹⁹

However, some changes in gRNA may lead to the loss of gene editing activity and limit the application of highly specific Cas9 mutants.¹²⁰ Therefore, gene editing efficiency can be improved by reducing off-target effects and maintaining effective target site editing activity of the CRISPR-Cas system, as well as by ensuring the compatibility between gene editing systems and various gRNA variants.

Several recently developed novel gene editing technologies have markedly improved gene editing efficiency, such as, a eukaryotic transposon-encoded first RNA-guided DNA-cutting enzyme (Fanzor) system has been recently verified that it can derive the gene editing in eukaryotes but not undergo collateral cleavage.^121,122 It enables precise gene editing. The novel gene editing technologies have several advantages, which must be investigated in the future. A recently developed CRISPR SpRYgests, which is a PAM-less in vitro system, can cleave DNA at any sequence, including sites refractory to wild-type SpCas9-mediated cleavage.¹³ Thus, CRISPR SpRYgests has potential applications in the molecular cloning field.¹³ Moreover, Durrant et al. discovered a series of novel large serine recombinases (LSRs) that can precisely insert a large DNA fragment (>7 kb) into the human genome without the involvement of host proteins and guide RNA.

As the average molecular weight of LSRs is two times lesser than that of Cas9, they can be easily delivered into cells.¹²³ Lampe et al. successfully performed a DSB-free DNA knock-in in mammals using the type I–F CAST system. This system, which was derived from Vibrio cholerae and comprises CRISPR-associated transposases, can mediate large DNA segment integration into the genome without the induction of DSBs.¹²⁴ Some transposons can also edit specific genes, such as Tol, ProtoRAG, IscB, Tn7, and IS200/IS605 transposons. These transposons are associated with high gene editing efficiencies and activities.^125

–129

Some transposons have been developed as novel gene editing tools, such as Tol2 transposon, Sleeping Beauty, and piggyBac.^130
–132 The ProtoRAG transposon has two effectors (RAG1 and RAG2) for DSB formation, which is the main difference compared with other transposon gene edit tools.^126,133,134 This may be used for new gene editing tool developing. The development of transposon gene editing tool may be a following worth noted direction in the future.

In conclusion, several factors affect the gene editing efficiency of the CRISPR-Cas system. Appropriate methods to improve the gene editing efficiency of the CRISPR-Cas system must be considered based on the application, cell types, DNA damage and repair pathways, and the forms and delivery modes of gene editing tools into cells.

Footnotes

AUTHORs' CONTRIBUTIONS

S.L., H.S. and J.L. conceived and designed the structure of this review, J.L., C.T., G.L., H.T., Y.W., S.L., and G.L. drafted the article. H.S., S.L., and W.Z. reviewed and edited the article, and supervised the study. All authors reviewed and approved the final version of this article.

AUTHOR DISCLOSURE

No competing financial interests exist.

FUNDING INFORMATION

This work was supported by National Natural Science Foundation of China (No. 32100705), Science and Technology Program of Guangzhou, China (Nos. 202201010036, 202201010154), Guangdong Basic and Applied Basic Research Foundation (No. 2022A1515012324), the Medical Scientific Research Foundation of Guangdong Province of China (No. A2021236).

The work is also granted by the National Key Clinical Specialty Construction Project (Clinical Pharmacy), and High Level Clinical Key Specialty (Clinical Pharmacy) in Guangdong Province, College Students' Innovation and Entrepreneurship Training Project of Guangdong Province (No. 202210573054), the Special Fund for the Cultivation of Scientific and Technological Innovation of College Students in Guangdong Province of China (No. pdjh2022b0270), the Special Fund for the Cultivation of National Natural Science Foundation of China in School of Clinical Pharmacy, Guangdong Pharmaceutical University (No. SCP2022-03), 2021 Open Project Fund of Guangdong Provincial Key Laboratory of Medicinal Functional Gene Research.

References

Wang

, Doudna

. CRISPR technology: A decade of genome editing is only the beginning. Science, 2023; 379(6629):eadd8643; doi: 10.1126/science.add8643

Jinek

, Chylinski

, Fonfara

, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012; 337(6096):816–821; doi: 10.1126/science.1225829

Jiang

, Doudna

. CRISPR-Cas9 structures and mechanisms. Annu Rev Biophys, 2017; 46(1):505–529.

