PEM-seq for Optimizing Gene Editing and Tracking DNA Repair

In 2019, a study titled "Optimizing genome editing strategy by primer-extension-mediated sequencing" was published in Cell Discovery. This research introduced a novel method for sensitively detecting CRISPR/Cas9 off-target sites through translocation capture and assessing its editing efficiency by quantifying incomplete Cas9-induced double-strand break (DSB) repair products. This technique, known as primer-extension-mediated sequencing (PEM-seq), enables simultaneous quantification of Cas9 editing efficiency, off-target activity, and chromosomal aberrations resulting from the editing process. Overall, PEM-seq represents a significant advancement in optimizing gene editing and tracking DNA repair mechanisms.

Background

CRISPR-Cas9, a gene editing tool with substantial potential, often exhibits off-target activity that can damage genomic sites with incomplete matches or lead to chromosomal rearrangements, thus limiting its therapeutic applications. CRISPR/Cas9 functions by pairing a guide RNA (gRNA) with a target DNA sequence, thereby anchoring Cas9 to the target gene and inducing a DNA DSB.

Efficient and precise gene editing is crucial for both clinical applications and the development of animal models, necessitating engineered nucleases with high editing capacity and low off-target activity. The method described in this paper, PEM-seq, is applicable for comprehensively assessing the editing capacity and specificity of nucleases.

Using PEM-seq, researchers explored Cas9 off-target sites and other aberrant chromosomal structures, including small insertions, large deletions, and genome-wide translocations. Furthermore, PEM-seq was employed to evaluate several widely used methods aimed at reducing Cas9 off-target activity. The results demonstrated that PEM-seq can thoroughly assess the editing efficiency and specificity of designed CRISPR/Cas9 strategies, thereby aiding in the selection of appropriate genome editing strategies for specific loci. Additionally, the researchers developed a new high-fidelity variant, termed further eCas9 (FeCas9), which exhibited significantly reduced off-target activity without compromising editing efficiency compared to wild-type (WT) Cas9.

Methods and Results

Experimental Design of PEM-seq

Building on the principles of DNA double-strand breaks and chromosomal translocations, the research group led by Hu Jiazhi developed PEM-seq, an enhanced and comprehensive quantitative method for assessing gene editing, based on an existing high-throughput sequencing technique (Hu et al., Nature Protocols, 2016).

PEM-seq captures Cas9-induced DSBs, including insertions, deletions, and genomic rearrangements. To enable quantification of these outcomes, the researchers employed primer extension, generating only one copy of the original template. The extended fragments were then isolated and connected to a bridging linker containing a 14 bp random molecular barcode (RMB) to tag each fragment, as illustrated in Figure 1a.

PEM-seq is capable of sensitively identifying off-target sites of Cas9 and accurately quantifying the cutting efficiency of CRISPR/Cas9 at target sites, thereby facilitating the identification of more efficient and safer Cas9 cutting sites.

PEM-seq's Sensitivity in Identifying CRISPR/Cas9 Off-Target Hotspots

To compare the sensitivity of PEM-seq and LAM-HTGTS in identifying off-target sites, the RAG1A locus in HEK293T cells was utilized. The data revealed that PEM-seq detected 53 off-target sites for Cas9 at the RAG1A locus, including 24 novel sites and 4 weak sites identified by LAM-HTGTS. Within 5 kb of the Cas9 cutting site, numerous abnormal chromosomal structures, accounting for 2.5% of total editing events, were observed, including inversions and deletions. Alarmingly, these abnormal structures extended up to 50 kb or more from the cutting site, posing a significant threat to genomic stability. These findings underscore the necessity of thorough pre-application assessment of Cas9.

The application of PEM-seq enables a comprehensive evaluation of Cas9 cutting efficiency and the resulting abnormal structures, thereby optimizing gene editing strategies to achieve maximum editing efficiency while minimizing off-target activity.

Figure 1: Detection of CRISPR/Cas9 off-target sites using PEM-seq.

The findings presented indicate that PEM-seq exhibits significantly higher sensitivity in detecting off-target sites compared to Linear Amplification-Mediated High-Throughput Genome-Wide Translocation Sequencing (LAM-HTGTS). Additionally, PEM-seq is highly adaptable and can be effectively applied to detect off-target sites in any cell line amenable to editing.

Detection of Chromosomal Abnormalities and Indel Analysis within 5 kb of Cas9 Cleavage Sites

PEM-seq has identified a substantial occurrence of chromosomal abnormalities within a 5 kb range surrounding the Cas9 cleavage sites. To evaluate the editing efficiency concerning the types of DSBs induced by Cas9, a comparative analysis was performed, as depicted in Figure 2a. The analysis revealed that the percentage of insertions and deletions (Indels) detected by PEM-seq is comparable to that identified by other detection methodologies. Further dissection of the CRISPR/Cas9-induced translocations and Indels demonstrated that the majority of Indels occurred within 20 base pairs of the target site, with large deletions predominantly situated within the 3-5 kb region downstream of the primer.

