Table 1.

Summary of chemical modifications of Cas9 crRNAs

Modification	Prosa	Consb	Reference	Key result	Cells	Assays
	Comav Inexpn Simple	Modstb	Gagnon32	18 5′-terminal DNA tolerated in crRNA	In vitro	In vitro
			Xue, Langer, Anderson33	10 5′-terminal DNA in crRNAs abolish off-target activity in Hepa1-6 and HEK293 cells. Optimized crRNA has 8 5′- and 16 3′-DNA	HEK293T cells constitutively expressing GFP and Cas9	GFP fluorescence, GUIDE-seq
	Comav Affinity	Modstb	Bennett, Cleveland42	Seven 5′-terminal 2′-F, part of optimized crRNA that reduced off-target activity	HEK293T, tracrRNA and Cas9 plasmid	Surveyor
			Anderson58	Structure-guided 2′-F modification of spacer is tolerated in highly modified sgRNA	In vivo (mouse liver)	TIDE
			Khvorova, Watts, Sontheimer59	Structure-guided modification of >80% as 2′-OMe or 2′-F in crRNA/tracrRNA increases activity	HEK293T cells constitutively expressing mCherry	mCherry fluorescence, TIDE
	Comav Inexpn Affinity Metstab	Steric	Bruhn, Porteus41	Three 3′- and 5′-terminal 2′-OMe slightly increased the activity of sgRNA	K562, CD34+ and T cells, Cas9 plasmid	T7EI, TIDE
			Anderson58	Structure-guided 2′-OMe modification is tolerated in invariable region (but not in spacer) of highly modified sgRNA	In vivo (mouse liver)	TIDE
			Khvorova, Watts, Sontheimer59	Structure-guided modification of >80% as 2′-OMe or 2′-F in crRNA/tracrRNA increases activity	HEK293T cells constitutively expressing mCherry	mCherry fluorescence, TIDE
	Affinity Metstab	Synth Costly Steric	Bennett, Cleveland42	Five (S)-cEt in shortened (12-nt) repeat region part of optimized crRNA that reduced off-target activity	HEK293T, tracrRNA and Cas9 plasmid	Surveyor
	Amidite Affinity Metstab	Toxic Costly Steric	Hubbard43	Up to six LNA are tolerated but less effective than BNANC in reducing off-target activity	U2OS and HeLa cells constitutively expressing Cas9	T7EI, NGS
	Affinity	Synth Costly Steric	Hubbard43	Up to six BNANC slightly reduce Cas9 on-target activity while broadly reducing off-target activity	U2OS and HeLa cells constitutively expressing Cas9	T7EI, NGS
	Comav Metstab Inexpn Simple Phrmkin	Lowaff Toxic Diast	Bennett, Cleveland42	Full PS (R = OH) modification of crRNA increased activity ~four-fold	HEK293T, tracrRNA and Cas9 plasmid	Surveyor
			Bruhn, Porteus41	Three 3′- and 5′-terminal MS (R = OMe) increased the activity of sgRNA	K562, CD34+ and T cells, Cas9 plasmid	T7EI, TIDE
			Anderson58	Structure-guided PS modification is tolerated at the 3′- and 5′-ends of highly modified sgRNA	In vivo (mouse liver)	TIDE
	Simple Metstab	Costly	Bruhn50	MP of phosphates 4, 5 and 7; 9-12; and 14 and 16 improved specificity >10-fold	K562, iPS, and CD34+ cells	NGS
	Simple Metstab	Costly	Bruhn, Porteus41	Three 3′- and 5′-terminal MSP strongly increased the activity of sgRNA	K562, CD34+ and T cells, Cas9 plasmid	T7EI, TIDE
	Metstab	Synth Costly	Rozners51	Individual AM1 were well tolerated in the PAM-distal region of crRNAs	HEK293 cells constitutively expressing Cas9	T7EI

^aAbbreviations for Pros: Affinity – enhanced affinity for complementary nucleic acids, Amidite – amidite is commercially available, Comav – commercially available as an RNA modification, Inexpn – inexpensive to manufacture, Metstab – enhanced metabolic stability, Phrmkin – improved pharmacokinetics, Simple – simple synthesis.

^bAbbreviations for Cons: Costly – high cost, Diast – diastereomeric mixtures lead to heterogeneous compounds, Lowaff – reduced affinity for complementary nucleic acids, Modstb – modest metabolic stability, Steric – steric bulk may interfere with RNA-protein interactions, Synth – multistep synthesis of monomers required, Toxic – potential toxicity concerns.