Iyer

, Suresh

, Guo

, et al. Precise therapeutic gene correction by a simple nuclease-induced double-stranded break. Nature, 2019; 568(7753):561–565; doi: 10.1038/s41586-019-1076-8

Boulton

, Jackson

. Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J, 1998; 17(6):1819–1828; doi: 10.1093/emboj/17.6.1819

Pâques

, Haber

. Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae . Microbiol Mol Biol Rev, 1999; 63(2):349–404; doi: 10.1128/mmbr.63.2.349-404.1999

Symington

. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev, 2002; 66(4):630–670, table of contents; doi: 10.1128/mmbr.66.4.630-670.2002

Fichter

, Setayesh

, Malik

. Strategies for precise gene edits in mammalian cells. Mol Ther Nucleic acids, 2023; 32:536–552; doi: 10.1016/j.omtn.2023.04.012

Jimenez-Sainz

, Jensen

. Imprecise medicine: BRCA2 variants of uncertain significance (VUS), the challenges and benefits to integrate a functional assay workflow with clinical decision rules. Genes, 2021; 12(5):780; doi: 10.3390/genes12050780

10.

Lee

, Neiditch

, Salus

, et al. RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination. Cell, 2004; 117(2):171–184; doi: 10.1016/s0092-8674(04)00301-0

11.

Nelson

, Hakim

, Ousterout

, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science, 2016; 351(6271):403–407.

12.

Liao

, Beisel

. The tracrRNA in CRISPR biology and technologies. Ann Rev Genet, 2021; 55:161–181; doi: 10.1146/annurev-genet-071719-022559

13.

Christie

, Guo

, Silverstein

, et al. Precise DNA cleavage using CRISPR-SpRYgests. Nat Biotechnol, 2023; 41(3):409–416; doi: 10.1038/s41587-022-01492-y

14.

, Xian

, Li

, et al. Rapid and user-friendly open-source CRISPR/Cas9 system for single- or multi-site editing of tomato genome. Horticult Res, 2019; 6:7; doi: 10.1038/s41438-018-0082-6

15.

Gaj

, Gersbach

, Barbas III

. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol, 2013; 31(7):397–405.

16.

Doudna

. The promise and challenge of therapeutic genome editing. Nature, 2020; 578(7794):229–236.

17.

Kocak

, Josephs

, Bhandarkar

, et al. Increasing the specificity of CRISPR systems with engineered RNA secondary structures. Nat Biotechnol, 2019; 37(6):657–666; doi: 10.1038/s41587-019-0095-1

18.

Schmid-Burgk

, Gao

, Li

, et al. Highly parallel profiling of Cas9 variant specificity. Mol Cell, 2020; 78(4):794–800.e8; doi: 10.1016/j.molcel.2020.02.023

19.

Huang

, Heins

, Miller

, et al. High-throughput continuous evolution of compact Cas9 variants targeting single-nucleotide-pyrimidine PAMs. Nat Biotechnol, 2023; 41(1):96–107; doi: 10.1038/s41587-022-01410-2

20.

Chen

, Lin-Shiao

, Trinidad

, et al. Decorating chromatin for enhanced genome editing using CRISPR-Cas9. Proc Natl Acad Sci U S A, 2022; 119(49):e2204259119; doi: 10.1073/pnas.2204259119

21.

Bravo

JPK

, Liu

, Hibshman

, et al. Structural basis for mismatch surveillance by CRISPR-Cas9. Nature, 2022; 603(7900):343–347; doi: 10.1038/s41586-022-04470-1

22.

Zetsche

, Gootenberg

, Abudayyeh

, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015; 163(3):759–771.

23.

Yan

, Hunnewell

, Alfonse

, et al. Functionally diverse type V CRISPR-Cas systems. Science, 2019; 363(6422):88–91.

24.

Konermann

, Lotfy

, Brideau

, et al. Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors. Cell, 2018; 173(3):665–676.e14.

25.

Cohen

, Boerwinkle

, Mosley Jr

, et al. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med, 2006; 354(12):1264–1272.

26.

