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CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing

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CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing International Journal of Molecular Sciences Article CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing Nina Reuven * , Julia Adler, Nadav Myers and Yosef Shaul * Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel; [email protected] (J.A.); [email protected] (N.M.) * Correspondence: [email protected] (N.R.); [email protected] (Y.S.) Abstract: The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has rev- olutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology- directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is “scarless” since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system’s utility in generating cell lines with biologically relevant Citation: Reuven, N.; Adler, J.; mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for Myers, N.; Shaul, Y. CRISPR selecting genome-edited cells requiring HDR. Co-Editing Strategy for Scarless Homology-Directed Genome Editing. Keywords: gene targeting; CRISPR/Cas9; genome editing; endogenous mutagenesis in cell lines; Int. J. Mol. Sci. 2021, 22, 3741. co-editing; scarless selection; temperature-sensitive cell lines https://doi.org/10.3390/ ijms22073741 Academic Editor: Vladimir Trifonov 1. Introduction Received: 31 January 2021 The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) Accepted: 26 March 2021 system and its application in a wide variety of organisms has revolutionized genome Published: 3 April 2021 editing. Cas, CRISPR-associated (Cas9), is a DNA endonuclease, which cleaves DNA specifically at the target site dictated by its associated guide RNA. Various methods have Publisher’s Note: MDPI stays neutral been developed to simplify the introduction of Cas9 and a programmable guide RNA with regard to jurisdictional claims in into the cells, including the modification of the guide so that it is expressed as a single published maps and institutional affil- guide RNA (sgRNA), encompassing the sequence specific to the targeted locus (guide) as iations. well as the tracrRNA portion necessary for interaction with the Cas9 protein [1–5]. The CRISPR/Cas9 system provides an elegant and efficient means of creating a specific double- strand break (DSB), which can then be exploited to edit the DNA at the break site. However, repair of the break depends on the endogenous DNA repair machinery. The predominant Copyright: © 2021 by the authors. pathway in most mammalian cells is nonhomologous end-joining (NHEJ), which is highly Licensee MDPI, Basel, Switzerland. efficient but error-prone, often making small insertions or deletions (indels) at the repair This article is an open access article site that can result in frameshift mutations leading to gene disruption (Figure1). Thus, the distributed under the terms and CRISPR system can be used to inactivate (knockout) genes by cutting the DNA and relying conditions of the Creative Commons on NHEJ to repair it inaccurately. To make specific template-directed edits in the genome, Attribution (CC BY) license (https:// the endogenous homologous recombination (HR) pathway is usually required (Figure1). creativecommons.org/licenses/by/ 4.0/). Int. J. Mol. Sci. 2021, 22, 3741. https://doi.org/10.3390/ijms22073741 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 16 Int. J. Mol. Sci. 2021, 22, 3741 by cutting the DNA and relying on NHEJ to repair it inaccurately. To make specific2 tem- of 15 plate-directed edits in the genome, the endogenous homologous recombination (HR) pathway is usually required (Figure 1). Figure 1. ClusteredClustered regularly regularly interspaced interspaced short short palindromic palindromic repeat repeat (CRISPR)/Cas9 (CRISPR)/Cas9 gene gene editing editing depends on on cellular cellular DNA DNA repair repair pathways. pathways. CRIS CRISPR/Cas9PR/Cas9 creates creates a double-strand a double-strand break break in the in the DNA at the specific specific location location dictated dictated by by the the gu guideide RNA RNA (sgRNA). (sgRNA). Cellular Cellular DNA DNA repair repair pathways pathways then repair the the break, break, with with nonhomologous nonhomologous end- end-joiningjoining