Academic journal article Genetics

Strategies for Efficient Genome Editing Using CRISPR-Cas9

Academic journal article Genetics

Strategies for Efficient Genome Editing Using CRISPR-Cas9

Article excerpt

PROGRAMMABLE DNA endonucleases have transformed the analysis of biological processes by enabling targeted genome editing in diverse species (reviewed in Urnov et al. 2010; Joung and Sander 2013; Carroll 2014; Chandrasegaran and Carroll 2016; Knott and Doudna 2018). These nucleases catalyze DNA breaks at specified sequences and trigger precise and imprecise repair outcomes via different pathways (Jasin and Haber 2016). Imprecise repair pathways introduce small-to-large insertions and deletions. Precise repair pathways insert specific DNA changes from either homologous chromosomes or exogenous, homologous templates. Our goal is to improve the frequency and fidelity of genome editing by investigating the rules governing repair pathway choices and repair outcomes.

The programmable endonuclease most widely deployed is the Streptococcus pyogenes CRISPR-associated protein 9 (SpCas9 orCas9) (reviewed in Mali etal. 2013a; Jiang and Doudna 2017). Cas9 is directed to its DNA target by a guide RNA that pairs with 20 bases of target DNA, called the spacer sequence (Mojica et al. 2009; Garneau et al. 2010; Jinek et al. 2012). The chief constraint limiting target choice is the requirement for an NGG motif to border the DNA sequence that is complementary to the spacer. The NGG motif is called the protospacer adjacent motif (PAM). The Cas9-guide RNA ribonucleoprotein (RNP) complex scans the genome, binds to PAM sequences, and melts the adjacent duplex DNA (Sternberg et al. 2014). If the neighboring nucleotides are complementary to the guide RNA, Cas9 undergoes a conformational change that activates its nuclease domains to make a DNA doublestrand break (DSB) (Anders et al. 2014; Sternberg et al. 2015).

These basic principles governing Cas9 targeting led to its widespread usage, but the repertoire of strategies that can be used to achieve desired genomic changes has some limitations. In this study, we develop strategies that overcome several impediments to achieve efficient Cas9 targeting and predictable DSB repair outcomes in the nematode Caenorhabditis elegans. These approaches can be exploited to improve genome editing across diverse plant and animal species.

We demonstrate that both imprecise and precise DNA repair from a single DSB is asymmetric, favoring repair in only one direction. We have exploited this property to establish guidelines for effective PAM choice and design of single-stranded repair templates to achieve high frequency insertion of desired changes within close proximity (30 bp) to a DSB and on a particular side of the DSB. We also devised efficient strategies to insert long non-homologous fragments of DNA (~10 kb) at DSB sites and to engineer small specific changes at considerable distance from a DSB (1.5 kb) or to incorporate a series of nucleotide substitutions throughout an entire locus, all without coinserting a selectable marker. These strategies are also useful for inserting DNA in sites such as AT-rich regions that are devoid of potential PAMs. These successes required that we optimize Cas9 delivery methods and guide RNA design.

We also expanded the repertoire of Cas9-dependent coconversion markers to be used in conjunction with the tools to edit targets of choice. These selectable markers, appropriate for diverse nematode species, enable the rapid identification of Cas9-edited animals that are also likely to have desired edits in the targets of choice. This approach is particularly useful when searching for edited targets that fail to cause visible phenotypes.

Finally, we devised and exploited an editing strategy to explore the timing, location, frequency, sex dependence, and categories of DSB repair events. We developed loci with allele-specific targets for Cas9 cleavage that can be contributed from either male or hermaphrodite germ cells during mating. We found that male sperm DNA was generally more permissive to Cas9 editing than hermaphrodite germ cell DNA. The frequency of recovering mutations in the locus of interest is higher if editable alleles of both the target locus and the co-conversion marker are contributed from the same parent during mating. …

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