The evolution of genome editing technology based on CRISPR (clustered regularly interspaced short palindromic repeats) system has led to a paradigm shift in biological research. CRISPR/Cas9-guide RNA complexes enable rapid and efficient genome editing in mammalian cells. This system induces double-stranded DNA breaks (DSBs) at target sites and most DNA breakages induce mutations as small insertions or deletions (indels) by non-homologous end joining (NHEJ) repair pathway. However, for more precise correction as knock-in or replacement of DNA base pairs, using the homology-directed repair (HDR) pathway is essential. Until now, many trials have greatly enhanced knock-in or substitution efficiency by increasing HDR efficiency, or newly developed methods such as Base Editors (BEs). However, accuracy remains unsatisfactory. In this review, we summarize studies to overcome the limitations of HDR using the CRISPR system and discuss future direction.
Genetically engineered mice are valuable subjects for developmental and pathomechanism studies. However, the traditional gene targeting method through embryonic stem cells (ESCs) has been time-consuming and costly. In 2013, the Jaenisch group introduced conducting gene modified mice in a one-step generation using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) genome engineering technology (1, 2). Since the CRISPR/Cas9-mediated system originated from the prokaryotic immune system (3–6), it enabled rapid and efficient genome editing in mammalian cells (7–11).
This system opened a new era in genome biology fields including animal, plants, and human genetic disease (12–15). Programmable endonuclease Cas9 with guide RNA (gRNA) induce DNA double-strand breaks (DSBs) on the target DNA sequences, and DSBs are repaired by non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathway, mainly (16–18). Among them, NHEJ is a predominant repair mechanism in higher eukaryotic cells or organisms. Therefore, after DSBs, NHEJ works dominantly and generates small insertions or deletions (indels), resulting in frame shifts at target genes eventually (19–21). Taking advantage of these characteristics, the efficient knock-out study through NHEJ pathway has been developed extensively in the genome editing field. However, since the NHEJ repair mechanism induces uncontrollable random mutations on target loci, NHEJ conjugated technologies showed limitations for precise genome editing, such as designated insertions and single-nucleotide substitutions (2, 22).
To overcome these limitations, many scientists made an effort to develop methods to insert donor template DNA using the HDR pathway, to perform precise gene editing. However, it was difficult to use HDR mechanism in gene editing unrestricted because of its extremely low efficiency. In mammalian cells, NHEJ is the major source of the DNA repair mechanism competing with the HDR pathway. Therefore, for more efficient HDR-mediated precise genome editing, numerous researchers have attempted to enhance HDR pathway or/and suppress NHEJ pathway by targeting key factors (23–25).
Recently, a new technology called base editors (BEs) has been introduced to overcome low accuracy of NHEJ and low efficiency of HDR. These powerful editing tools can change single nucleotide without DNA DSBs in cells (26, 27). BEs are composed of catalytically impaired Cas9 variant with deaminase classified as cytosine base editors (CBEs) and adenine base editors (ABEs), allowing direct conversion from C to T or A to G (28–30). Recent reports showed that various applications using base editors enable single nucleotide substitutions in mammalian genome successfully (31–35). Although it is clear that base-editing technique is an innovative development, limitations remain in the case of single base substitution, as well as insufficient accuracy/efficacy
In this review, we will report recently developed methods for precise gene editing as enhanced HDR-mediated gene engineering and direct base editing in mammal species. Diverse strategies to increase HDR efficiency are introduced. One is optimization of the HDR pathway by controlling the length of homology arms of template donor DNA. Another is the inhibition of NHEJ pathway which competes with HDR. Additionally, we also introduce BEs, a method for tailored single nucleotide substitution.
The most precise genome editing method is utilizing HDR mechanism to insert artificial DNA sequences to target locus or to induce single-nucleotide substitutions. However, the efficiency of HDR pathway in nature is extremely low (2, 36–38). Recently, several studies reported new methods to overcome low efficiency by optimizing template donor DNA. Researchers modulated the length of homology arms and types of donor DNA, such as single strand DNA (ssDNA) or double strand DNA (dsDNA) (Table 1). Renaud
NHEJ mediated genome editing induces random mutations such as small indels on target sites. Therefore, these kinds of mutations led the frame shift on targeted genes and is proper for knock-out studies but not for inducing precise mutations, such as point mutations or knock-in studies. Conversely, HDR repair system is good in generating precise point mutations and for inserting external artificial DNA sequences. However, low efficiency has always been a major obstacle to broad use. A number of studies have attempted to increase HDR efficiency by regulating DSBs repair mechanisms (Fig. 1A). It is well known that NHEJ and HDR pathways are in competition (49–51). Several studies have shown that suppression of key molecules involved in the NHEJ pathway could increase efficiency of HDR. Many proteins are known to be relevant with NHEJ pathway including Ku heterodimers (Ku70/80), DNA-dependent protein kinase catalytic subunits (DNA-PKcs), DNA ligase IV, the X-ray repair cross-complementing protein 4 (XRCC4), and the XRCC4-like factor (XLF) as core complexes (52–54). Among these related proteins Chu
