Impact of the size of CRISPR-Cas gene cassettes on the packaging efficiency and transduction titer of lentiviral vectors

doi:10.21203/rs.3.rs-1458483/v1

Download PDF

Research Article

Impact of the size of CRISPR-Cas gene cassettes on the packaging efficiency and transduction titer of lentiviral vectors

https://doi.org/10.21203/rs.3.rs-1458483/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

CRISPR-Cas technology has revolutionized the use of genome-editing in studies on diverse biological topics. Lentiviral vectors (LVs) have been widely used for the generation of stably transduced cells that durably express the Cas protein and guide RNA (gRNA), the key components of the CRISPR-Cas editing system. This delivery system allows sustained gene expression in dividing and non-dividing cells in vitro and in vivo and LVs can be pseudo-typed with a variety of envelope proteins to establish a broad tropism for a range of target cells. A major challenge on the use of LVs in clinical settings is the requirement to obtain vector stocks of a sufficiently high titer. The endonuclease encoded by Streptococcus pyogenes Cas9 (SpCas9) is the most widely used CRISPR endonuclease for genome engineering, but it has an important limitation because of its relatively large size (∼4.1 kb coding sequence) that may hamper the delivery by means of viral vector systems. Smaller Cas endonucleases have been developed more recently, but these systems still need to be validated in specific experimental settings.

Results

This work aimed to assess the impact of the CRISPR-Cas transgene size on the production of LVs and their ability to transduce cells. We observed that the LV transduction efficiency measured on the SupT1 T cell line dropped dramatically for RNA transcripts approaching 8 kb, e.g. a further increase of 1 kb reduced the transduction efficiency by more than 5-fold. As the number of produced virus particles was not affected, this means that the use of larger transgenes may cause the production of more empty virion particles.

Conclusions

This study confirmed that the transfer efficiency decreased for CRISPR-Cas genes above a certain size. One of the strategies to avoid the restricted RNA packaging can be the use of smaller CRISPR-Cas systems or the inclusion of truncated versions of the regulatory sequences like the promoters that are needed for efficient expression of the transgenes. This work has implications for the design of effective CRISPR-Cas editing therapies that use viral vectors, LVs in particular.

Viral vector

lentiviral vector

transduction titer

CRISPR-Cas

gene therapy

HIV

CRISPR-Cas is a relatively simple, yet effective technique for genome editing in mammalian cells. The RNA-guided Cas endonucleases of bacterial origin have been optimized to create efficient tools for the manipulation of DNA genomes of mammalian cells. This has accelerated fundamental research and enabled the clinical translation of novel therapeutic strategies to successfully treat genetic and other diseases [1, 2]. In fact, the considerable diversity of the prokaryotic CRISPR-Cas systems is currently driving a true biotechnological revolution. Most of these systems require two small RNAs, CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA), which were combined into a single guide RNA (gRNA) to simplify the use [3]. Other systems do not require tracrRNA, e.g. the Cas12a system is guided by only a single crRNA [4, 5]. The prototype Cas9 enzyme cleaves double-stranded DNA (dsDNA), but Cas12 is additionally able to edit single-stranded DNA (ssDNA) and Cas13 cleaves exclusively RNA [4, 6, 7]. The distinct cleavage specificities of different Cas homologs trigger different DNA repair pathways of the host cell, for instance non-homologous end joining (NHEJ), that result in distinct editing lesions [8]. Thus, selection of the appropriate CRISPR-Cas system is key and depends on the specific application that one has in mind. Selection of the appropriate gene delivery system is another critical variable that should be addressed early.

