TABLE 4.

CRISPR-Cas9 component format advantages, limits and advances.

Format	Advantages	Limitations	Advances demonstrated in any species (rat in bold)
Cas9
Plasmid	No limit on insert size Easy engineering High expression	Delayed activity Mosaicism Increased off-targets Delayed activity	Cas9 protein allowing rapid and more efficient editing (Kim et al., 2014; Ménoret et al., 2015) Large editing toolbox variants (Table 3) Improved chromatin accessibility (Chen F. et al., 2017; Ding et al., 2019) Cas9 engineered to activate repair pathways (Charpentier et al., 2018; Tran et al., 2019) Cas9 engineering to be degraded in G1 (Gutschner et al., 2016; Charpentier et al., 2018; Lomova et al., 2019)
mRNA	Expression faster than plasmid Limit mosaicism and off-targets	Delayed activity In vitro transcription efficiency/toxicity
Protein	Ready to cut Limit mosaicism and off-targets Affordable and high quality	Crystallization at high dose In vivo stability potentially immunogenic
gRNA
Plasmid	No limit on insert size Easy to engineer	Delayed activity	Chemical modification (Kim S. et al., 2018; Filippova et al., 2019) Essential sequence, secondary structures and functional modules of gRNA (Briner et al., 2014; Kartje et al., 2018) Overlapping gRNA (Jang et al., 2018) gRNA engineering to activate repair pathways (Nakade et al., 2018; Tran et al., 2019)
IVT sgRNA	Easy to produce and use Flexible in sequence and length Efficient	Time-consuming production Induced immune responses Limited in chemical modification
Synthetic sgRNA	Affordable and high quality Chemical modifications Ready to use Efficient	Order full sgRNA for each project Long RNA synthesis Difficulties in adding fluorophore for tracking
Synthetic dgRNA	Short RNA synthesis Low cost and high quality Same tracrRNA for all project Chemical modifications Fluorophores added for tracking Efficient	crRNA & tracrRNA hybridization in vitro
DNA donor
ssODN	Low cost synthesis High efficacy for mutation or short KI	Limited in length to 200nt	DNA synthesis progresses (Hao et al., 2020) Chemical modification (Renaud et al., 2016; Liang X. et al., 2017; Yu et al., 2020) Insertion close to cut site (Inui et al., 2014; Liang X. et al., 2017) 3′ overhang DNA donor (Liang X. et al., 2017; Hirotsune et al., 2020) Carry to cut site by Cas9 (Ma et al., 2017; Aird et al., 2018; Gu et al., 2018; Ling et al., 2020; Wang Z. et al., 2020) Carry to cut site by gRNA (Carlson-Stevermer et al., 2017; Lee et al., 2017) Carry to cut site by DNA donor engineering (Nguyen et al., 2020) DNA donor in vivo excision from plasmid (Aida et al., 2016; Yao et al., 2017; Zhang et al., 2017)
lsDNA	Usable for long KI	Limited in length Difficult to produce Mutated KI Expensive to synthesize
dsDNA	Usable for long KI Easy to produce and engineer No limit on insert size	Few random insertions
Plasmid	Usable for long KI Easy to produce and engineer No limit on insert size	Few random insertions

IVT, in vitro–transcribed; gRNA, guide RNA; sgRNA, single gRNA; dgRNA, dual gRNA; ssODN, single-stranded oligonucleotides; lsDNA, long single-stranded DNA; dsDNA, linear double-stranded DNA.