Özcan

, Krajeski

, Ioannidi

, et al. Programmable RNA targeting with the single-protein CRISPR effector Cas7-11. Nature, 2021; 597(7878):720–725.

27.

, Liang

, Wilusz

. CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells. Nucleic Acids Res, 2022; 50(11):e65.

28.

, Xu

, Guo

, et al. The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model. Genome Biol, 2023; 24(1):20; doi: 10.1186/s13059-023-02860-w

29.

Rees

, Liu

. Base editing: Precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet, 2018; 19(12):770–788.

30.

Gaudelli

, Komor

, Rees

, et al. Programmable base editing of A• T to G• C in genomic DNA without DNA cleavage. Nature, 2017; 551(7681):464–471.

31.

Kim

, Ryu

, Kim

, et al. Highly efficient RNA-guided base editing in mouse embryos. Nat Biotechnol, 2017; 35(5):435–437; doi: 10.1038/nbt.3816

32.

Komor

, Kim

, Packer

, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 2016; 533(7603):420–424.

33.

Tsai

, Joung

. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat Rev Genet, 2016; 17(5):300–312; doi: 10.1038/nrg.2016.28

34.

Richter

, Zhao

, Eton

, et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol, 2020; 38(7):883–891; doi: 10.1038/s41587-020-0453-z

35.

Yamano

, Nishimasu

, Zetsche

, et al. Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell, 2016; 165(4):949–962; doi: 10.1016/j.cell.2016.04.003

36.

Hillary

, Ceasar

. A review on the mechanism and applications of CRISPR/Cas9/Cas12/Cas13/Cas14 proteins utilized for genome engineering. Mol Biotechnol, 2023; 65(3):311–325; doi: 10.1007/s12033-022-00567-0

37.

Gootenberg

, Abudayyeh

, Lee

, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, 2017; 356(6336):438–442; doi: 10.1126/science.aam9321

38.

Gootenberg

, Abudayyeh

, Kellner

, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science, 2018; 360(6387):439–444; doi: 10.1126/science.aaq0179

39.

Tong

, Huang

, Xiao

, et al. High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects. Nat Biotechnol, 2023; 41(1):108–119; doi: 10.1038/s41587-022-01419-7

40.

Broughton

, Deng

, Yu

, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol, 2020; 38(7):870–874; doi: 10.1038/s41587-020-0513-4

41.

Cong

, Ran

, Cox

, et al. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013; 339(6121):819–823; doi: 10.1126/science.1231143

42.

Lin-Shiao

, Pfeifer

, Shy

, et al. CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells. Nucleic Acids Res, 2022; 50(3):1256–1268.

43.

Liang

, Xu

, Zhang

, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell, 2015; 6(5):363–372; doi: 10.1007/s13238-015-0153-5

44.

Hsu

, Lander

, Zhang

. Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014; 157(6):1262–1278; doi: 10.1016/j.cell.2014.05.010

45.

, Dai

, Wang

, et al. Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing. Nucleic Acids Res, 2021; 49(2):969–985; doi: 10.1093/nar/gkaa1251

46.

Wang

, Cheng

JKW

, Zhang

, et al. Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects. Nucleic Acids Res, 2021; 49(6):e36; doi: 10.1093/nar/gkaa1264

47.

Dumitrache

, Hu

, Son

, et al. Trex2 enables spontaneous sister chromatid exchanges without facilitating DNA double-strand break repair. Genetics, 2011; 188(4):787–797.

48.

Yang

, Ren

, Yu

, et al. Methods favoring homology-directed repair choice in response to CRISPR/Cas9 induced-double strand breaks. Int J Mol Sci, 2020; 21(18):6461.

49.

, Shi

, Guo

, et al. Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR–Cas9 and ssODN in human cancer cells. Cell Biosci, 2018; 8(1):1–15.

50.

Kragelund

, Weterings

, Hartmann-Petersen

, et al. The Ku70/80 ring in Non-Homologous End-Joining: easy to slip on, hard to remove. Front Biosci (Landmark edition), 2016; 21(3):514–527; doi: 10.2741/4406

51.

Scott

, Francis-Newton

, Marsh

, et al. A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death. Cell Chem Biol, 2021; 28(6):835–847. e5.

52.