Vector systems based on retroviruses have been a popular delivery tool because they exhibit the unique property of integration into the genome of the host cell, which ensures the prolonged presence of the transgene. A replication-defective vector based on the human immunodeficiency virus (HIV) is called a lentiviral vector (LV). LVs are an efficient tool for transgene delivery to achieve stable and long-term expression in eukaryotic cells and have been successfully used for clinical gene therapy applications [9, 10]. LV-based vectors have a particular advantage over other retroviral systems in that they can infect both dividing and non-dividing cells. For some difficult-to-transduce cell types such as stem cells, LVs can greatly improve the transduction efficiency and thereby increase the probability of transgene integration into the genome of the target cells. However, safety concerns were raised after some initial oncogenesis events were described in trials with retroviral vectors [11, 12]. Following the development of leukemia in a few patients, the gene therapy field shifted more towards the use of LV. Initially, there was a similar concern that LV could also be oncogenic. LV-induced insertional mutagenesis was described in proliferative hematopoietic stem cell (HSCs) and tumors were observed in LV-treated mice [13, 14]. Researchers pursued different strategies to optimize these vectors and a safe third-generation LV was developed for clinical use [15–17]. For instance, whereas HIV encodes 5 essential proteins, only 3 genes (gag, pol, rev) were retained in this LV, which are actually expressed from separate plasmids to avoid the chance of generation of replication-competent virus by recombination [18–20]. The requirement for the HIV transcriptional regulator tat was circumvented by the use of a novel, constitutively active promoter and the HIV env (envelope) function was replaced by the G protein of vesicular stomatitis virus (VSV-G) to allow transduction of other cell types. In addition, all genes were codon-optimized to boost their expression, which also reduced the sequence similarity to the replication-competent virus. A complete 4-plasmid system was created by inclusion of the transfer plasmid that produces a transcript that is selectively packaged in the LV particles, followed by conversion into dsDNA that eventually is integrated in the genome of the transduced cells (Fig. 1A). Note that the LV RNA genome is packaged as a dimeric RNA in assembling virion particles.

A) We used a third-generation lentiviral vector that consists of 4 components: the transfer plasmid encodes the transgene cassette, the envelope plasmid encodes the Vesicular Stomatitis Virus (VSV) glycoprotein (G), and two packaging plasmids that encode Rev and Gag-Pol. Ψ is the packaging signal on the vector RNA. RRE is the Rev-responsive element and cPPT is the central polypurine tract, signals that play an important role in enhancing transgene expression and vector transduction, respectively. B) Lentiviral vectors that encode the CRISPR-Cas components: the CRISPR endonucleases RfxCas13d, CjCas9, SaCa9, AsCas12b and SpCas9 and their respective gRNA scaffolds were cloned in LV to yield constructs 1+, 2+, 3+, 4 + and 5+, respectively. The U6 promoter and gRNA scaffolds were deleted to make constructs 1-, 2-, 3-, 4- and 5-.

The LV can in principle transfer any chosen CRISPR-Cas system into any target cell type. However, LVs have a limited packaging capacity for the transgene RNA transcript and previous studies revealed that the LV titer decreases with increasing transgene size [21–23]. Kumar et al. demonstrated that there is a reduction in transduction titer of approximately 3-fold per kilobases (kb) increase in RNA cargo size for inserts over 5 kb, whereas the titer dropped approximately 100-fold for inserts between 6 and 12.5 kb [21]. Likewise, Canté-Barret et al. demonstrated that the length of the viral RNA is a critical factor that affects the efficiency of LV-mediated gene transfer [22]. Whereas the LV transduction efficiency on human and mouse stem cells dropped dramatically for viral RNAs that approached 6 kb, a modest reduction of their size of 0.6 kb rescued the transduction efficiency by more than threefold. Consistently, Sweeney et al. showed that the LV titer drops with increasing genome size [24]. In general, genome sizes close to or exceeding that of the natural HIV template (9.8 kb) result in decreased packaging capacity and consequently a low transduction efficiency. The relatively large size of Cas systems thus presents a serious challenge for packaging in LV systems. For instance, a simple combination of the Streptococcus pyogenes endonuclease (SpCas9) with a gRNA cassette already makes a transgene of at least 4.2 kb. Additional regulatory elements like promoters, enhancers, regulatory RNA motifs and GFP or another reporter gene do easily make a vector that is significantly larger than 9 kb. The precise size limitations for packaging of CRISPR/Cas systems in LV systems have not been determined yet. Therefore, the aim of this work was to assess the impact of Cas genome size on LV production, the packaging of the transgene RNA and the transduction efficiency. We therefore packaged five Cas genes encoding endonucleases of difference size, with and without the matching g/crRNA cassette. We included in this study the prototype Staphylococcus aureus Cas9 system (SaCas9, 3.2 kb RNA) that produces indels at a similar efficiency as SpCas9 [25], but also the Cas9 system from Campylobacter jejuni (CjCas9, 3.0 kb RNA) that efficiently edits genes in mouse muscle and eye tissue [26], and the Cas12a and Cas13d systems that represent smaller Cas enzymes with just 3.9 and 2.9 kb RNA, respectively [27, 28].

