Strategies for Efficient Genome Editing Using CRISPR-Cas9

Abstract

The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and nonmodel organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes for both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-strand breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans. We also devised strategies to insert long (10 kb) exogenous sequences and incorporate multiple nucleotide substitutions at a considerable distance from DSBs. We expanded the repertoire of co-conversion markers appropriate for diverse nematode species. These selectable markers enable rapid identification of Cas9-edited animals also likely to carry edits in desired targets. Lastly, we explored the timing, location, frequency, sex dependence, and categories of DSB repair events by developing loci with allele-specific Cas9 targets that can be contributed during mating from either male or hermaphrodite germ cells. We found a striking difference in editing efficiency between maternally and paternally contributed genomes. Furthermore, imprecise repair and precise repair from exogenous repair templates occur with high frequency before and after fertilization. Our strategies enhance Cas9-targeting efficiency, lend insight into the timing and mechanisms of DSB repair, and establish guidelines for achieving predictable precise and imprecise repair outcomes with high frequency.

Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans

“…The nucleotides at the top represent the sequence of the 169-bp ssODN repair template consisting of the 99-bp mCherry 1-3 sequence flanked by 35-bp homology arms. (Farboud et al 2019). The lower efficiency observed when inserting this larger fragment could also be due to an inefficient crRNA.…”

“…Simultaneous editing of other genes is commonly used as positive control of Cas9 activity. Several co-CRISPR strategies are being followed to produce visible phenotypes or to repair lethal mutations (Kim et al 2014;Ward 2015;Farboud et al 2019). The editing of dpy-10 is widely used for generating dominant phenotypes and for being suitable for any genetic background (Arribere et al 2014;Kim et al 2014).…”

“…However, the challenge currently lies in inserting larger fragments of DNA, such as the integration of fluorescent reporters into endogenous loci or gene replacements. Even though it has been demonstrated that this can be achieved by targeting vectors with homology arms (Dickinson et al 2013;Schwartz and Jorgensen 2016;Farboud et al 2019;McDiarmid et al 2018), this method requires additional time to construct the correct plasmid and, in some cases, remove the markers from the genome. An alternative option is to use a PCR product with 35-bp flanking sites that are homologous to the insertion site as a repair template (Paix et al 2015).…”

Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasiteSchistosoma mansoni

“…In addition, off-target effects are common, in particular when long dsRNA molecules are used, and the gene silencing is typically not heritable unless a RNAi-based construct is employed as a transgene expressed in the germ line [12]. Therefore, to truly examine gene function across the life-cycle, transgenesis-based approaches that are available in model organisms [13,14] need to be developed for S. mansoni, including the ability to create genetically-modified parasite strains with homozygous gene knock-outs, and specific gene mutations.…”

Peel-1 negative selection promotes screening-free CRISPR-Cas9 genome editing in Caenorhabditis elegans

“…replacing C. elegans genes with genes from other species, deleting entire open reading frames, or deleting several adjacent genes simultaneously) [24,25]. However, several methods based on linear DNA repair templates have recently been developed that allow for efficient generation of small edits (typically up to fluorophore-sized insertions) in C. elegans without the need for cloning [2,22,[39][40][41][42][43][44]. While these approaches currently require manual screening/isolation and/or PCR validation to identify genome-edited animals and have lower efficiency for large edits, with further optimization, peel-1-DMS could be adapted to these strategies to allow cloning-and screening-free genome editing.…”