Zhang

J-P

, Li

X-L

, Li

G-H

, et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol, 2017; 18(1):1–18.

53.

Lin

, Staahl

, Alla

, et al. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife, 2014; 3:e04766.

54.

Richardson

, Ray

, DeWitt

, et al. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol, 2016; 34(3):339–344.

55.

Zhang

, Wang

, Liu

, et al. Multiple copies of a linear donor fragment released in situ from a vector improve the efficiency of zinc-finger nuclease-mediated genome editing. Gene Ther, 2014; 21(3):282–288.

56.

Gutierrez-Triana

, Tavhelidse

, Thumberger

, et al. Efficient single-copy HDR by 5′modified long dsDNA donors. Elife, 2018; 7:e39468.

57.

Roche

, Gytz

, Hussain

, et al. Double-stranded biotinylated donor enhances homology-directed repair in combination with Cas9 monoavidin in mammalian cells. CRISPR J, 2018; 1(6):414–430.

58.

Lee

, Mackley

, Rao

, et al. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. Elife, 2017; 6;e25312; doi: 10.7554/eLife.25312

59.

Ruff

, Koh

, Keskin

, et al. Aptamer-guided gene targeting in yeast and human cells. Nucleic Acids Res, 2014; 42(7):e61; doi: 10.1093/nar/gku101

60.

, Zhuang

, Hu

, et al. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res, 2017; 27(4):578–581; doi: 10.1038/cr.2017.29

61.

Farbiak

, Cheng

, Wei

, et al. All-in-one dendrimer-based lipid nanoparticles enable precise HDR-mediated gene editing in vivo. Adv Mater (Deerfield Beach, Fla), 2021; 33(30):e2006619; doi: 10.1002/adma.202006619

62.

Yang

, Guell

, Byrne

, et al. Optimization of scarless human stem cell genome editing. Nucleic Acids Res, 2013; 41(19):9049–9061; doi: 10.1093/nar/gkt555

63.

Savic

, Ringnalda

, Lindsay

, et al. Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. Elife, 2018; 7; doi: 10.7554/eLife.33761

64.

Naeem

, Majeed

, Hoque

, et al. Latest developed strategies to minimize the off-target effects in CRISPR-Cas-mediated genome editing. Cells, 2020; 9(7):1608.

65.

, Javidi-Parsijani

, Makani

, et al. Delivering SaCas9 mRNA by lentivirus-like bionanoparticles for transient expression and efficient genome editing. Nucleic Acids Res, 2019; 47(8):e44; doi: 10.1093/nar/gkz093

66.

Yin

, Song

C-Q

, Suresh

, et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat Biotechnol, 2017; 35(12):1179–1187.

67.

Zhang

, Shen

, Li

, et al. Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing. Theranostics, 2021; 11(2):614–648; doi: 10.7150/thno.47007

68.

Cheng

, Zhang

, Ding

. CRISPR/Cas9 delivery system engineering for genome editing in therapeutic applications. Pharmaceutics, 2021; 13(10):1649;; doi: 10.3390/pharmaceutics13101649

69.

Taharabaru

, Yokoyama

, Higashi

, et al. Genome editing in a wide area of the brain using dendrimer-based ternary polyplexes of Cas9 ribonucleoprotein. ACS Appl Mater Interfaces, 2020; 12(19):21386–21397; doi: 10.1021/acsami.9b21667

70.

Pickar-Oliver

, Gersbach

. The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol, 2019; 20(8):490–507; doi: 10.1038/s41580-019-0131-5

71.

Roy

, Zhao

, Yang

, et al. CRISPR/Cascade 9-mediated genome editing-challenges and opportunities. Front Genet, 2018; 9:240; doi: 10.3389/fgene.2018.00240

72.

Liu

, Chen

, Xie

, et al. Versatile and efficient in vivo genome editing with compact Streptococcus pasteurianus Cas9. Mol Ther, 2022; 30(1):256–267; doi: 10.1016/j.ymthe.2021.06.013

73.

Zhang

, Wang

, Li

, et al. Establishment of an integrated CRISPR/Cas9 plasmid system for simple and efficient genome editing in Medaka in vitro and in vivo. Biology, 2023; 12(2); doi: 10.3390/biology12020336

74.