being being the the predominant predominant pathway pathway in in most most mammalian cells. Editing a gene according to an exogenously added donor DNA template re- mammalian cells. Editing a gene according to an exogenously added donor DNA template requires quires homology-directed repair (HDR), active during the S/G2 cell cycle phase. Fusing a recruit- homology-directed repair (HDR), active during the S/G2 cell cycle phase. Fusing a recruitment ment domain for cellular repair factors, such as the MRN complex, increases HDR-dependent CRISPRdomain forediting. cellular Illustrated repair factors,is Cas9 suchwith asa 126aa the MRN MRN-recruiting complex, increases domain HDR-dependentfrom HSV-1 UL12 CRISPR fused atediting. the N- Illustrated or C-terminus is Cas9 of withCas9 a(U 126aaN and MRN-recruiting UC, respectively). domain Figure from created HSV-1 with UL12 Biorender.com. fused at the N- or C-terminus of Cas9 (UN and UC, respectively). Figure created with Biorender.com. The HR pathway is mostly restricted to the S/G2 cell cycle phase; thus, the window for thisThe type HR pathwayof editing is is mostly narrow. restricted Various to approaches the S/G2 cell have cycle been phase; used thus, to boost the windowHDR in mammalianfor this type cells of editing in order is narrow.to improve Various the yiel approachesd of HDR have edited been cells used (reviewed to boost in HDR [6,7]). in mammalian cells in order to improve the yield of HDR edited cells (reviewed in [6,7]). Strategies implemented have included inhibiting NHEJ or inducing HDR genetically and Strategies implemented have included inhibiting NHEJ or inducing HDR genetically and pharmacologically [8–10] and by restricting Cas9 cleavage to the S/G2 cell cycle phase by pharmacologically [8–10] and by restricting Cas9 cleavage to the S/G2 cell cycle phase by regulating the timing of its degradation or potentially by using an activatable Cas9 [11,12]. regulating the timing of its degradation or potentially by using an activatable Cas9 [11,12]. Additionally, extra domains have been added to Cas9 that help to promote the HDR pro- Additionally, extra domains have been added to Cas9 that help to promote the HDR cess [13,14] or that function as recruiting domains for the cellular HR machinery [15]. Our process [13,14] or that function as recruiting domains for the cellular HR machinery [15]. laboratory has shown that chimeric Cas9 proteins with recruitment domains fused to ei- Our laboratory has shown that chimeric Cas9 proteins with recruitment domains fused to ther the N-(UN Cas9) or C- (UC Cas9) terminus of Cas9 promoted a two-fold increase in either the N-(U Cas9) or C- (U Cas9) terminus of Cas9 promoted a two-fold increase in HDR-dependentN editing (FigureC 1) [16]. This short 126aa domain, derived from the HSV- HDR-dependent editing (Figure1)[ 16]. This short 126aa domain, derived from the HSV-1 1 UL12 protein, recruits the endogenous MRN complex (Mre11, Rad50, Nbs1). UL12 protein, recruits the endogenous MRN complex (Mre11, Rad50, Nbs1). Since the efficiency of CRISPR editing, even with enhancements, is often low, proto- Since the efficiency of CRISPR editing, even with enhancements, is often low, protocols colsto select to select for edited for edited cells havecells beenhave developed.been developed. When When using using co-editing co-editing to insert to ainsert selectable a se- lectablemarker inmarker addition in addition to editing to aediting gene ofa gene interest of interest (GOI), the(GOI), selected the selected cells will cells be will highly be highlyenriched enriched for the for second the second desired desired mutation mutation as well as [17 well]. Here [17]. weHere show we show another another strategy strat- to egyselect to forselect successfully for successfully edited cells,edited using cells, reversion using reversion of an endogenous of an endogenous temperature-sensitive temperature- (ts)sensitive mutation (ts) mutation as the selectable as the selectable marker. Wemarker. tested We this tested strategy this strategy on a cell on line a thatcell line we hadthat previouslywe had previously developed, developed, with a with ts mutation a ts mutation in the in essential the essentialTAF1 TAF1gene gene [16]. [16]. TAF1 TAF1
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