Major DNA repair pathways, NHEJ and HDR are not always activated during all cell cycle stages. NHEJ dominates over all M, G1, S, and G2 phases, while HDR can only compete with NHEJ, during S and G2 phases. HDR is down regulated during M phase and G1 phase (59–61). Various small molecules exert their effects by controlling such stages in part (Fig. 1B). Li
More than 50% of human pathogenic mutations are point mutations or single nucleotide polymorphisms (SNPs) (26). As the importance of precise medicine arises, accurate single nucleotide substitutions in the genome have been required for pathology or mechanistic studies. However, in the beginning of the CRISPR technology, specific nucleotide substitutions at desired target sites could only be induced by an HDR-based CRISPR system, despite its low efficiency. To overcome such limitation, new tools called Base Editors (BEs) were developed to induce single-nucleotide substitution, which do not need a template donor DNA (Fig. 2A and 2B) (28–30). Because these techniques do not introduce DSBs, they never use DNA repair mechanisms as NHEJ, MMEJ, or HDR pathways. BEs were composed of nuclease activity deficient Cas9, nickase Cas9 (nCas9) or dead Cas9 (dCas9), and cytidine deaminase or adenine deaminase. They enable conversion of C to T, or A to G, and vice versa. They are newly-developed methods not affected by HDR efficiency in case of inducing substitutions. These tools were verified through various research groups and applied to many other organisms, including mice and rabbits (31, 32, 66, 67). The substitution efficiency was higher than the HDR mechanism. However, the unique characteristic of BEs, such as base editing window which indicates the specific region occurring substitution, could be a limitation to inducing single-nucleotide substitution to the exact target base pair. So, some researchers attempted to change the base editing window. One study induced some mutations at cytidine deaminase domains to narrow the base editing window for more specific substitutions (68). Conversely, to extend coverage of BE systems, some researchers demonstrated that using the extended guided RNA could extend coverage of BEs and using Cas9 variants with different protospacer adjacent motif (PAM) sequences, such as xCas9 and VQR variants (32, 69, 70). There remain several improvements in the BE system. Accuracy and efficacy have not been satisfied for clinical demands and knock-in of external DNA sequences are impossible.
CRISPR/Cas9 mediated genome engineering applicable to a variety of organisms is crucial as a tool for research and clinical applications. In this review, we showed efforts to increase efficiency of HDR, one of the genetic manipulation strategies, for accurate and specific targeted knock-in. Recent efforts to improve HDR efficiency have focused on controlling the homology arm length, or suppressing the NHEJ pathway using small molecules. In particular, the Tild-CRISPR method, a method of controlling donor DNA homology arm length, is expected to greatly improve the efficiency of HDR. Based on these results, HDR efficiency is expected to be enhanced by combining NHEJ pathway inhibition with small molecules and the control of homology arm length. Additionally, the BEs (nucleotide substitution methods for specific target sites) are expected to be applied to studies of clinical pathology mechanism by allowing tailored point mutation. Recently, development of gene editing technology has suggested the possibility of clinical application as a genetic disease therapeutic agent. However, accuracy of gene correction fails to meet clinical demands and additionally, the stable
This study was supported by the Chung Yang, Cha Young Sun & Jang Hi Joo Memorial fund, Korea university grant (K1804351), and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) of Korea (NRF-2018M3A9H3021707, NRF-2018R1D1A1B07048434, and NRF-2014M3A9D5A01075128).
The authors have no conflicting interests.
Regulation of homology arm of donor DNA to enhance knock-in efficiency
|Species||Methods||Donor DNA||Insertion size||HA size||Reference|
|Rat, Mouse||Zygote||Microinjection||ssODN (chemical modifications: phosphorothioate or LNA)||~100 bp||~100 bp||39|
|Human||HEK293, iPSC||Transfection, electroporation||ssODN (silent mutations)||100 bp/400 bp||50 bp||40|
|Mouse||Zygote||Microinjection||ssODN||527 bp/893 bp||55 bp/103 bp||41|
|Cas9 mRNA or protein/gRNA (crRNA + tracrRNA) (Easi-CRISPR method)|
|Human||HEK293T, U2-OS||Transfection||ssODN (13 bp PCV recognition sequences at 5’-end)||50 bp||75 bp||43|
|Cas9 protein/gRNA (PCV-Cas9 fusion)|
|Mouse||Zygote, ESC||Microinjection||ssODN||~42 bp
|Cas9 mRNA/gRNA||dsDNA (Linearization)|
|Human||HEK293T||Transfection||dsDNA (Linearization)||~1.5 kb||~25 bp||44, 45|
|Plasmid (PITCh method)|
|Mouse, monkey||Zygote||Microinjection, mRNA/gRNA||dsDNA (Linearization)||700 bp/6.1 kb||800 bp||46, 47|
|E14.5 embryo||In utero electroporation, Cas9 mRNA/gRNA|
|Adult mouse||Hydrodynamic injection, Cas9 mRNA/gRNA|
|Mouse, Human||Zygote||Microinjection||dsDNA (Linearization or PCR amplification)||~2 kb||800 bp||48|
|E14.5 embryo||Cas9 mRNA/gRNA (Tild method)|
|In utero electroporation|
|Mouse||2-cell stage embryo||Microinjection, Cas9 mRNA/gRNA (2C-HR-CRISPR with a biotin-Streptavidin approach)||dsDNA (PCR amplification)||717 bp/1.4 kb||100 bp/3 kb||42|
HA: Homology arm, iPSC: induced Pluripotent Stem Cell, ESCs: embryonic stem cells, gRNA: guide RNA, ssODNs: single-stranded oligo DNA nucleotides, dsDNA: double-strand DNA, Easi: Efficient additions with ssDNA inserts, PVC: Porcine Circovirus 2, PITCh: Precise Integration into Target Chromosome, Tild: targeted integration with linearized dsDNA, 2C-HR: two-cell homologous recombination.