LV production is not affected by increasing size of the transgene cassette

LVs with CRISPR-Cas transgene cassettes were produced by co-transfection of human embryonic kidney (HEK293T) cells with a standard set of three packaging plasmids (Fig. 1A). We selected five CRISPR-Cas systems of different size for this study: RfxCas13d, CjCas9, SaCas9, AsCas12a and SpCas9 (Fig. 1B). The Cas genes were equipped with the P2A peptide that connects the Cas open reading frames to that of the green fluorescent protein (eGFP), which triggers subsequent “self-cleavage” of the polyprotein. The CRISPR-Cas endonucleases were expressed from the constitutive and strong EF-1α promoter. A second set of five LVs was made with an additional gRNA expression cassette driven by the Polymerase-III U6 promoter. We named these LV constructs 1–5 with the – or + sign dependent on gRNA addition. Sizes of the Cas and gRNA cassettes and the “transgene” transcripts that should be packaged in the LV particles is plotted in Fig. 2A. These values range from 6.7 kb for construct 1- (RfxCas13d) to 8.7 kb for constructs 5+ (SpCas9 + gRNA). This set represents a significant fraction of the currently available gene editing systems. Note that all these novel LV designs stay below the RNA transcript length of the original HIV pathogen (9.8 kb).

A) Sizes of the Cas genes and the corresponding gRNA cassettes are shown (in bp). The length of the corresponding mRNA transcripts with and without gRNA cassette are listed (in nt). B) Virion production by the LV constructs was quantitated in the culture supernatant using CA-p24 ELISA. Shown is the mean value ± SD from independent triplicate transfections. Variability in lentivirus production were assessed using One-way analysis of variance (ANOVA) and the Brown-Forsythe test. Significance was set at p < 0.05.

To compare virion production capacity among LVs carrying different CRISPR-Cas systems, lentivirus secreted in the culture supernatant was collected at two days post-transfection and either stored or concentrated some 20-fold using Vivaspin6 100,000 MWCO centrifugation filters that should retain mature LV particles of ~ 90 nm diameter. The level of capsid p24 (CA-p24) protein in the culture medium was quantitated with ELISA before and after the concentration step (Fig. 2B). Note that this Gag protein and its processing product CA-p24 are made from one of the co-transfected “packaging” plasmids. Thus, one would ideally expect to see no effect of changes in the transgene construct on the level of LV production, and that is indeed what we observed. We scored an approximately 20-fold higher CA-p24 level upon filter-mediated concentration, but again no effect of the different Cas transgenes on the level of LV production, both for the non-concentrated and concentrated sample set. These results confirm that LV production is independent of the transgene size.

The LV transduction titer is significantly reduced for larger CRISPR-Cas transgenes

Next, we determined the transduction titer of the concentrated LV stocks on the SupT1 T cell line by measuring eGFP transgene expression of transduced cells by fluorescence-activated cell sorting (FACS). The transduction titer varied over 3 logs for this set of constructs, from approximately 10⁴ to 10⁷ transduction units (TU)/ml. We observed a strong inverse correlation (R²:0.48, P:0.02) between the transgene size, which spans from 6.7 to 8.7 kb, and the LV transduction titer (Fig. 3A).

A) The LV transduction titer is attenuated with increasing RNA genome size. The points indicate the LV titer of the indicated vectors. Values represent the average from three independent replicates and error bars show the standard deviation. Simple linear regression was plotted, the R squared value (0.48) and the intersection with the X-axis at 15.8 kb are shown. B) We plotted the LV titer versus CA-p24 production. The R squared of 0.02 is indicated, demonstrating an absence of any correlation.