Figure 2: Evaluation of CRISPR/Cas9 editing efficiency via PEM-seq.

The data indicate that PEM-seq reliably quantifies the substantial accumulation of junctions at target sites. Additionally, PEM-seq accurately detects aberrant DSB repair products accumulating around the target region. This capability significantly contributes to the assessment of CRISPR/Cas9 editing efficiency.

Reduced Off-Target Activity and Editing Efficiency of Cas9 Nickase

Cas9 nickase exhibits lower off-target activity, albeit with a concomitant reduction in target editing efficiency. It has been reported that generating two adjacent DNA nicks using Cas9 nickase minimizes off-target damage. Experimental validation, corroborated by PEM-seq, revealed that the editing efficiency of Cas9 nickase for the RAG1 gene is only half that of Cas9, with activity observed exclusively on RAG1A.

Figure 3: Cas9 nickase exhibits reduced off-target activity, accompanied by a decrease in on-target editing efficiency.

The results demonstrate that Cas9 nickase reduces off-target damage in the genome-editing process, albeit at the expense of editing efficiency.

FeCas9: A High-Fidelity Cas9 Variant with Low Off-Target Activity

To mitigate the off-target activity associated with CRISPR/Cas9, the research group led by Hu Jiazhong performed combinatorial screening of mutation sites within existing Cas9 variants. Through PEM-seq, they identified a variant, FeCas9, which exhibits cutting efficiency comparable to that of wild-type Cas9 (WT Cas9) but with significantly lower off-target frequency. The following observations were made:

a) The application of FeCas9 mirrors that of traditional Cas9, with the introduction of the D1135E31 mutation into enhanced Cas9 (eCas9) yielding the FeCas9 variant.

b) FeCas9 demonstrates higher specificity compared to eCas9.

c) The editing efficiency of FeCas9 on the RAG1A gene is comparable to that of WT Cas9 and eCas9.

d) FeCas9 consistently exhibits very low off-target activity across all tested loci, without any significant change in editing efficiency.

e) Regarding editing specificity, FeCas9 outperforms eCas9 at all tested loci.

Factors such as target sequence, Cas9 variant, cell type, and Cas9 activation time all influence the editing outcome. Under these conditions, FeCas9 presents itself as a promising candidate for genome editing applications.

Figure 4: Editing efficiency and specificity of Cas9 variants.

Evaluation of AcrIIA4 Inhibitor's Efficacy in Suppressing Cas9 Off-Target Activity

The effectiveness of the AcrIIA4 inhibitor in suppressing the off-target activity of Cas9 has been shown to be suboptimal. The widely used Cas9 inhibitor, AcrIIA4, was evaluated for its ability to block Cas9 activity at the RAG1A locus in HEK293T cells using PEM-seq.

Figure 5: AcrIIA4's limited role in minimizing Cas9 off-target effects, showing incomplete inhibition.

The results indicate that the off-target editing efficiency was only reduced by a factor of 1.7 to 4.6, which is not as significant as the reduction in on-target editing efficiency. This suggests that AcrIIA4 is more effective at blocking Cas9 target activity.

Research Conclusions

In comparison to currently employed methods for evaluating CRISPR/Cas9 editing efficiency, PEM-seq offers two distinct advantages:

Potential in Genomic Stability Research: PEM-seq holds significant potential in the study of genomic stability. The process of genome repair is intricately linked to DNA repair mechanisms. Traditional methods often rely on Polymerase Chain Reaction (PCR) or molecular cloning to obtain repair information, which is limited by small sample sizes and inherent biases.

Provision of Large Quantitative Datasets: PEM-seq can quantitatively assess the various stages of DNA repair, thereby enabling a detailed depiction of the process from DNA damage to repair. This allows for the development of more accurate models of DNA repair mechanisms.

Limitations of PEM-seq

Despite its advantages, PEM-seq also presents certain limitations:

Dependence on Bait DSBs: PEM-seq relies heavily on bait DSBs to capture genome-wide translocations. The current version of PEM-seq is not well-suited for evaluating the editing specificity of mutations directly generated within the genome.

Identification but Not Quantification of Off-Targets: While PEM-seq can identify off-target sites of CRISPR/Cas9, it is unable to quantify the frequency of these off-target events.

Future Directions

Overall, the application of PEM-seq is still in an exploratory phase. There is a need for further in-depth research to fully elucidate its mechanisms and potential applications.