Seki

, Rutz

. Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells. J Exp Med, 2018; 215(3):985–997; doi: 10.1084/jem.20171626

75.

Zuris

, Thompson

, Shu

, et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol, 2015; 33(1):73–80; doi: 10.1038/nbt.3081

76.

Rosenblum

, Gutkin

, Kedmi

, et al. CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy. Sci Adv, 2020; 6(47):eabc9450; doi: 10.1126/sciadv.abc9450

77.

Liang

, Potter

, Kumar

, et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol, 2015; 208:44–53; doi: 10.1016/j.jbiotec.2015.04.024

78.

Liu

, He

, Xu

, et al. A dual-targeting delivery system for effective genome editing and in situ detecting related protein expression in edited cells. Biomacromolecules, 2018; 19(7):2957–2968; doi: 10.1021/acs.biomac.8b00511

79.

Liu

, Wan

, Wang

, et al. A boronic acid-rich dendrimer with robust and unprecedented efficiency for cytosolic protein delivery and CRISPR-Cas9 gene editing. Sci Adv, 2019; 5(6):eaaw8922; doi: 10.1126/sciadv.aaw8922

80.

Mout

, Ray

, Yesilbag Tonga

, et al. Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano, 2017; 11(3):2452–2458.

81.

Yue

, Zhou

, Cheng

, et al. Graphene oxide-mediated Cas9/sgRNA delivery for efficient genome editing. Nanoscale, 2018; 10(3):1063–1071; doi: 10.1039/c7nr07999k

82.

Shen

, Liu

, He

, et al. PEGylated graphene oxide-mediated protein delivery for cell function regulation. ACS Appl Mater Interfaces, 2012; 4(11):6317–6323; doi: 10.1021/am3019367

83.

Yang

, Tang

, Jiang

, et al. Nanoscale ATP-responsive zeolitic imidazole framework-90 as a general platform for cytosolic protein delivery and genome editing. J Am Chem Soc, 2019; 141(9):3782–3786; doi: 10.1021/jacs.8b11996

84.

Stephens

, Lauron

, Kashentseva

, et al. Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9. J Controll Release, 2019; 298:128–141; doi: 10.1016/j.jconrel.2019.02.009

85.

Statkute

, Wang

ECY

, Stanton

. An optimized CRISPR/Cas9 adenovirus vector (AdZ-CRISPR) for high-throughput cloning of sgRNA, using enhanced sgRNA and Cas9 variants. Hum Gene Ther, 2022; 33(17–18):990–1001; doi:10.1089/hum.2021.120

86.

Chen

, Gonçalves

. Engineered viruses as genome editing devices. Mol Ther, 2016; 24(3):447–457; doi: 10.1038/mt.2015.164

87.

Samulski

, Muzyczka

. AAV-mediated gene therapy for research and therapeutic purposes. Annu Rev Virol, 2014; 1(1):427–451; doi: 10.1146/annurev-virology-031413-085355

88.

Kotterman

, Schaffer

. Engineering adeno-associated viruses for clinical gene therapy. Nat Rev Genet, 2014; 15(7):445–451; doi: 10.1038/nrg3742

89.

Tran

, Graf

, Wulf-Goldenberg

, et al. CRISPR-Cas9-mediated ELANE mutation correction in hematopoietic stem and progenitor cells to treat severe congenital neutropenia. Mol Ther, 2020; 28(12):2621–2634; doi: 10.1016/j.ymthe.2020.08.004

90.

Martin

, Ikeda

, Cromer

, et al. Highly efficient and marker-free genome editing of human pluripotent stem cells by CRISPR-Cas9 RNP and AAV6 donor-mediated homologous recombination. Cell Stem Cell, 2019; 24(5):821–828.e5; doi: 10.1016/j.stem.2019.04.001

91.

Kumar

, Keller

, Makalou

, et al. Systematic determination of the packaging limit of lentiviral vectors. Hum Gene Ther, 2001; 12(15):1893–1905; doi: 10.1089/104303401753153947

92.

Ailles

, Naldini

. HIV-1-derived lentiviral vectors. Curr Topics Microbiol Immunol, 2002; 261:31–52; doi: 10.1007/978-3-642-56114-6_2

93.