A size reduction of 1 kb improved the transduction efficiency by more than 5-fold, which is consistent with previous work [21–24]. The two smallest CRISPR-Cas systems CjCas9 and RfxCas13d did yield the highest transduction titer, and the largest SpCas9 and AsCas12a systems exhibit the lowest titer. The two RfxCas13d constructs (1- and 1+) exhibited an approximately ten-fold lower titer than the two CjCas9 vectors of similar size (2- and 2+). Parameters other than transgene size may also influence the transduction efficiency. For instance, the relatively low packaging efficiency of the 1- and 1 + constructs when compared to the LV constructs 2- and 2 + may relate to a small difference in GC-content of the transgene transcripts (50% and 53%, respectively). Linear regression of the observed effects suggest that the transduction titer will drop to values around zero for transgenes with an insert around 15.8 kb (Fig. 3A). This value could be a rough estimate of the upper size of RNA transcripts that can be packaged, albeit rather inefficiently, in a LV particle. It is indeed likely that a reduced packaging efficiency is witnessed for RNA genome sizes that exceed the RNA genome size of the HIV pathogen (9.8 kb) [24].

We also analyzed the impact of the extra guide RNA cassette on the titer of these five LV particles. Most + constructs experience a small drop in transduction titer when compared to the matching - constructs (Fig. 3A). Interestingly, it seems that the biggest negative impact is apparent for the largest LV constructs 4 and 5, which are obviously closer to the upper size of the packaged RNA. For completeness, we also plotted the LV titer versus LV production as expressed in CA-p24 values (Fig. 3B), confirming that level of LV production is not sensitive to the RNA genome size.

Large RNA transcripts are less efficiently packaged in LV particles

We assume that the transduction titer of the larger LV transgenes declines because the RNA transcripts become too big to be efficiently encapsidated inside the virion particles, thus causing a reduced RNA content of these virus particles. To measure this, the viral RNA present in the virus-containing culture supernatant was isolated and reverse transcribed into cDNA. These cDNA products were amplified by PCR using primers targeting the gag gene to obtain a relative measure of the amount of transgene RNA present in the LV particles (Fig. 4A). PCR products were analyzed on ethidium bromide-stained 1% agarose gels and DNA bands were quantified using ImageJ software (Fig. 4B). Consistent with the previous results, we scored an inverse correlation between the size of the viral RNA transcript and the amount of RNA present in culture supernatant (Fig. 4C), implying sub-optimal RNA encapsidation for the extended transcripts. To explore whether the amount of packaged RNA directly affects the LV titer, we plotted these values, which revealed a simple positive correlation (Fig. 4D). Linear regression revealed that the LV system would lose most of its RNA packaging capacity when the length of the gene insert exceeds 10 kb, which is roughly the genome size of infectious HIV. These results are also fully consistent with the reduction in LV titer observed for RNA transcripts above 9.8 kb (Fig. 3A, 10⁴ TU/ml at 10 kb).

A) Schematic of the 5’ end of the lentiviral vector with the position of forward and reverse PCR primers marked with arrows. B) Viral RNA was isolated from 5 ml culture supernatant with produced HIV particles. The packaged LV RNA was reverse transcribed into cDNA, which was PCR-amplified and the PCR products were separated on a 1% agarose gel and visualized by ethidium bromide. DNA bands were quantified as a measure of the packaged LV RNA. The construct name is displayed at the top. C) The impact of the LV transgene size (in kb) on the LV RNA content or packaging efficiency. Linear regression was performed (intersection of X-axis at 10.0 kb) and the R squared value is indicated. D) The LV titer is plotted versus the level of packaged LV RNA, showing a linear correlation with an R squared value of 0.37.

