Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated

Clearly defined error bars
State explicitly what error bars represent (e.g. SD, SE, CI) Our web collection on statistics for biologists may be useful.

Software and code
Policy information about availability of computer code

Data collection
Provide a description of all commercial, open source and custom code used to collect the data in this study, specifying the version used OR state that no software was used.

Data analysis
Provide a description of all commercial, open source and custom code used to analyse the data in this study, specifying the version used OR state that no software was used.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

April 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
No sample size calculation was performed. Most of our experiments involved testing whether Nanoblades mediate genome editing in cultured cells where sample size is not relevant. Experiments involving animals did not require statistical analysis of the results as we were mainly testing for the ability of Nanoblades to mediate genome editing. We therefore did not perform any sample size calculation.
Data exclusions We did not exclude any data in any of the experiments performed in cultured cells.
Regarding the experiments consisting in retro-orbital injection of Nanoblades in FRG mice ( Figure 5), we decided to exclude results from 2 controls of the second biological replicate (Figure 5b bottom panel) to improve the clarity of the figure. Indeed, all negative controls were negative as expected, so we decided to display only 2 out of the 4 controls performed. The data is available upon request.

Replication
Most experiments performed in cultured cells were performed in three independent biological replicates. Experiments involving the generation of transgenic animals ( Figure 4 and Supplementary Figure 5) were performed in two independent injections of individuals. As we were looking for a binary response (gene inactivation or not) and did not plan on doing any statistical analysis, the number of injected embryos was defined in each replicate by the number of embryos available. Experiments involving retro-orbital injection of Nanoblades in FRG mice ( Figure 5) were performed in 2 independent biological replicates, the first consisting in the injection of 8 mice (3 controls and 5 aiming at disrupting the Hpd gene) for the first replicate and 14 mice for the second biological replicate (6 controls and 8 aiming at disrupting the HPD gene). Again, we were looking for a binary response (gene editing or not) so the number of animals used in each experiment was mainly defined by their availability. Dose dependent editing of HEK293 presented in Figure 1c corresponds to a single replicate where the Cas9 amount within Nanoblades was dosed by Elisa, but the experiment was performed in additional replicates to show a dose response in genome editing. Control experiments to monitor the co-delivery of cellular proteins by Nanoblades (presented in Supplementary Figure 2c and d) were performed in two independent replicates.
Randomization Our experiments did not involve testing on a large number of individuals or groups of individuals. As such randomization of samples is not relevant to our study.

Blinding
For all animal experiments, the persons extracting total DNA and monitoring genome-editing did not have access to group allocation until we obtained the results from our tests (they only had access to the animal identification number).
Reporting for specific materials, systems and methods Obtaining unique materials All plasmids required to produce Nanoblades are readily available upon request to the authors and will be also available through addgene. The plasmid coding for the genetically engineered Baboon R-less envelope that is important to produce efficient Nanoblades is available upon request from Els Verhoeyen (els.verhoyen@ens-lyon.fr). Nanoblades are protected by a patent (WO2017068077 A1) and therefore all commercial applications derived from our materials must be licensed.

Authentication
Most of our cell lines were bought from commercial vendors specifically for this project. We therefore did not perform any authentication test. U2OS cells used in Figure 1 have not been authenticated.

Mycoplasma contamination
All immortalized cell lines used in this study were tested negative for mycoplasma contamination using a commercial kit (MycoAlert™ Mycoplasma Detection Kit, catalog number: LT07).

April 2018
Commonly misidentified lines (See ICLAC register) None of the cell lines used in our study are listed in the ICLAC database of commonly misidentified cell lines.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Wild animals
The study did not involve the use of wild animals.

Field-collected samples
The study did not involve samples collected from the field.