Ramadier

, Chalumeau

, Felix

, et al. Combination of lentiviral and genome editing technologies for the treatment of sickle cell disease. Mol Ther, 2022; 30(1):145–163; doi: 10.1016/j.ymthe.2021.08.019

94.

Wang

, Ma

, Rodríguez Labrada

, et al. Recent advances in lentiviral vectors for gene therapy. Sci China Life Sci, 2021; 64(11):1842–1857; doi: 10.1007/s11427-021-1952-5

95.

Park

, Oh

, Shim

, et al. In vivo neuronal gene editing via CRISPR–Cas9 amphiphilic nanocomplexes alleviates deficits in mouse models of Alzheimer's disease. Nat Neurosci, 2019; 22(4):524–528.

96.

Lostalé-Seijo

, Louzao

, Juanes

, et al. Peptide/Cas9 nanostructures for ribonucleoprotein cell membrane transport and gene edition. Chem Sci, 2017; 8(12):7923–7931.

97.

Zhang

, Baxter

, Ren

, et al. Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. Nat Biotechnol, 2023. [Epub ahead of print]; doi: 10.1038/s41587-023-01756-1

98.

Sun

, Ji

, Hall

, et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew Chem (International ed in English), 2015; 54(41):12029–12033; doi: 10.1002/anie.201506030

99.

, Shen

, Zhang

, et al. Heritable multiplex genetic engineering in rats using CRISPR/Cas9. PLoS One, 2014; 9(3):e89413; doi: 10.1371/journal.pone.0089413

100.

Dever

, Bak

, Reinisch

, et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature, 2016; 539(7629):384–389.

101.

Cho

, Ryu

J-Y

, Lee

HAR

, et al. Lecithin nano-liposomal particle as a CRISPR/Cas9 complex delivery system for treating type 2 diabetes. J Nanobiotechnol, 2019; 17(1):1–12.

102.

Gauss

, Lieber

. DEAE-dextran enhances electroporation of mammalian cells. Nucleic Acids Res, 1992; 20(24):6739.

103.

Bakrania

, Variya

, Patel

. Role of β-interferon inducer (DEAE-Dextran) in tumorigenesis by VEGF and NOTCH1 inhibition along with apoptosis induction. Front Pharmacol, 2017; 8:930; doi: 10.3389/fphar.2017.00930

104.

Kenjo

, Hozumi

, Makita

, et al. Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice. Nat Commun, 2021; 12(1):7101; doi: 10.1038/s41467-021-26714-w

105.

Chu

, Rios

, Huang

, et al. CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18. Gene Ther, 2015; 22(10):822–829; doi: 10.1038/gt.2015.53

106.

Wang

, Zhang

, Gao

. CRISPR-based therapeutic genome editing: Strategies and in vivo delivery by AAV vectors. Cell, 2020; 181(1):136–150.

107.

Bak

, Porteus

. CRISPR-mediated integration of large gene cassettes using AAV donor vectors. Cell Rep, 2017; 20(3):750–756.

108.

Lee

, Bishop

, Zhang

, et al. Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new era of personalized medicine. Genes Dis, 2017; 4(2):43–63; doi: 10.1016/j.gendis.2017.04.001

109.

C-F

, Chen

G-J

, Luo

Y-L

, et al. Rational designs of in vivo CRISPR-Cas delivery systems. Adv Drug Deliv Rev, 2021; 168:3–29.

110.

Kotterman

, Chalberg

, Schaffer

. Viral vectors for gene therapy: Translational and clinical outlook. Annu Rev Biomed Eng, 2015; 17:63–89.

111.

Wilbie

, Walther

, Mastrobattista

. Delivery aspects of CRISPR/Cas for in vivo genome editing. Accounts Chem Res, 2019; 52(6):1555–1564.

112.

Luis

. The old and the new: Prospects for non-integrating lentiviral vector technology. Viruses, 2020; 12(10):1103; doi: 10.3390/v12101103

113.

Ottaviano

, Georgiadis

, Gkazi

, et al. Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia. Sci Transl Med, 2022; 14(668):eabq3010; doi: 10.1126/scitranslmed.abq3010

114.