This CRISPR-Cas research allowed us to develop a better understanding of the limitations of RNA packaging in the LV system. The production of a significant portion of empty LV particles without transgene RNA should ideally be avoided as they will have no therapeutic meaning. Empty virion particles may also compete with transgene-loaded virions for binding to the appropriate receptor, thereby reducing the chance of transgene transfection. Additional unwanted effects could be exerted by unwanted empty LV particles as they have the ability to enter the target cells, induce an innate immune response etc. We measured that the efficiency of packaging of differently sized RNA transgenes determines the transduction titer and thus the delivery efficiency of the LV system. The maximum size of RNA cargo in LVs was estimated around 10 kb, providing a reference for choosing the appropriate CRISPR-Cas system for a specific gene therapy application. This study confirmed that the transfer efficiency decreased for CRISPR-Cas inserts of increasing size. Remarkably, the LV transduction efficiency measured on the SupT1 T cell line dropped dramatically for RNA transcripts approaching 8 kb as a further increase of 1 kb reduced the transduction efficiency by more than 5-fold. Even though we observed a striking inverse correlation between gene transfer efficiency and the insert size, this likely will not represent a perfect correlation as other properties of the packaged RNA may also have an effect. For instance, we speculate that the low GC-content of the LV 1- and 1 + constructs may explain their low/high packaging efficiency when compared to constructs 2- and 2+.

CRISPR-Cas based strategies provide an effective method to edit genomes, thereby stimulating novel biological research and future clinical applications in a wide range of areas. The field of CRISPR-Cas biotechnology is developing rapidly and an increasing number of novel CRISPR-Cas systems have been discovered or engineered for use in mammalian cells. A number of CRISPR-Cas based clinical trials are currently ongoing or have been planned to start in the near future [29–31], e.g. guiding the future clinical use of somatic cell editing. A safe and effective system for the in vivo delivery of CRISPR-Cas reagents remains one of the most challenging aspects of clinical gene editing. It is likely that the success of CRISPR-Cas based therapies will improve further by development of optimized and fine-tuned vectors to deliver the CRISPR-Cas payloads. Over decades, researchers have developed a variety of methods for DNA transfer such as electroporation [32], microinjection [33, 34] or viral vectors. Microinjection has no apparently limitation for the size of the DNA molecules to be transferred, but only a very limited number of individual cells can be injected. Electroporation is feasible in vitro or ex vivo, but challenging to be approved for clinical use because electroporation can induce significant changes in the transcriptome that can trigger downregulation of metabolic processes [35]. In addition, electroporation can cause tissue damage when not used properly [36]. Viral vectors were engineered for delivery of genetic cargo to a wide range of cell lines in vitro and tissues in vivo [37, 38].

For further improvement, one of the strategies to avoid the RNA packaging limitation will be the use of smaller CRISPR-Cas systems as shown here or the inclusion of truncated versions of the polymerase promoters and/or regulatory elements needed for efficient expression of the CRISPR-Cas components [39, 40]. However, two rather bulky promoters seem required to drive the expression of the small gRNA transcript by RNA polymerase III and the lengthy mRNA transcript encoding the Cas9 endonuclease by RNA polymerase II. Recently, we reported that the H1 promoter has dual-polymerase activity (Pol II and Pol III), which was subsequently used for the design of a single-promoter CRISPR-Cas9 system [23]. This size reduction strategy of the transgene provided a significant titer advantage in the lentiviral vector over the regular CRISPR system. Another strategy to reduce the size of gene expression cassettes is to express polyprotein products from a single promoter, which are subsequently split into separate domains, each with a specialized function. A popular fusion tool is the P2A peptide that can be cleaved by a specific protease, and we demonstrated that the H1 promoter could efficiently drive the expression of such a polyprotein [23]. Such vector improvements become even more important when vector production needs to be scaled up in order to allow clinical studies and possibly commercialization.

Plasmids and constructs

The lentiviral vector Lenti-saCas9-Puro (Addgene plasmid #96920) containing the human codon-optimized SaCas9 expression cassette was used as backbone. The puromycin resistant gene was substituted for eGFP gene between the BspEI and XbaI sites to generate construct 3+ (Fig. 2). The CjCas9, AsCas12a and SpCas9 cassettes were amplified by PCR using High-Fidelity DNA Polymerase (Thermo Fisher) from Addgene plasmid #68338, #84739 [41] and #63592 [42], respectively. The corresponding gRNA scaffolds were amplified from Addgene plasmid #89753 [26], #84739 [41] and #48138 [43], respectively. The SaCas9 cassette of construct 3 + was substituted by each Cas cassette using the NheI and BamHI restriction enzyme sites and the gRNA cassette was substituted for each g/crRNA cassette using the NheI and Acc65I restriction enzyme sites to build constructs 2+, 4 + and 5+, respectively. For construct 1+, the full CRISPR-RfxCas13d cassette, containing the RfxCas13d gene, EF-1α-core promoter and gRNA driven by the U6 promoter was removed from the Addgene plasmid #138147[28] with Acc65I and BamHI restriction enzymes and inserted into the Acc65I and BamHI sites of Lenti-saCas9-Puro using T4 DNA Ligase (Thermo Fisher). Then, the U6 promoter and the RNA scaffolds of each CRISPR-Cas system between the Acc65I and NheI sites were removed from all 1–5 + constructs to make the corresponding 1-5- set.

Lentiviral vector production and transduction

The LV was produced and titrated as described previously [44]. HEK293T cells were cultured in DMEM (Life Technologies) complemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin at 37˚C and 5% CO₂. LV was produced by co-transfection of packaging plasmids pSYNGP [45], pRSV-rev and pVSV-g [46] and an equimolar amount of LV plasmid with Lipofectamine 2000 (Invitrogen). After transfection, the medium was replaced with OptiMEM (Invitrogen). After two days, the LV-containing supernatant was collected, filtered (0.45 µm) and concentrated approximately 20-fold using Vivaspin6 100,000 MWCO centrifugal filter units (Sartorius) at 10,000 rpm for at least 1 h at 4˚C. The CA-p24 protein level was measured before and after concentration of the LV stock by an in-house CA-p24 ELISA assay. SupT1 cells (ATCC CRL-1942) were grown in advanced RPMI 1640 (GIBCO BRL) supplemented with L-glutamine, 1% FCS, 1% penicillin/streptomycin and transduced with concentrated lentiviral vectors (LV). The LV titer was determined three days post-transduction by quantitating GFP-expression using flow cytometry. Transduced cells were washed with fluorescence-activated cell sorting (FACS) buffer (PBS with 2% FCS), and the percentage of cells expressing GFP was measured by a BD LSR Fortessa interfaced with the FACS-Diva software. The mean fluorescence intensity (MFI) of GFP was analyzed by FlowJo version X software. The transduction titer was calculated following the formula: Transducing units per mL (TU/ml) = cell number x ((percentage of fluorescent cells/100)/dilution factor of the vector).

Viral RNA level measurement

The LV was produced by co-transfection of 5 µg of a LV-construct with the three packaging plasmids in 2×10⁶ HEK293T cells in a T25 flask. After two days, virion particles in the culture supernatant were concentrated some 20-fold using Vivaspin6 100,000 MWCO centrifugation filters and the viral RNA extracted by mixing 0.2 ml supernatant with 1 ml Trizol at room temperature for 5 min. Viral RNA was extracted with 1/5th volume of Chloroform (Sigma-Aldrich). The sample was centrifuged at 12,000x g at 4°C for 12 min and the upper water phase was collected and 0.5 ml isopropanol was added to precipitate the RNA. Samples were incubated at room temperature for 10 min and then centrifuged at 12,000x g for 10 min at 4°C. The RNA pellet was collected and washed with 75% ethanol and centrifuged at 7,500x g for 5 min at 4°C. The RNA was dried and dissolved in 10 µl RNase-free water. 8 µl of isolated viral RNA was used for reverse transcription using iScript™ Reverse Transcription kit (BIO-RAD). 1 µl of cDNA products were quantified by PCR using primers targeting the gag gene (Fwd: 5’-ACGATTCGCAGTTAATCCTGG-3’ and Rev: 5’-ACTTTTGTTTTGCTCTTCCT-3’) and DreamTaq DNA polymerase (Thermo Fisher) Cycling conditions were 95°C for 5 min and 20 cycles of 95°C for 15 s, 55°C for 15 s and 72°C for 15 s. Triplicates were included for all samples PCR products were analyzed on ethidium bromide-stained 1% agarose gels in 0.5x Tris-borate-EDTA (TBE) (Thermo Fisher Scientific) and electrophoresed at 90 V for at least 1 h. DNA bands were quantified using ImageJ software.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9 (GraphPad). Results are presented as the mean values ± SD. Comparisons between vectors was analyzed by one-way ANOVA and the Brown-Forsythe test. Significance was set at p < 0.05. For the linear regression analysis, we considered variables relevant when R squared was greater than 0.3.