Kreitz

, Friedrich

, Guru

, et al. Programmable protein delivery with a bacterial contractile injection system. Nature, 2023; 616(7956):357–364; doi: 10.1038/s41586-023-05870-7

115.

, Dai

, Wang

. Vaccines delivered by integration-deficient lentiviral vectors targeting dendritic cells induces strong antigen-specific immunity. Vaccine, 2010; 28(41):6675–6683; doi: 10.1016/j.vaccine.2010.08.012

116.

Dong

, Kantor

. Lentiviral vectors for delivery of gene-editing systems based on CRISPR/Cas: Current state and perspectives. Viruses, 2021; 13(7); doi: 10.3390/v13071288

117.

Chavez

, Rane

, Chen

, et al. Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus. Nat Biomed Eng, 2023; 7(5):661–671; doi: 10.1038/s41551-023-01037-x

118.

Liang

, Potter

, Kumar

, et al. Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA. J Biotechnol, 2017; 241:136–146.

119.

Manghwar

, Lindsey

, Zhang

, et al. CRISPR/Cas System: Recent advances and future prospects for genome editing. Trends Plant Sci, 2019; 24(12):1102–1125; doi: 10.1016/j.tplants.2019.09.006

120.

Hsu

, Scott

, Weinstein

, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol, 2013; 31(9):827–832; doi: 10.1038/nbt.2647

121.

Bao

, Jurka

. Homologues of bacterial TnpB_IS605 are widespread in diverse eukaryotic transposable elements. Mobile DNA, 2013; 4(1):12; doi: 10.1186/1759-8753-4-12

122.

Saito

, Xu

, Faure

, et al. Fanzor is a eukaryotic programmable RNA-guided endonuclease. Nature, 2023; 620(7974):660–668; doi: 10.1038/s41586-023-06356-2

123.

Durrant

, Fanton

, Tycko

, et al. Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome. Nat Biotechnol, 2023; 41(4):488–499; doi: 10.1038/s41587-022-01494-w

124.

Lampe

, King

, Halpin-Healy

, et al. Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases. Nat Biotechnol, 2023; doi: 10.1038/s41587-023-01748-1

125.

Koga

. Transposition mechanisms and biotechnology applications of the medaka fish Tol2 transposable element. Adv Biophys, 2004; 38:161–180.

126.

Huang

, Tao

, Yuan

, et al. Discovery of an active RAG transposon illuminates the origins of V(D)J recombination. Cell, 2016; 166(1):102–114; doi: 10.1016/j.cell.2016.05.032

127.

Han

, Xiao

, Wang

, et al. Development of miniature base editors using engineered IscB nickase. Nat Methods, 2023; 20(7):1029–1036; doi: 10.1038/s41592-023-01898-9

128.

Faure

, Saito

, Benler

, et al. Modularity and diversity of target selectors in Tn7 transposons. Mol Cell, 2023; 83(12):2122–2136.e10; doi: 10.1016/j.molcel.2023.05.013

129.

Altae-Tran

, Kannan

, Demircioglu

, et al. The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases. Science, 2021; 374(6563):57–65; doi: 10.1126/science.abj6856

130.

Sandoval-Villegas

, Nurieva

, Amberger

, et al. Contemporary transposon tools: A review and guide through mechanisms and applications of sleeping beauty, piggyBac and Tol2 for genome engineering. Int J Mol Sci, 2021; 22(10); doi: 10.3390/ijms22105084

131.

Klem

, Gray

, Lovely

. The Zebrafish Tol2 System: A modular and flexible gateway-based transgenesis approach. J Vis Exp, 2022; 189; doi: 10.3791/64679

132.

Muñoz-López

, García-Pérez

. DNA transposons: Nature and applications in genomics. Curr Genomics, 2010; 11(2):115–128; doi: 10.2174/138920210790886871

133.

Liu

, Yuan

, Gao

, et al. Functional regulation of an ancestral RAG transposon ProtoRAG by a trans-acting factor YY1 in lancelet. Nat Commun, 2020; 11(1):4515; doi: 10.1038/s41467-020-18261-7

134.

Liu

, Zhang

, Liu

, et al. Structural insights into the evolution of the RAG recombinase. Nat Rev Immunol, 2022; 22(6):353–370; doi: 10.1038/s41577-021-00628-6