Campylobacter jejuni (CjCas9)

CRISPR RNA (crRNA)

double-stranded DNA (dsDNA)

G protein of vesicular stomatitis virus (VSV-G)

guide RNA (gRNA)

hematopoietic stem cell (HSCs)

human immunodeficiency virus (HIV)

kilobases (kb)

Lentiviral vectors (LVs)

non-homologous end joining (NHEJ)

single-stranded DNA (ssDNA)

Staphylococcus aureus Cas9 system (SaCas9)

Streptococcus pyogenes Cas9 (SpCas9)

trans-acting CRISPR RNA (tracrRNA)

Ethics approval and consent to participate

Not applicable

Consent to publish

Not applicable

Availability of data and materials

Not applicable

Competing interests

The authors declare no competing interests.

Author’s information

Ben Berkhout is an Associate Editor on the journal and a Guest Editor for the collection

Funding

MF is the recipient of a Chinese Government Scholarship (CSC). This work was supported by NIH RO1 grant 1R01AI145045IH (to BB and EHC).

Authors' Contributions

MF and EHC designed the experiments. MF conducted the experiments. MF, EHC and BB analysed the data and drafted the manuscript.

Acknowledgements

Not applicable

Eyquem, J., et al., Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature, 2017. 543(7643): p. 113–117.
Frangoul, H., et al., CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia. N Engl J Med, 2021. 384(3): p. 252–260.
Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816–21.
Zetsche, B., et al., Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 2015. 163(3): p. 759–71.
Shmakov, S., et al., Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol Cell, 2015. 60(3): p. 385–97.
Cox, D.B.T., et al., RNA editing with CRISPR-Cas13. Science, 2017. 358(6366): p. 1019–1027.
Hsu, P.D., E.S. Lander, and F. Zhang, Development and applications of CRISPR-Cas9 for genome engineering. Cell, 2014. 157(6): p. 1262–1278.
Gao, Z., et al., Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA. Nucleic Acids Res, 2020. 48(10): p. 5527–5539.
Magrin, E., et al., Long-term outcomes of lentiviral gene therapy for the beta-hemoglobinopathies: the HGB-205 trial. Nat Med, 2022. 28(1): p. 81–88.
Naldini, L., et al., In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 1996. 272: p. 263–267.
Howe, S.J., et al., Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest, 2008. 118(9): p. 3143–3150.
Hacein-Bey-Abina, S., et al., LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science, 2003. 302(5644): p. 415–9.
Wu, C. and C.E. Dunbar, Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity. Front Med, 2011. 5(4): p. 356–71.
Themis, M., et al., Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice. Mol Ther, 2005. 12(4): p. 763–71.
Dull, T., et al., A third-generation lentivirus vector with a conditional packaging system. J Virol, 1998. 72(11): p. 8463–71.
Cavazzana-Calvo, M., et al., Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature, 2010. 467(7313): p. 318–22.
Thompson, A.A., et al., Gene Therapy in Patients with Transfusion-Dependent beta-Thalassemia. N Engl J Med, 2018. 378(16): p. 1479–1493.
Dishart, K.L., et al., Third-generation lentivirus vectors efficiently transduce and phenotypically modify vascular cells: implications for gene therapy. J Mol Cell Cardiol, 2003. 35(7): p. 739–48.
Naldini, L., D. Trono, and I.M. Verma, Lentiviral vectors, two decades later. Science, 2016. 353(6304): p. 1101–2.
Dull, T., et al., A third-generation lentivirus vector with a conditional packaging system. J Virol, 1998. 72(11): p. 8463–8471.
Kumar, M., et al., Systematic determination of the packaging limit of lentiviral vectors. Hum Gene Ther 2001. 12(15): p. 1893–1905.
Cante-Barrett, K., et al., Lentiviral gene transfer into human and murine hematopoietic stem cells: size matters. BMC Res Notes, 2016. 9: p. 312.
Gao, Z., E. Herrera-Carrillo, and B. Berkhout, A Single H1 Promoter Can Drive Both Guide RNA and Endonuclease Expression in the CRISPR-Cas9 System. Mol Ther Nucleic Acids, 2019. 14: p. 32–40.
Sweeney, N.P. and C.A. Vink, The impact of lentiviral vector genome size and producer cell genomic to gag-pol mRNA ratios on packaging efficiency and titre. Mol Ther Methods Clin Dev, 2021. 21: p. 574–584.
Tan, Y., et al., Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity. Proc Natl Acad Sci U S A, 2019. 116(42): p. 20969–20976.
Kim, E., et al., In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun, 2017. 8: p. 14500.
Jacobi, A., et al., Engineered AsCas12a Variants with Enhanced Activity and Broadened PAM Compatibility. Molecular Therapy, 2019. 27(4): p. 351–351.
Wessels, H.H., et al., Massively parallel Cas13 screens reveal principles for guide RNA design. Nature Biotechnology, 2020.
Zhou, H., et al., Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease in Mice. Cell, 2020. 181(3): p. 590–603.e16.
Platt, R.J., et al., CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell, 2014. 159(2): p. 440–55.
Wang, C.H., et al., CRISPR-engineered human brown-like adipocytes prevent diet-induced obesity and ameliorate metabolic syndrome in mice. Sci Transl Med, 2020. 12(558).
Ma, X., et al., Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells. Nat Commun, 2018. 9(1): p. 1303.
Hur, J.K., et al., Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol, 2016. 34(8): p. 807–8.
Wu, H., et al., Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems. Cell Mol Life Sci, 2018. 75(19): p. 3593–3607.
Cromer, M.K., et al., Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34(+) Hematopoietic Stem and Progenitor Cells. Mol Ther, 2018. 26(10): p. 2431–2442.
Potter, H. and R. Heller, Transfection by Electroporation. Curr Protoc Mol Biol, 2018. 121: p. 9.3.1–9.3.13.
Kotterman, M.A. and D.V. Schaffer, Engineering adeno-associated viruses for clinical gene therapy. Nat Rev Genet, 2014. 15(7): p. 445–51.
Joglekar, A.V. and S. Sandoval, Pseudotyped Lentiviral Vectors: One Vector, Many Guises. Hum Gene Ther Methods, 2017. 28(6): p. 291–301.
Gao, Z., et al., Extinction of all infectious HIV in cell culture by the CRISPR-Cas12a system with only a single crRNA. Nucleic Acids Res, 2020.
Gao, Z., et al., Engineered miniature H1 promoters with dedicated RNA polymerase II or III activity. J Biol Chem, 2021. 296: p. 100026.
Zetsche, B., et al., Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nature Biotechnology, 2017. 35(1): p. 31–34.
Chen, S., et al., Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell, 2015. 160(6): p. 1246–60.
Ran, F.A., et al., Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 2013. 8(11): p. 2281–2308.
Ter Brake, O., et al., Silencing of HIV-1 with RNA interference: a multiple shRNA approach. Mol Ther 2006. 14(6): p. 883–892.
Kotsopoulou, E., et al., A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene. J Virol 2000. 74: p. 4839–4852.
Zufferey, R., et al., Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol, 1998. 72(12): p. 9873–9880.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Impact of the size of CRISPR-Cas gene cassettes on the packaging efficiency and transduction titer of lentiviral vectors

Status:

Version 1

Abstract

Figures

Introduction

Results

LV production is not affected by increasing size of the transgene cassette

The LV transduction titer is significantly reduced for larger CRISPR-Cas transgenes

Large RNA transcripts are less efficiently packaged in LV particles

Discussion

Materials And Methods

Plasmids and constructs

Lentiviral vector production and transduction

Viral RNA level measurement

Statistical analysis

Abbreviations

Declarations

Competing interests

References

Additional Declarations

Status:

Version 1